U.S. patent application number 11/444107 was filed with the patent office on 2007-02-22 for methods for producing micrornas.
This patent application is currently assigned to Cold Spring Harbor Laboratory. Invention is credited to Ross Dickins, Gregory J. Hannon, Scott W. Lowe.
Application Number | 20070044164 11/444107 |
Document ID | / |
Family ID | 37943875 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070044164 |
Kind Code |
A1 |
Dickins; Ross ; et
al. |
February 22, 2007 |
Methods for producing microRNAs
Abstract
The invention relates to recombinant vectors for inducible
and/or tissue specific expression of double-stranded RNA molecules
that interfere with the expression of a target gene. In certain
embodiments, the invention relates to the use of Tet
(tetracycline)-responsive RNA Polymerase II (Pol II) promoters
(e.g., TetON or TetOFF) to direct inducible knockdown in certain
cells of an integrated or an endogenous gene, such as p53. The
invention also relates to a method for producing transgenic animals
(e.g., mice) expressing inducible (such as tetracycline-regulated),
reversible, and/or tissue-specific double-stranded RNA molecules
that interfere with the expression of a target gene.
Inventors: |
Dickins; Ross; (Cold Spring
Harbor, NY) ; Hannon; Gregory J.; (Huntington,
NY) ; Lowe; Scott W.; (Cold Spring Harbor,
NY) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Cold Spring Harbor
Laboratory
Cold Spring Harbor
NY
|
Family ID: |
37943875 |
Appl. No.: |
11/444107 |
Filed: |
May 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60686135 |
May 31, 2005 |
|
|
|
Current U.S.
Class: |
800/14 ; 435/325;
435/6.11; 435/6.16; 514/44R; 536/23.2 |
Current CPC
Class: |
C12N 2799/027 20130101;
A01K 2217/05 20130101; A61P 43/00 20180101; C12N 2830/008 20130101;
C12N 2830/003 20130101; C12N 2310/14 20130101; C12N 2840/203
20130101; C12N 2330/30 20130101; A01K 2217/20 20130101; C12N
2310/53 20130101; A01K 67/0271 20130101; C07K 14/4746 20130101;
A01K 2267/0331 20130101; C12N 2310/111 20130101; C12N 15/111
20130101; C12N 15/1135 20130101; A01K 2267/03 20130101; C12N
2800/30 20130101; C12N 2830/006 20130101; C07K 14/82 20130101; A01K
67/0275 20130101; C12N 15/8509 20130101; A01K 2227/105 20130101;
C12N 2320/50 20130101 |
Class at
Publication: |
800/014 ;
514/044; 435/006; 435/325; 536/023.2 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; A61K 48/00 20070101 A61K048/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] Work described herein was funded, in whole or in part, by
Mouse Models of Human Cancer Consortium Grant No. 25480211. The
United States government has certain rights in the invention.
Claims
1. An artificial nucleic acid construct comprising an RNA
Polymerase II (Pol II) promoter operably linked to a coding
sequence for expressing a precursor molecule for an siRNA, said
siRNA inhibiting the expression of a target gene, wherein the
nucleic acid construct directs the expression of the precursor
molecule and/or the siRNA, and substantially inhibits the
expression of the target gene when stably integrated into a host
cell genome.
2. The nucleic acid construct of claim 1, wherein said Pol II
promoter is an inducible promoter, a tissue-specific promoter,
and/or a developmental stage-specific promoter.
3. The nucleic acid of claim 2, wherein the inducible promoter is a
tetracyclin-responsive promoter.
4. The nucleic acid construct of claim 3, wherein the
tetracyclin-responsive promoter is a TetON promoter, the
transcription from which promoter is activated at the presence of
tetracyclin (tet), doxycycline (Dox), or a tet analog.
5. The nucleic acid construct of claim 3, wherein the
tetracyclin-responsive promoter is a TetOFF promoter, the
transcription from which promoter is turned off at the presence of
tetracyclin (tet), doxycycline (Dox), or a tet analog.
6. The nucleic acid construct of claim 2, wherein the Pol II
promoter is an LTR promoter or a CMV promoter.
7. The nucleic acid construct of claim 2, wherein the precursor
molecule is a precursor microRNA.
8. The nucleic acid construct of claim 7, wherein the precursor
microRNA (miR) is an artificial miR comprising coding sequence for
said siRNA for said target gene.
9. The nucleic acid construct of claim 8, wherein the miR comprises
a backbone design of microRNA-30 (miR-30).
10. The nucleic acid construct of claim 8, wherein the miR
comprises a backbone design of miR-15a, -16, -19b, -20, -23a, -27b,
-29a, -30b, -30c, -104, -132s, -181, -191, -223.
11. The nucleic acid construct of claim 2, wherein the precursor
molecule is a short hairpin RNA (shRNA).
12. The nucleic acid construct of claim 1, wherein a single
integrated copy of the nucleic acid construct is sufficient for
substantially inhibiting the expression of the target gene.
13. The nucleic acid construct of claim 1, further comprising an
enhancer for the Pol II promoter.
14. The nucleic acid construct of claim 1, further comprising a
reporter gene under the control of a second promoter.
15. The nucleic acid construct of claim 14, wherein the second
promoter and the reporter gene is downstream of (3'-to) the coding
sequence for the precursor molecule.
16. The nucleic acid construct of claim 15, wherein the reporter
gene is translated from an internal ribosomal entry site (IRES)
between a second promoter and the reporter gene.
17. The nucleic acid construct of claim 1, further comprising at
least one selectable marker.
18. The nucleic acid construct of claim 1, further comprising a
reporter gene, wherein the coding sequence for expressing the
precursor molecule is embeded or inserted into the 5'-UTR
(untranslated region), 3'-UTR, or an intron of the reporter
gene.
19. The nucleic acid construct of claim 1, further comprising a Pol
III promoter upstream of the coding sequence for expressing the
precursor molecule.
20. The nucleic acid construct of claim 1, wherein the target gene
is associated with a disease condition selected from cancer or
infectious disease.
21. The nucleic acid construct of claim 20, wherein the target gene
is over-expressed or abnormally active in the disease.
22. The nucleic acid construct of claim 20, wherein the target gene
is an oncogene or an antagonist/inhibitor or dominant negative
mutation of a tumor suppressor gene.
23. A cell comprising the nucleic acid construct of claim 1.
24. The cell of claim 23, which is a mammalian cell.
25. The cell of claim 23, wherein the Pol II promoter is an
inducible promoter, and wherein the cell further comprises an
additional construct for expressing an activator or an inhibitor of
the inducible promoter.
26. The cell of claim 25, wherein the inducible promoter is a
tet-responsive promoter, and wherein the additional construct
encodes tTA or rtTA.
27. A non-human mammal comprising the cell according to claim
23.
28. The non-human mammal of claim 27, which is a chimeric
mammal.
29. The non-human mammal of claim 27, which is a transgenic
mammal.
30. A method for inhibiting the expression of a target gene of
interest in a cell, comprising introducing a construct according to
claim 1 into the cell, wherein the siRNA molecule derived from the
precursor molecule is specific for the target gene.
31. The method of claim 30, further comprising inhibiting at least
one additional target gene(s) of interest in the cell by
introducing at least one additional constructs according to claim 1
into the cell, wherein each of the siRNA molecules derived from the
precursor molecules are specific for the additional target genes,
respectively.
32. A method for treating a gene-mediated disease, comprising
introducing into an individual having the disease a construct
according to claim 1, where the siRNA derived from the precursor
molecule is specific for the gene mediating the disease.
33. A method of validating a gene as a potential target for
treating a disease, comprising: (1) introducing a construct
according to claim 1 into a cell associated with the disease,
wherein the siRNA molecule derived from the precursor molecule is
specific for the gene; (2) assessing the effect of inhibiting the
expression of the gene on one or more disease-associated phenotype;
wherein a positive effect on at least one disease-associated
phenotype is indicative that the gene is a potential target for
treating the disease.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application Ser. No. 60/686,135, entitled "METHODS
FOR PRODUCING MICRORNAS," and filed on May 31, 2005. The teachings
of the entire referenced application is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] RNA interference (RNAi) has been used to silence the
expression of a target gene. RNAi is a sequence-specific
post-transcriptional gene silencing mechanism triggered by
double-stranded RNA (dsRNA). It causes degradation of mRNAs
homologous in sequence to the dsRNA. The mediators of the
degradation are 21-23-nucleotide small interfering RNAs (siRNAs)
generated by cleavage of longer dsRNAs (including hairpin RNAs) by
DICER, a ribonuclease III-like protein. Molecules of siRNA
typically have 2-3-nucleotide 3' overhanging ends resembling the
RNAse III processing products of long dsRNAs that normally initiate
RNAi. When introduced into a cell, they assemble an endonuclease
complex (RNA-induced silencing complex), which then guides target
mRNA cleavage. As a consequence of degradation of the targeted
mRNA, cells with a specific phenotype of the suppression of the
corresponding protein product are obtained (e.g., reduction of
tumor size, metastasis, angiogenesis, and growth rates).
[0004] The small size of siRNAs, compared with traditional
antisense molecules, prevents activation of the dsRNA-inducible
interferon system present in mammalian cells. This helps avoid the
nonspecific phenotypes normally produced by dsRNA larger than 30
base pairs in somatic cells. See, e.g., Elbashir et al., Methods
26:199-213 (2002); McManus and Sharp, Nature Reviews 3:737-747
(2002); Hannon, Nature 418:244-251 (2002); Brummelkamp et al.,
Science 296:550-553 (2002); Tuschl, Nature Biotechnology 20:446-448
(2002); U.S. Application US2002/0086356 A1; WO 99/32619; WO
01/36646; and WO 01/68836.
SUMMARY OF THE INVENTION
[0005] One aspect of the invention provides an artificial nucleic
acid construct comprising an RNA Polymerase II (Pol II) promoter
operably linked to a coding sequence for expressing a precursor
molecule for an siRNA, the siRNA inhibiting the expression of a
target gene, wherein the nucleic acid construct directs the
expression of the precursor molecule and/or the siRNA, and
substantially inhibits the expression of the target gene when the
artificial nucleic acid construct is stably integrated into a host
cell genome.
[0006] In certain embodiments, the Pol II promoter is an inducible
promoter, a tissue-specific promoter, or a developmental
stage-specific promoter. For example, the inducible promoter may be
a tetracyclin-responsive promoter, including commercially available
TetON promoter (the transcription from which promoter is activated
at the presence of tetracyclin (tet), doxycycline (Dox), or tet
analog), or the TetOFF promoter (the transcription from which
promoter is turned off at the presence of tetracyclin (tet),
doxycycline (Dox), or a tet analog), e.g., those from Clontech,
Inc.
[0007] In other embodiments, the inducible promoter may be selected
from: a promoter operably linked to a lac operator (LacO), a
LoxP-stop-LoxP system promoter, or a GeneSwitch.TM. or T-REx.TM.
system promoter (Invitrogen), or equivalents thereof with identical
or substantially similar mechanisms.
[0008] In yet other embodiments, the Pol II promoter can be any
art-recognized Pol II promoters, such as an LTR promoter or a CMV
promoter.
[0009] In certain embodiments, the precursor molecule may be a
precursor microRNA, such as an artificial miR comprising coding
sequence for the siRNA for the target gene. For example, the miR
may comprise a backbone design of microRNA-30 (miR-30).
Alternatively, the miR may comprise a backbone design of miR-15a,
-16, -19b, -20, -23a, -27b, -29a, -30b, -30c, -104, -132s, -181,
-191, -223. See US 2005/0075492A1 (incorporated herein by
reference).
[0010] In other embodiments, the precursor molecule may be a short
hairpin RNA (shRNA).
[0011] The constructs of the instant invention is highly potent,
and a single integrated copy of the subject nucleic acid construct
is sufficient for substantially inhibiting the expression of the
target gene. "Substantially inhibiting" as used herein includes
inhibiting at least about 20%, or about 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 99% or close to 100% of the expression or mRNA
and/or protein of the target gene.
[0012] The constructs of the invention may further comprise an
enhancer for the Pol II promoter.
[0013] The constructs of the invention may further comprise a
reporter gene under the control of a second promoter, such as a
luciferase, a fluorescent protein (e.g. GFP, RFP, YFP, BFP, etc.),
or an enzyme, or any other art-recognized reporter whose physical
presence and/or activity can be readily assessed using an
art-recognized method.
[0014] In certain embodiments, the second promoter and the reporter
gene can be downstream of (3'-to) the coding sequence for the
precursor molecule. In other embodiments, the reporter gene is
translated from an internal ribosomal entry site (IRES) between a
second promoter and the reporter gene.
[0015] In other embodiments, the coding sequence for expressing the
precursor molecule may be embedded or inserted into the 5'-UTR
(5'-untranslated region), 3'-UTR, or an intron of the reporter
gene.
[0016] The constructs of the invention may further comprise at
least one selectable marker, such as puromycin, zeocin, hygromycin,
or neomycin, etc.
[0017] The constructs of the invention may further comprise a Pol
III promoter upstream of the coding sequence for expressing the
precursor molecule.
[0018] The constructs of the invention can be used to inhibit the
expression of a number of different target genes. In certain
embodiments, the target gene is associated with a disease condition
such as cancer or infectious disease. For example, the target gene
may be over-expressed or abnormally active in the disease. In
addition, the target gene may be an oncogene or an
antagonist/inhibitor or dominant negative mutation of a tumor
suppressor gene.
[0019] Another aspect of the invention provides a cell comprising
any of the subject nucleic acid constructs.
[0020] In certain embodiments, the cell may be a mammalian
cell.
[0021] In certain embodiments, the cell may be a tissue culture
cell (e.g., a primary cell, or a cell from an established cell
line), a cell in vivo, or a cell manipulated ex vivo.
[0022] If the Pol II promoter is an inducible promoter, the cell
may further comprise an additional construct for expressing an
activator or an inhibitor of the inducible promoter. For example,
if the inducible promoter is a tet-responsive promoter, the
additional construct may encode tTA or rtTA. If the inducible
promoter is a LacO-responsive promoter, the additional construct
may encode LacI. If the inducible promoter is a LoxP-stop-LoxP
system promoter, the additional construct may encode a Cre
recombinase, which may be under the transcriptional control of an
inducible promoter, a developmental stage-specific promoter, or a
tissue-specific promoter.
[0023] Another aspect of the invention provides a non-human mammal
comprising any of the subject cells described above. In certain
embodiments, the non-human mammal may be a chimeric mammal some of
whose somatic or germ cells are subject cells as described above.
Alternatively, the non-human mammal may be a transgenic mammal all
of whose somatic or germ cells are subject cells described
above.
[0024] Another aspect of the invention provides a method for making
a subject chimeric non-human mammal as described above, comprising
introducing a construct according to any of the subject nucleic
acid constructs into an embryonic stem (ES) cell and generating a
chimeric mammal from the ES cell.
[0025] Another aspect of the invention provides a method for making
a subject transgenic non-human mammal described above, comprising
mating a subject chimeric non-human mammal described above with
another animal from the same species.
[0026] Another aspect of the invention provides a method for
inhibiting the expression of a target gene of interest in a cell,
comprising introducing a subject construct into the cell, wherein
the siRNA molecule derived from the precursor molecule is specific
for the target gene.
[0027] In certain embodiments, the method further comprises
inhibiting at least one additional target gene(s) of interest in
the cell by introducing at least one additional constructs
according to any one of the subject nucleic acid constructs into
the cell, wherein each of the siRNA molecules derived from the
precursor molecules are specific for the additional target genes,
respectively.
[0028] Another aspect of the invention provides a method for
treating a gene-mediated disease, comprising introducing into an
individual having the disease a construct according to any of the
subject nucleic acid constructs, where the siRNA derived from the
precursor molecule is specific for the gene mediating the
disease.
[0029] Another aspect of the invention provides a method of
validating a gene as a potential target for treating a disease,
comprising: (1) introducing a construct according to any one of the
subject nucleic acid constructs described herein into a cell
associated with the disease, wherein the siRNA molecule derived
from the precursor molecule is specific for the gene; (2) assessing
the effect of inhibiting the expression of the gene on one or more
disease-associated phenotype; wherein a positive effect on at least
one disease-associated phenotype is indicative that the gene is a
potential target for treating the disease.
[0030] In certain embodiments, the gene is over-expressed or
abnormally active in disease cells or tissues. Alternatively, the
gene may be downstream of and is activated by a second gene
over-expressed or abnormally active in disease cells or tissues. In
addition, the product of the gene antagonizes an suppressor of a
second gene over-expressed or abnormally active in disease cells or
tissues.
[0031] In certain embodiments, the cell may be a tissue culture
cell, such as a primary cell isolated from diseased tissues, or
from an established cell line derived from diseased tissues.
[0032] In other embodiments, the cell is within diseased tissues,
and step (2) above comprises evaluating one or more symptoms of the
disease.
[0033] In certain embodiments, the cell may be one from a
transgenic animal, such as one comprising any of the subject
nucleic acid constructs.
[0034] For example, in a transgenic animal with a transgene
comprising any of the subject nucleic acid constructs, the
transgene may encode a precursor molecule, which, upon processing,
generates a siRNA specific for the candidate target gene.
Preferably, the expression of the precursor molecule is inducible,
reversible, and/or tissue-specific.
[0035] In certain embodiments, the method further comprises
assessing the side effect, if any, of knocking down the expression
of the target gene in one or more tissues/organs other than the
diseased tissue, wherein the target gene is a valid target if the
side effect, if any, is acceptable to a person of skill in the
respective art (e.g., when validating a drug target, such side
effects resulting from impairment of the target gene function in
other tissues must be acceptable to a physician or
veterinarian).
[0036] In certain embodiments, the expression of the gene may be
inducibly inhibited by a subject construct, or inducibly activated
by turning down the expression of a subject construct.
[0037] It is also contemplated that all embodiments of the
invention, including those specifically described for different
aspects of the invention, can be combined with any other
embodiments of the invention as appropriate.
[0038] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows effective knockdown via single copy expression
of miR30-based shRNAs from a retroviral LTR promoter. (A) Schematic
representation of predicted RNA folds for simple stem/loop and
miR30 design shRNAs. Note extensive predicted folding for the 300
nt pre-miR30 RNA. Folds were generated using mfold. (B) Retroviral
vectors used to deliver shRNAs to mammalian cells. Provirus layouts
are shown to indicate promoter activity of the integrated virus.
Active promoters are shown as open arrows, with two inverted black
arrows representing shRNA stem sequences. (C) Western blot analysis
for p53 expression of NIH3T3 cells transduced with the retroviral
vectors shown in B and selected in puromycin. A tubulin blot is
shown as a loading control. (D) Colony formation assay for the
cells shown in (C). Cells were seeded in 6 well plates at 2500
cells/well, and allowed to grow for 10 days before harvesting. (E)
Western blot analysis for p53 expression in NIH3T3 cells transduced
at less than 5% efficiency (assessed by GFP FACS; not shown) with
the retroviral vectors shown in (B). A tubulin blot is shown as a
loading control. Similar results were obtained in other cell types
including wild type and p19ARF-null MEFs (data not shown).
[0040] FIG. 2 shows that RNA polymerase II-driven shRNAs can
effectively promote tumorigenesis and chemotherapy resistance in
vivo. (A) Kaplan-Meier curve showing mouse survival following
adoptive transfer of E.mu.-Myc HSCs infected with LTR-driven Bim
shRNAs. (B) Western blot showing reduced BimEL and BimL expression
in E.mu.-Myc lymphomas expressing Bim shRNAs. Control: archived
tumors arising from E.mu.-Myc HSCs (on either a wild type,
ARF.sup.+/- or p53.sup.+/- background; not shown) were used as
controls for Bim expression. (C) Kaplan-Meier curves showing
tumor-free survival (left) and overall survival (right) for mice
harboring p19ARF-null lymphomas infected with either the
LMP-p53.1224 retrovirus or vector control. Tumor-bearing mice were
given a single 10 mg/kg dose of adriamycin at day zero. (D) Flow
cytometry analysis of GFP expression in lymphoma cells harvested
from the mice in (A). Representative histograms show the percent of
GFP-positive cells at the time of treatment (left) and after tumor
relapse (right).
[0041] FIG. 3 shows stable and regulatable shRNA expression from a
tet-responsive RNA polymerase II promoter. (A) Provirus layout of
the SIN-TREmiR30-PIG (TMP) retroviral vector. (B) Western blot
analysis of Rb expression in HeLa-tTA cells infected with
TMP-Rb.670. Cells were treated with 100 ng/mL Dox for 4 days prior
to harvesting. Control uninfected HeLa-tTA cells treated with Dox
are also shown. (C) Rb expression in homogeneous cultures derived
from single-cell clones of HeLa-tTA cells infected at single copy
with TMP-Rb.670. Cells were cultured in normal, Dox-free medium
prior to harvesting. con=control uninfected HeLa-tTA cells. (D) Dox
dose/response analysis of Rb expression in HeLa-tTA clone Rb.670C.
Cells were cultured for 8 days in the indicated Dox concentration
prior to harvesting. Control uninfected HeLa-tTA cells (con)
cultured with or without Dox are also shown. Note the presence of a
non-specific band in the GFP immunoblot, running just below GFP.
(E) Rb expression in HeLa-tTA clone Rb.670C cells over time in
response to shifting into or out of Dox. Cells were cultured
without Dox (left panels) or in 100 ng/mL Dox (right panels) for
eight days prior to shifting them into 100 ng/mL Dox or Dox-free
medium, respectively. Again, note the presence of a faint
non-specific band in the GFP immunoblot. Similar results were
observed for all Rb.670 clones showing good Rb knockdown in
Dox-free medium (C, above), with some clonal variation in kinetics.
In addition, similar results were obtained using other mishRNAs
targeting Rb and PTEN (data not shown). (F) Rb expression in
homogeneous cultures derived from single-cell clones of U2OS-rtTA
cells infected at around 1% efficiency with TMP-Rb.670. Cells were
cultured in 1000 ng/mL Dox for several days prior to harvesting.
(G) Dox dose/response analysis of Rb expression in U2OS-rtTA clone
Rb.670R5 cells. Cells were cultured for 8 days in the indicated Dox
concentration prior to harvesting. Control uninfected U2OS-rtTA
cells cultured with or without Dox are also shown. (H) Rb
expression in U2OS-rtTA clone Rb.670R5 cells in response to Dox
treatment. Cells were cultured without Dox for eight days prior to
shifting them into 1000 ng/mL Dox. Note that Rb.670R5 cells express
some GFP in Dox-free medium, and Rb levels are slightly decreased
compared with controls, indicating slightly leaky expression from
the TRE-CMV promoter in this particular clone.
[0042] FIG. 4 shows reversible p53 knockdown in primary MEFs. (A)
Colony formation assays of wild type MEFs doubly infected with
TMP-p53.1224 and tTA. Cells were seeded in 6 well plates at 5000
cells/well, and grown for 8 days before harvesting. Upper wells
contained Dox-free medium, whereas lower wells contained 100 ng/mL
Dox. Positive control p53-null MEFs are shown, as are negative
control wild type MEFs infected with TMP-p53.1224 alone or with
TMP-PTEN.1010 plus tTA. (B) Western blot analysis of p53 and GFP
expression in cells expanded from a single-cell clone of wild type
MEFs infected with TMP-p53.1224 and tTA (WtT cells). Cells were
cultured in 100 ng/mL Dox for various times prior to harvesting.
(C) Morphology and GFP fluorescence of WtT cells originally plated
at colony formation density, and cultured in Dox-free medium (upper
panels) or 100 ng/mL Dox (lower panels). Right panel: SA-.beta.-gal
staining of WtT cells cultured in Dox-free medium (upper) or 100
ng/mL Dox (lower). (D) Left panel: Colony formation assay for WtT
cells cultured for 8 days in 100 ng/mL Dox, then seeded in Dox free
medium (upper well) or 100 ng/mL Dox (lower well). Right panel:
Colony formation assay of cells equivalent to those in the upper
well of the left panel (formerly Dox-treated, dormant WtT cells
after extended culture in Dox-free medium). Cells were seeded and
harvested as in (A). (E) Morphology, GFP fluorescence, and
SA-.beta.-gal staining of WtT cells infected with Ras and cultured
in normal medium (upper panels) or 100 ng/mL Dox (lower panels).
(F) Western blot analysis of p53 and GFP expression in WtT cells
infected with Ras. Cells were cultured in 100 ng/mL Dox for various
times prior to harvesting.
[0043] FIG. 5 shows regulated p53 knockdown in tumors. (A) GFP and
standard imaging of representative tumor-bearing nude mice, with
Dox treatment commencing at day 0 (lower panels). Untreated
controls are shown (upper panels). (B) Representative tumor growth
curves for WtT-Ras tumors in an untreated mouse (open squares), or
a mouse treated for 10 days with Dox (filled circles indicate Dox
treatment) commencing at day 0. Each data point is the average
volume of 2 tumors for a single mouse. Similar results were
obtained for 8 different WtT-Ras clones, with slightly differing
kinetics. (C) Representative tumor growth curves for WtT-E1A/Ras
tumors in an untreated mouse (open squares), or a mouse treated for
7 days with Dox (filled circles indicate Dox treatment) commencing
at day 0. Each data point is the average volume of 2 tumors for a
single mouse. Insets show GFP status of a single tumor at various
times. (D) Histological analysis of cell morphology and apoptosis
in representative nude mouse tumors harvested from untreated mice
or mice treated with Dox for several days. (E) Western blot
analysis of p53 and GFP expression in representative WtT-Ras and
WtT-E1A/Ras tumors harvested from untreated mice or mice treated
with Dox for several days. Cultured WtT-Ras cells treated with Dox
are shown as a control.
[0044] FIG. 6 shows an siRNA northern blot of tissues isolated from
animals of various genotypes, probed with a labelled
oligonucleotide that hybridizes to the p53.1224 siRNA. The
individual lanes are: M: Molecular weight marker; 1: LAP-tTA liver;
2: LAP-tTA; TRE-1224 liver; 3: LAP-tTA; TRE-1224 liver after 4 days
Doxycycline administration; 4: LAP-tTA spleen; 5: LAP-tTA; TRE-1224
spleen; 6: LAP-tTA; TRE-1224 spleen after 4 days Doxycycline
administration; 7: Eu-myc; TRE-1224 mouse 4-1 spleen; 8: Eu-myc;
TRE-1224 mouse 6-4 spleen; 9: Eu-myc; Eu-tTA; TRE-1224 mouse #1
spleen; 10: Eu-myc; Eu-tTA; TRE-1224 mouse #2 spleen; 11: Eu-myc;
Eu-tTA; TRE-1224 mouse #1-2 spleen; 12 & 13: Spleen (12) and
lymph node (13) from a tumor-bearing nude mouse recipient of Eu-myc
lymphoma cells; 14 & 15: Spleen (14) and lymph node (15) from a
tumor-bearing nude mouse recipient of Eu-myc; Eu-tTA; TRE-1224
lymphoma cells; 16 & 17: Spleen (16) and lymph node (17) from a
tumor-bearing nude mouse recipient of Eu-myc; Eu-tTA; TRE-1224
lymphoma cells, after 14 days Doxycycline administration.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0045] RNA interference (RNAi) is normally triggered by double
stranded RNA (dsRNA) or endogenous microRNA precursors (pre-mRNAs).
Since its discovery, RNAi has emerged as a powerful genetic tool
for suppressing gene expression in mammalian cells. Stable gene
knockdown can be achieved by expression of synthetic short hairpin
RNAs (shRNAs), traditionally from RNA polymerase III promoters.
[0046] The instant invention generally relates to the use of RNA
Polymerase II promoters to express microRNA (mRNA) precursors
and/or short hairpin RNAs (shRNAs), either in vitro, ex vivo, or in
vivo, especially from as few as one single stably integrated
expression construct. The single expression construct may be stably
transfected/infected into a target cell, or may be a germline
transgene. Transgenic animals with the subject RNAi constructs,
which may be regulated to express mishRNA in an inducible,
reversible, and/or tissue-specific manner, can be used to establish
valuable animal models for certain disease, such as those
associated with loss-of-function of certain target genes. The
ability to control both the timing (e.g., at certain developmental
stages) and location (e.g., tissue-specific) of target gene
knock-down, including the ability to reverse the course of
induction/inactivation, renders the subject system a powerful tool
to study gene function and disease progression. Such animal models
or cells thereof may also be used for drug screening or
validation.
[0047] In certain embodiments, Pol II promoters controls the
transcription of the subject mRNA/shRNA coding sequence. In
general, any Pol II compatible promoters may be used for the
instant invention.
[0048] In certain embodiments, various inducible Pol II promoters
may be used to direct precursor mRNA/shRNA expression. Exemplary
inducible Pol II promoters include the tightly regulatable Tet
system (either TetOn or TetOFF), and a number of other inducible
expression systems known in the art and/or described herein. The
tet systems allows incremental and reversible induction of
precursor mRNA/shRNA expression in vitro and in vivo, with no or
minimal leakiness in precursor mRNA/shRNA expression. Such
inducible system is advantages over the existing unidirectional
Cre-lox strategies. Other systems of inducible expression may also
be used with the instant constructs and methods.
[0049] In certain embodiments, expression of the subject mRNA/shRNA
may be under the control of a tissue specific promoter, such as a
promoter that is specific for: liver, pancreas (exocrine or
endocrine portions), spleen, esophagus, stomach, large or small
intestine, colon, GI tract, heart, lung, kidney, thymus,
parathyroid, pineal glan, pituitory gland, mammary gland, salivary
gland, ovary, uterus, cervix (e.g., neck portion), prostate,
testis, germ cell, ear, eye, brain, retina, cerebellum, cerebrum,
PNS or CNS, placenta, adrenal cortex or medulla, skin, lymph node,
muscle, fat, bone, cartilage, synovium, bone marrow, epithelial,
endothelial, vescular, nervous tissues, etc. The tissue specific
promoter may also be specific for certain disease tissues, such as
cancers. See Fukazawa et al., Cancer Research 64: 363-369, 2004
(incorporated herein by reference).
[0050] Any tissue specific promoters may be used in the instant
invention. Merely to illustarte, Chen et al. (Nucleic Acid
Research, Vol. 34, database issue, pages D104-D107, 2006) described
TiProD, the Tissue-specific Promoter Database (incorporated herein
by reference). Specifically, TiProD is a database of human promoter
sequences for which some functional features are known. It allows a
user to query individual promoters and the expression pattern they
mediate, gene expression signatures of individual tissues, and to
retrieve sets of promoters according to their tissue-specific
activity or according to individual Gene Ontology terms the
corresponding genes are assigned to. The database have defined a
measure for tissue-specificity that allows the user to discriminate
between ubiquitously and specifically expressed genes. The database
is accessible at tiprod.cbi.pku dot edu.cn:8080/index.html. It
covers most (if not all) the tissues described above.
[0051] In certain embodiments, if the reversibly inducible systems
of the invention are used, the subject shRNAs are not designed to
target the promoter regions of a target gene to avoid irreversible
TGS.
[0052] In certain embodiments, artificial mRNA constructs based on,
for example, miR30 (microRNA 30), may be used to express precursor
mRNA/shRNA from single/low copy stable integration in cells in
vivo, or through germline transmission in transgenic animals. For
example, Silva et al. (Nature Genetics 37: 1281-88, 2005,
incorporated herein by reference) have described extensive
libraries of pri-miR-30-based retroviral expression vectors that
can be used to down-regulate almost all known human (at least
28,000) and mouse (at least 25,000) genes (see RNAi Codex, a single
database that curates publicly available RNAi resources, and
provides the most complete access to this growing resource,
allowing investigators to see not only released clones but also
those that are soon to be released, available at http://codex.cshl
dot edu). Although such libraries are driven by Pol III promoters,
they can be easily converted to the subject Pol II-driven promoters
(see Methods in Dickins et al., Nat. Genetics 37: 1289-95, 2005;
also see page 1284 in Silva et al., Nat. Genetics 37: 1281-89,
2005).
[0053] In certain embodiments, even a single copy of stably
integrated precursor mRNA/shRNA construct results in effective
knockdown of a target gene.
[0054] In certain embodiments, the inducible Tet system, coupled
with the low-copy integration feature of invention, allows more
flexible screening applications, such as in screening for
potentially lethal shRNAs or synthetic lethal shRNAs.
[0055] In certain embodiments, the subject precursor mRNA cassette
may be inserted within a gene encoded by the subject vector. For
example, the subject precursor mRNA coding sequence may be inserted
with an intron, the 5'- or 3'-UTR of a reporter gene such as GFP,
etc.
[0056] In certain embodiments, cultured cells, such as wild type
mouse fibroblasts or primary cells can be switched from
proliferative to senescent states simply through regulated
knockdown of p53 using the subject constructs and methods.
[0057] The constructs and methods of the invention is advantageous
in several respects.
[0058] In one respect, stable precursor mRNA/shRNA expression may
be effected through retroviral or lentiviral delivery of the
mRNA/shRNAs, which is shown to be effective at single copy per
cell. This allows very effective stable gene expression regulation
at extremely low copy number per cell (e.g. one per cell), thus
vastly advantageous over systems requiring the introduction of a
large copy number of constructs into the target cell by, for
example, transient transfection.
[0059] Compare to transfection where there are multiple copies
(such as multiple episomal copies) of the shRNA construct, and the
LTR is active, the instant system is preferable for stable
expression of the shRNA.
[0060] Using the instant system, Applicants have discovered rapid
and coordinated entry into senescence upon re-establishment of wild
type p53 expression in p53 defective cells. Such an observation
would not have been possible using previous technologies.
[0061] Another useful feature of the invention is that it is
compatible with an established miR30 mRNA/shRNA library, which
contains designed mRNA/shRNA constructs targeting almost all human
and mouse genes. Any specific member of the library can be readily
cloned (such as by PCR) into the vectors of the instant invention
for Pol II-driven regulated and stable expression.
[0062] Other vector designs with different promoters have shown
dependence on position of transcriptional start and stop sites. The
subject method/system apparently has no such stringent
requirements.
[0063] Applicants have also discovered that promoter interference
between Pol II and Pol III promoters may prevent efficient
transcription of encoded shRNA, while the use of mRNA precursor has
largely overcome this problem.
[0064] Another aspect of the invention provides a method for drug
target validation. The outcome of inhibiting the function of a
gene, especially the associated effect in vivo, is usually hard to
predict. Gene knock-out experiments offer valuable data for this
purpose, but is expensive, time consuming, and potentially
non-informative since many genes are required for normal
development, such that loss-of-function mutation in such genes
causes embryonic lethality. Using the methods of the instant
invention, especially the inducible expression regulation system of
the invention, any potential drug target/candidate gene for
therapeutic intervention may be tested first by selectively up-
and/or down-regulating their expression in vitro, ex vivo, or in
vivo, and determining the effect of such regulated expression,
especially in vivo effects on an organism. If disruption of the
normal expression pattern of a candidate gene shows desired
phenotypes in vitro and/or in vivo, the candidate gene is chosen as
a target for therapeutic intervention. Various candidate compounds
can then be screened to identify inhibitors or activators of such
validated targets.
[0065] Another aspect of the invention provides a method to
determine the effect of coordinated expression regulation of two or
more genes. For example, mRNA/shRNA constructs for two more target
genes may be introduced into a target cell (e.g., by stable
integration) or an organism (e.g., by viral vector infection or
transgenic techniques), and their expression may be individually or
coordinately regulated using the inducible and/or tissue specific
or developmental specific promoters according to the instant
invention. Since different inducible promoters are available, the
expression of the two or more target genes may be regulated either
in the same or opposite direction (e.g., both up- or
down-regulating, or one up one down, etc.). Such experiments can
provide useful information regarding, inter alia, genetic
interaction between related genes.
[0066] In certain embodiments, the instant invention allows highly
efficient knockdown of a target gene from a single (retroviral)
integration event, thus providing a highly efficient means for
certain screening applications. For example, the instant system and
methods may be used to test potentially lethal mRNA/shRNAs or
synthetic lethal mRNA/shRNAs.
[0067] The invention also provide a method to treat certain cancer,
especially those cancer overexpressing Ras pathway genes (e.g., Ras
itself) and having impaired p53 function, comprising introducing
into such cells an active p53 gene or gene product to induce
senescence and/or apoptosis, thereby killing the cancer cells, or
at least inhibit cancer progression and/or growth.
[0068] The general feature of the invention having been described,
the following section provides certain illustrative aspects of the
invention that may be combined in specific embodiments. Other
similar or equivalent art-recognized methods may also be readily
adapted for use in the instant invention.
II. MicroRNA and RNAi Design
[0069] DNA vectors that express perfect complementary short
hairpins RNAs (shRNAs) are commonly used to generate functional
siRNAs. However, the efficacy of gene silencing mediated by
different short-hairpin derived siRNAs may be inconsistent, and a
substantial number of short-hairpin siRNA expression vectors can
trigger an anti-viral interferon response (Nature Genetics 34: 263,
2003). 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 microRNA (mRNA)
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 mRNA hairpin and its flanking sequences.
[0070] MicroRNAs (miRNAs) are endogenously encoded
.about.22-nt-long RNAs that are generally expressed in a highly
tissue- or developmental-stage-specific fashion and that
post-transcriptionally regulate target genes. More than 200
distinct mRNAs having been identified in plants and animals, these
small regulatory RNAs are believed to serve important biological
functions by two prevailing modes of action: (1) by repressing the
translation of target mRNAs, and (2) through RNA interference
(RNAi), that is, cleavage and degradation of mRNAs. In the latter
case, miRNAs function analogously to small interfering RNAs
(siRNAs). Importantly, miRNAs are expressed in a highly
tissue-specific or developmentally regulated manner and this
regulation is likely key to their predicted roles in eukaryotic
development and differentiation. Analysis of the normal role of
mRNAs will be facilitated by techniques that allow the regulated
over-expression or inappropriate expression of authentic mRNAs in
vivo, whereas the ability to regulate the expression of siRNAs will
greatly increase their utility both in cultured cells and in vivo.
Thus one can design and express artificial microRNAs based on the
features of existing microRNA genes, such as the gene encoding the
human miR-30 microRNA. These miR30-based shRNAs have complex folds,
and, compared with simpler stem/loop style shRNAs, are more potent
at inhibiting gene expression in transient assays.
[0071] miRNAs are first transcribed as part of a long, largely
single-stranded primary transcript (Lee et al., EMBO J. 21:
4663-4670, 2002). This primary mRNA transcript is generally, and
possibly invariably, synthesized by RNA polymerase II (pol II) and
therefore is normally polyadenylated and may be spliced. It
contains an .about.80-nt hairpin structure that encodes the mature
22-nt mRNA as part of one arm of the stem. In animal cells, this
primary transcript is cleaved by a nuclear RNaseIII-type enzyme
called Drosha (Lee et al., Nature 425: 415-419, 2003) to liberate a
hairpin mRNA precursor, or pre-miRNA, of 65 nt, which is then
exported to the cytoplasm by exportin-5 and the GTP-bound form of
the Ran cofactor (Yi et al., Genes Dev. 17: 3011-3016, 2003). Once
in the cytoplasm, the pre-miRNA is further processed by Dicer,
another RNaseIII enzyme, to produce a duplex of 22 bp that is
structurally identical to an siRNA duplex (Hutvagner et al.,
Science 293: 834-838, 2001). The binding of protein components of
the RNA-induced silencing complex (RISC), or RISC cofactors, to the
duplex results in incorporation of the mature, single-stranded mRNA
into a RISC or RISC-like protein complex, whereas the other strand
of the duplex is degraded (Bartel, Cell 116: 281-297, 2004).
[0072] The miR-30 architecture can be used to express mRNAs or
siRNAs from pol II promoter-based expression plasmids. See also
Zeng et al., Methods in Enzymology 392: 371-380, 2005 (incorporated
herein by reference).
[0073] FIG. 2B of Zeng (supra) shows the predicted secondary
structure of the miR-30 precursor hairpin ("the miR-30 cassette").
Boxed are extra nucleotides that were added originally for
subcloning purposes (Zeng and Cullen, RNA 9: 112-123, 2003; Zeng et
al., Mol. Cell 9: 1327-1333, 2002). They represent XhoI-BglII sites
at the 50 end and BamHI-XhoI sites at the 30 end. These appended
nucleotides extend the minimal miR-30 precursor stem shown by
several basepairs, similar to the in vivo situation where the
primary miR-30 precursor is transcribed from its genomic locus (Lee
et al., Nature 425: 415-419, 2003), and an extended stem of at
least 5 bp is essential for efficient miR-30 production. Based on
the numbering in FIG. 2B, mature miR-30 is encoded by nucleotides
44 to 65 and anti-miR-30 by nucleotides 3 to 25 of this precursor.
In the simplest expression setting, the cytomegalovirus (CMV)
immediate early enhancer/promoter may be used to transcribe the
miR-30 cassette. The cassette is preceded by a leader sequence of
approximately 100 nt and followed by approximately 170 nt before
the polyadenylation site (Zeng et al., Mol. Cell 9: 1327-1333,
2002). These lengths are arbitrary and can be longer or shorter.
Mature 22-nt miR-30 can be made from such constructs.
[0074] Several other authentic mRNAs have been over-expressed by
using analogous RNA pol II-based expression vectors or even pol
III-dependent promoters (Chen et al., Science 303: 83-86, 2004;
Zeng and Cullen, RNA 9: 112-123, 2003). Expression simply requires
the insertion of the entire predicted mRNA precursor stem-loop
structure into the expression vector at an arbitrary location.
Because the actual extent of the precursor stem loop can sometimes
be difficult to accurately predict, it is generally appropriate to
include .about.50 bp of flanking sequence on each side of the
predicted 80-nt mRNA stem-loop precursor to be sure that all
cis-acting sequences necessary for accurate and efficient Drosha
processing are included (Chen et al., Science 303: 83-86,
2004).
[0075] In an exemplary embodiment, to make the miR-30 expression
cassette, the sequence from +1 to 65 (excluding the 15-nt terminal
loop of the miR-30 cassette, FIG. 2B of Zeng) may be replaced as
follows: the sequence from nucleotides 39 to 61, which is perfectly
complementary to a target gene sequence, will act as the active
strand during RNAi. The sequence from nucleotides 2 to 23 is thus
designed to preserve the double-stranded stem in the miR-30-target
cassette, but nucleotide +1 is now a C, to create a mismatch with
nucleotide 61, a U, just like nucleotides 1 and 65 in the miR-30
cassette (FIG. 2B). Because the 30 arm of the stem (miR-30-target)
is the active component for RNAi, changes in the 50 arm of the stem
will not affect RNAi specificity. A 2-nt bulge may be present in
the stem region of the authentic miR-30 precursor (FIG. 2B of
Zeng). A break in the helical nature of the RNA stem may help ward
off nonspecific effects, such as induction of an interferon
response (Bridge et al., Nat. Genet. 34: 263-264, 2003) in
expressing cells. This may be why mRNA precursors almost invariably
contain bulges in the predicted stem. The miR-30 cassette in FIG.
2A of Zeng is then substituted with the miR-30-target cassette, and
the resulting expression plasmid can be transfected into target
cells.
[0076] The use of pol II promoters, especially when coupled with an
inducible expression system (such as the TetOFF system of Clontech)
offers flexibility in regulating the production of mRNAs in
cultured cells or in vivo. Selection of stable cell lines leads to
less leaky expression in the absence of the activator or presence
of doxycycline, and therefore a stronger induction.
[0077] In certain embodiments, it would be advantageous if the
antisense strand, for example, of the above miR-30-target construct
is preferentially made as a mature mRNA, because its opposite
strand does not have any known target. The relative basepairing
stability at the 50 ends of an siRNA duplex is a strong determinant
of which strand will be incorporated into RISC and hence be active
in RNAi; the strand whose 50 end has a weaker hydrogen bonding
pattern is preferentially incorporated into RISC, the RNAi effecter
complex (Khvorova et al., Cell 115: 209-216, 2003; Schwarz et al.,
Cell 115: 208-299, 2003). This same principle can also be applied
to the design of DNA vector-based siRNA expression strategies,
including the one described here. However, for artificial mRNAs,
the fact that the internal cleavage sites by Drosha and Dicer
cannot be precisely predicted at present adds a degree of
uncertainty as a 1- or 2-nt shift in the cleavage site can generate
rather different hydrogen bonding patterns at the 50 ends of the
resulting duplex, thus changing which strand of the duplex
intermediate is incorporated into RISC. This is in contrast to the
situation with synthetic siRNA duplexes, which have defined ends.
On the other hand, any minor heterogeneity at the ends of an
artificial mRNA duplex intermediate might not be a problem, as the
mRNAs would still be perfectly complementary to their target.
[0078] The role of internal loop, stem length, and the surrounding
sequences on the expression of mRNAs from miR-30-derived cassettes
may also be systematically examined to optimize expression of the
miR-based shRNA. Such analyses may suggest design elements that
would maximize the yield of the intended RNA products. On the other
hand, some heterogeneity could be inevitable. In addition to the
50-end rule, specific residues at some positions within an siRNA
may also enhance siRNA function (Reynolds et al., Nat. Biotech. 22:
326-330, 2004).
[0079] In general, picking a target region with more than 50% AU
content and designing a weak 50 end base pair on the antisense
strand would be a good starting point in the design of any
artificial mRNA/siRNA expression plasmid (Khvorova et al., Cell
115: 209-216, 2003; Reynolds et al., Nat. Biotech. 22: 326-330,
2004; Schwarz et al., Cell 115: 208-299, 2003).
[0080] In certain embodiments, expression of the miR-30 cassette
may be in the antisense orientation, especially when the cassette
is to be used in lentiviral or retroviral vectors. This is partly
because mRNA processing may result in the degradation of the
remainder of the primary mRNA transcript.
[0081] In other embodiments, vectors may contain inserts expressing
more than one mRNAs. In such constructs, the fact that each mRNA
stem-loop precursor is independently excised from the primary
transcript by Drosha cleavage to give rise to a pre-miRNA allows
simultaneous expression of several artificial or authentic mRNAs by
a tandem array on a precursor RNA transcript.
[0082] Genome wide libraries of shRNAs based on the miR30 precursor
RNA have also been generated. Each member of such libraries target
specific human or mouse genes, and may be readily converted to the
vectors/expression systems of the instant invention. The following
section describes the design of such libraries.
[0083] Paddison et al. (Nature Methods 1(2): 163-67, 2004,
incorporated herein by reference) have described a genome-wise
library of shRNAs based on the miR30 precursor RNA, which may be
adapted for use in the instant invention. The described vector
pSHAG-MAGIC2 (pSM2) is roughly equivalent to pSHAG-MAGIC1 as
described in Paddison et al. Methods Mol. Biol. 265: 85-100 (2004),
incorporated herein by reference. The few notable exceptions
include: the new cloning strategy is based on the use of a single
oligonucleotide that contains the hairpin and common 5' and 3' ends
as a PCR template (see FIG. 2 of Paddison, Nature Methods 1(2):
163-67, 2004). The resulting PCR product is then cloned into the
hairpin cloning site of the pSM2 vector, which drives miR-30-styled
hairpins by the human U6 promoter. Inserts from this library may be
excised (see Example below) and cloned into the instant vectors for
Pol II-driven expression of the same miR-30-styled hairpins. This
allows the instant methods to be coupled with the existing library
of miR-30-style constructs that contains most human and mouse
genes.
[0084] Paddison also describes the detailed methods for designing
22-nucleotide sequences (targeting a target gene) that can be
inserted into the precursor mRNA, PCR protocols for amplification,
and relevant critical steps and trouble-shootings, etc. (all
incorporated herein by reference).
[0085] MicroRNAs (including the siRNA products and artificial
microRNAs as well as endogenous microRNAs) have potential for use
as therapeutics as well as research tools, e.g. analyzing gene
function. As a general method, the mature microRNA (miR) of the
invention, especially those non-miR-30 based microRNA constructs of
the invention may also be produced according to the following
description.
[0086] In certain embodiments, 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.
[0087] 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 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.
[0088] 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.
[0089] 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.
[0090] "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 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.
[0091] 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.
[0092] 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.
[0093] 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 through a linker, additional nucleotides or other
molecules.
[0094] 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.
[0095] 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.
[0096] In an alternative embodiment, useful interfering RNAs can be
designed with a number of software programs, e.g., the OligoEngine
siRNA design tool available at wwv.olioengine.com. The siRNAs of
this invention may range about, e.g., 19-29 basepairs in length for
the double-stranded portion. In some embodiments, the siRNAs are
hairpin RNAs having an about 19-29 bp stem and an about 4-34
nucleotide loop. Preferred siRNAs are highly specific for a region
of the target gene and may comprise any about 19-29 bp fragment of
a target gene mRNA that has at least one, preferably at least two
or three, bp mismatch with a nontarget gene-related sequence. In
some embodiments, the preferred siRNAs do not bind to RNAs having
more than 3 mismatches with the target region.
III. Expression Vectors and Host Cells
[0097] 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 expression elements include at least one promoter, such as a
Pol II promoter, which may direct the expression of the operably
linked microRNA precursor (e.g. the shRNA encoding sequence). 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.
[0098] RNA polymerase III (Pol III) transcription units normally
encode the small nuclear RNA U6 (see Tran et al., BMC Biotechnology
3: 21, 2003, incorporate herein by reference), or the human RNAse P
RNA Hi. However, RNA polymerase II (Pol II) transcription units
(e.g., units containing a CMV promoter) is preferred for use with
inducible expression. It will be appreciated that in the vectors of
the invention, the subject shRNA encoding sequence may be operably
linked to a variety of other promoters.
[0099] In some embodiments, the promoter is a type II tRNA promoter
such as the tRNAVa promoter and the tRNAmet promoter. These
promoters may also be modified to increase promoter activity. In
addition, enhancers can be placed near the promoter to enhance
promoter activity. Pol II enhancer may also be used for Pol III
promoters. For example, an enhancer from the CMV promoter can be
placed near the U6 promoter to enhance U6 promoter activity (Xia et
al., Nuc Acids Res 31, 2003).
[0100] In certain embodiments, the subject Pol II promoters are
inducible promoters. Exemplary inducible Pol II systems are
available from Invitrogen, e.g., the GeneSwitch.TM. or T-REx.TM.
systems; from Clontech (Palo Alto, Calif.), e.g., the TetON and
TetOFF systems.
[0101] An exemplary Tet-responsive promoter is described in WO
04/056964A2 (incorporated herein by reference). See, for example,
FIG. 1 of WO 04/056964A2. In one construct, a Tet operator sequence
(TetOp) is inserted into the promoter region of the vector. TetOp
is preferably inserted between the PSE and the transcription
initiation site, upstream or downstream from the TATA box. In some
embodiments, the TetOp is immediately adjacent to the TATA box. The
expression of the subject shRNA encoding sequence is thus under the
control of tetracycline (or its derivative doxycycline, or any
other tetracycline analogue). Addition of tetracycline or Dox
relieves repression of the promoter by a tetracycline repressor
that the host cells are also engineered to express.
[0102] In the TetOFF system, a different tet transactivator protein
is expressed in the tetOFF host cell. The difference is that
Tet/Dox, when bind to an activator protein, is now required for
transcriptional activation. Thus such host cells expressing the
activator will only activate the transcription of an shRNA encoding
sequence from a TetOFF promoter at the presence of Tet or Dox.
[0103] An alternative inducible promoter is a lac operator system,
as illustrated in FIG. 2A of WO 04/056964 A2 (incorporated by
reference). Briefly, a Lac operator sequence (LacO) is inserted
into the promoter region. The LacO is preferably inserted between
the PSE and the transcription initiation site, upstream or
downstream of the TATA box. In some embodiments, the LacO is
immediately adjacent to the TATA box. The expression of the RNAi
molecule (shRNA encoding sequence) is thus under the control of
IPTG (or any analogue thereof). Addition of IPTG relieves
repression of the promoter by a Lac repressor (i.e., the LacI
protein) that the host cells are also engineered to express. Since
the Lac repressor is derived from bacteria, its coding sequence may
be optionally modified to adapt to the codon usage by mammalian
transcriptional systems and to prevent methylation. In some
embodiments, the host cells comprise (i) a first expression
construct containing a gene encoding a Lac repressor operably
linked to a first promoter, such as any tissue or cell type
specific promoter or any general promoter, and (ii) a second
expression construct containing the dsRNA-coding sequence operably
linked to a second promoter that is regulated by the Lac repressor
and IPTG. Administration of IPTG results in expression of dsRNA in
a manner dictated by the tissue specificity of the first
promoter.
[0104] Yet another inducible system, a LoxP-stop-LoxP system, is
illustrated in FIGS. 3A-3E of WO 04/056964 A2 (incorporated by
reference). The RNAi vector of this system contains a
LoxP-Stop-LoxP cassette before the hairpin or within the loop of
the hairpin. Any suitable stop sequence for the promoter can be
used in the cassette. One version of the LoxP Stop-LoxP system for
Pol II is described in, e.g., Wagner et al., Nucleic Acids Research
25:4323-4330, 1997. The "Stop" sequences (such as the one described
in Wagner, sierra, or a run of five or more T nucleotides) in the
cassette prevent the RNA polymerase III from extending an RNA
transcript beyond the cassette. Upon introduction of a Cre
recombinase, however, the LoxP sites in the cassette recombine,
removing the Stop sequences and leaving a single LoxP site. Removal
of the Stop sequences allows transcription to proceed through the
hairpin sequence, producing a transcript that can be efficiently
processed into an open-ended, interfering dsRNA. Thus, expression
of the RNAi molecule is induced by addition of Cre.
[0105] In some embodiments, the host cells contain a Cre-encoding
transgene under the control of a constitutive, tissue-specific
promoter. As a result, the interfering RNA can only be inducibly
expressed in a tissue-specific manner dictated by that promoter.
Tissue-specific promoters that can be used include, without
limitation: a tyrosinase promoter or a TRP2 promoter in the case of
melanoma cells and melanocytes; an MMTV or WAP promoter in the case
of breast cells and/or cancers; a Villin or FABP promoter in the
case of intestinal cells and/or cancers; a RIP promoter in the case
of pancreatic beta cells; a Keratin promoter in the case of
keratinocytes; a Probasin promoter in the case of prostatic
epithelium; a Nestin or GFAP promoter in the case of CNS cells
and/or cancers; a Tyrosine Hydroxylase, S100 promoter or
neurofilament promoter in the case of neurons; the
pancreas-specific promoter described in Edlund et al., Science 230:
912-916, 1985; a Clara cell secretory protein promoter in the case
of lung cancer; and an Alpha myosin promoter in the case of cardiac
cells.
[0106] Cre expression also can be controlled in a temporal manner,
e.g., by using an inducible promoter, or a promoter that is
temporally restricted during development such as Pax3 or Protein 0
(neural crest), Hoxal (floorplate and notochord), Hoxb6
(extraembryonic mesoderm, lateral plate and limb mesoderm and
midbrain-hindbrain junction), Nestin (neuronal lineage), GFAP
(astrocyte lineage), Lck (immature thymocytes). Temporal control
also can be achieved by using an inducible form of Cre. For
example, one can use a small molecule controllable Cre fusion, for
example a fusion of the Cre protein and the estrogen receptor (ER)
or with the progesterone receptor (PR). Tamoxifen or RU486 allow
the Cre-ER or Cre-PR fusion, respectively, to enter the nucleus and
recombine the LoxP sites, removing the LoxP Stop cassette. Mutated
versions of either receptor may also be used. For example, a mutant
Cre-PR fusion protein may bind RU486 but not progesterone. Other
exemplary Cre fusions are a fusion of the Cre protein and the
glucocorticoid receptor (GR). Natural GR ligands include
corticosterone, cortisol, and aldosterone. Mutant versions of the
GR receptor, which respond to, e.g., dexamethasone, triamcinolone
acetonide, and/or RU38486, may also be fused to the Cre
protein.
[0107] In certain embodiments, additional transcription units may
be present 3' to the shRNA portion. For example, an internal
ribosomal entry site (IRES) may be positioned downstream of the
shRNA insert, the transcription of which is under the control of a
second promoter, such as the PGK promoter. The IRES sequence may be
used to direct the expression of a operably linked second gene,
such as a reporter gene (e.g., a fluorescent protein such as GFP,
BFP, YFP, etc., an enzyme such as luciferase (Promega), etc.). The
reporter gene may serve as an indication of infection/transfection,
and the efficiency and/or amount of mRNA transcription of the
shRNA-IRES-reporter cassette/insert. Optionally, one or more
selectable markers (such as puromycin resistance gene, neomycin
resistance gene, hygromycin resistance gene, zeocin resistance
gene, etc.) may also be present on the same vector, and are under
the transcriptional control of the second promoter. Such markers
may be useful for selecting stable integration of the vector into a
host cell genome.
[0108] Certain exemplary vectors useful for expressing the
precursor microRNAs are shown in the examples. Thus the invention
encompasses the nucleotide sequence of such vectors as well as
variants thereof.
[0109] In general, variants typically will share at least 40%
nucleotide identity with any of the described vectors, 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. More
preferably the preferred variants have at least 80% and, most
preferably, at least 90% sequence homology to the described
sequences.
[0110] 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. Such variants may be further
subject to functional testing such that variants that substantially
preserve the desired/relevant function of the original vectors are
selected/identified.
[0111] 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 II 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.
[0112] One useful inducible expression system that can be adapted
for use in the instant invention is the Tet-responsive system,
including both the TetON and TetOFF embodiments.
[0113] TetOn system is a commercially available 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. The Clontech
TetON system includes the pRev-TRE vector, which can be packaged
into retrovirus and used to infect a Tet-On cell line expressing
the reverse tetracycline-controlled transactivator (rtTA). Once
introduced into the TetON host cell, the shRNA insert can then be
inducibly expressed in response to varying concentrations of the
tetracycline derivate doxycycline (Dox).
[0114] 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.
[0115] 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;
lentiviruses; and RNA viruses such as any retrovirus. One can
readily employ other unnamed vectors known in the art.
[0116] 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).
[0117] Exemplary vectors are disclosed herein and in US
2005/0075492 A2 (incorporated herein by reference) and WO 04/056964
A2 (incorporated herein by reference).
[0118] The invention also encompasses host cells transfected with
the subject vectors, especially host cell lines with stably
integrated shRNA constructs. In certain embodiments, the subject
host cell contains only a single copy of the integrated construct
expressing the desired shRNA (optionally under the control of an
inducible and/or tissue specific promoter). Host cells include for
instance, cells (such as primary 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, etc.). Exemplary cells
include: NIH3T3 cells, MEFs, 293 or 293T cells, CHO cells,
hematopoietic stem/progenitor cells, cancer cells, etc.
IV. Methods of Using
[0119] In certain aspects, methods of the invention comprise
contacting and introducing into a target cell with a subject vector
capable of expressing a precursor microRNA as described herein, to
regulate the expression of a target gene in the cell. 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 a target protein in the target cell. Accumulation
of the protein may be effected in a number of different ways. For
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.
[0120] 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.
[0121] 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, etc.
[0122] 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.
[0123] 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 example, 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.
[0124] 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.
[0125] 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).
[0126] 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.
[0127] 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.
[0128] 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;
Poxyiridae (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).
[0129] 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 palladium,
Treponema pertenue, Leptospira, Rickettsia, and Actinomyces
israelli.
[0130] 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.
[0131] The vectors of this invention can be delivered into host
cells via a variety of methods, including but not limited to,
liposome fusion (transposomes), infection by viral vectors, and
routine nucleic acid transfection methods such as electroporation,
calcium phosphate precipitation and microinjection. In some
embodiments, the vectors are integrated into the genome of a
transgenic animal (e.g., a mouse, a rabbit, a hamster, or a
nonhuman primate). Diseased or disease-prone cells containing these
vectors can be used as a model system to study the development,
maintenance, or progression of a disease that is affected by the
presence or absence of the interfering RNA.
[0132] Expression of the mRNA/siRNA introduced into a target cell
may be confirmed by art-recognized techniques, such as Northern
blotting using a nucleic acid probe. For cell lines that are more
difficult to transfect, more extracted RNA can be used for
analyses, optionally coupled with exposing the film longer. Once
expression of the mRNA/siRNA is confirmed, the DNA construct can
then be tested for RNAi efficacy against a cotransfected construct
encoding the target protein or directly against an endogenous
target. In the latter case, one preferably should have a clear idea
of transfection efficiency and of the half-life of the target
protein before performing the experiment.
V. Pharmaceutical Use and Methods of Administration
[0133] 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 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.
[0134] 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, or
symptoms thereof. Such terms also include prevention of
disease/condition in, for example, subjects/individuals predisposed
to such diseases/conditions, or at high risk of developing such
diseases/conditions.
[0135] 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.
[0136] 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.
[0137] 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 affect 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.
[0138] 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.
[0139] 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 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.
[0140] 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.
[0141] 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.
[0142] Injections can be e.g., intravenous, intradermal,
subcutaneous, intramuscular, or interperitoneal. The composition
can be injected interdermally for treatment or 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.
[0143] 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 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.
[0144] 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).
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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 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.
[0151] 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.
[0152] 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, 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.
[0153] 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.
[0154] 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.
VI. Exemplary Uses
[0155] Drug Target Validation
[0156] Good drugs are potent and specific; that is, ideally, they
must have strong effects on a specific biological pathway or tissue
(such as the disease tissue), while having minimal effects on all
other pathways or all other tissues (e.g., healthy tissues).
Confirmation that a compound inhibits the intended target (drug
target validation) and the identification of undesirable secondary
effects are among the main challenges in developing new drugs.
[0157] Modern drug screening typically requires tremendous amounts
of time and financial resources. Ideally, before even committing to
such an extensive drug development program to identify a drug, one
would like to know whether the intended drug target would even make
a good target for treating a disease. That is, whether antagonizing
the function of the intended target (such as a disease-associated
oncogene or survival gene), would be sufficient/effective to treat
the disease, and whether such treatment would bear an acceptable
risk or side effect. For example, if a cancer is determined to be
caused by an activating mutation in the Ras pathway, or caused by
abnormal activity of a survival gene such as Bcl-2, the subject
system can be used to generate animal models for drug target
validation. Specifically, one can generate a transgenic mouse with
the subject tet-responsive mishRNA expression, with the mishRNA
targeting a gene that is a potential drug target (i.e., Ras or
Bcl-2 in this example). Tumors with various initiating lesions can
then be made in the mouse, and the mishRNA can be switched on in
the tumor (if, for example, a tet-ON regulator is used). Such
mishRNA expression mimicks the action of a (yet to be identified)
drug that would interfere with that target. If knocking down the
target gene is effective to reverse or stall the course of the
disease, the target gene is a valid target.
[0158] Optionally, the mishRNA transgene can be switched on in a
number of tissues or organs, or even in the whole organism, in
order to verify the potential side effects of the (yet to be
identified) drug on other healthy tissues/organs.
[0159] Thus another aspect of the invention provides an animal
useful for drug target validation, comprising a germline transgene
encompassing the sugject artificial nucleic acid, which
transcription is driven by a Pol II promoter. The expression of the
encoded precursor molecule (such as one based on the miR30-design)
leads to an siRNA that targets a candidate drug target. Optionally,
the precursor molecule is expressed in an inducible, reversible,
and/or tissue-specific manner.
[0160] In a related aspect, the invention provides a method for
drug target validation, comprising antagonizing the function of a
candidate drug target (gene) using a subject cell or animal (e.g.,
a transgenic animal) encompassing the sugject artificial nucleic
acid, either in vitro or in vivo, and assessing the ability of the
encoded precursor molecule to reverse or stall the disease progress
or a particular phenotype associated with a pathological condition.
Optionally, the method further comprises assessing any side effects
of inhibiting the function of the target gene on one or more
healthy organs/tissues.
[0161] Animal Disease Model
[0162] The subject nucleic acid constructs enables one to switch on
or off a target gene or certain target genes (e.g., by using
crossing different lines of transgenic animals to generate
multiple-transgenic animals) inducibly, reversibly, and/or in a
tissue-specific manner. This would faciliate conditional knock-out
or turning-on of any target gene(s) in a tissue-specific manner,
and/or during a specific developmental stage (e.g., embryonic,
fetal, neonatal, postnatal, adult, etc.). Animals bearing such
transgenes may be treated, such as by providing a tet analog in
drinking water, to turn on or off certain genes to allow certain
diseases to develop/manifest. Such system and methods are
particularly useful, for example, to analize the role of any known
or suspected tumor suppressor genes in the maintenance of
immortalized or transformed states, and in continued tumor growth
in vivo.
[0163] In certain embodiments, the extent of gene knock-down may be
controlled to achieve a desired level of gene expression. Such
animals or cell (healthy or diseased) may be used to study disease
progress, response to certain treatment, and/or screening for drug
leads.
[0164] The ability of the subject system to use a single genomic
copy of the Pol II promoter-driven mishRNA cassette to control gene
expression is particularly valuable for complex library
screening.
[0165] The subject gene knock-down by expression of shRNA-mirs may
be very similar to overexpression of protein-coding cDNAs. Thus any
expression systems allowing targeted, regulated and tissue-specific
expression, which have traditionally be limited to gene
overexpression studies, may now be adapted for loss-of-function
studies, especially when combined with the available genome-wide,
sequence-verified banks of miR-30-based shRNAs targeting model
organisms, such as human and mouse.
EXAMPLES
[0166] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Introduction
[0167] In contrast to RNA Polymerase II promoters which are used by
genes encoding proteins, RNA Polymerase III (Pol III) promoters,
such as U6 and H1 promoters, normally drive the transcription of
several endogenous small nuclear RNAs (snRNAs). For this reason,
Pol III promoters have been widely adopted to drive transcription
of synthetic short hairpin RNAs (shRNAs) in cells and animals.
Applicants and others have used shRNAs driven by U6 promoters to
achieve stable knockdown of target genes. Delivery of Pol III
promoter-shRNA cassettes by retroviral transduction of mammalian
cells results in stable suppression of target gene expression.
[0168] However, shRNA driven by Pol III promoters has certain
practical problems. First of all, unlike Pol II promoters, the Pol
III promoters do not lend themselves to regulation. Secondly, such
Pol III-driven shRNAs can be ineffective inhibitors of their target
mRNAs when expressed from a single-copy vector.
[0169] Here, Applicants have used certain RNA Polymerase II (Pol
II) expression systems to allow potent and regulatable RNAi in
mammalian cells. Applicants have shown that miR30 design shRNAs
expressed from the LTR promoter of an integrated retrovirus
suppress target genes more effectively than when expressed from an
RNA polymerase III promoter, even when expressed from a single-copy
in the genome (e.g., from a stably transfected or a transgenic
copy). Furthermore, regulated shRNA expression was also achieved by
using inducible/reservible Pol II promoters, such as a
Tet-responsive Pol II promoter.
Example I
RNA Polymerase III Promoters are Sufficient for Stable shRNA
Expression
[0170] In order to identify a preferred retroviral vector for
delivery of promoter-shRNA cassettes into mammalian cells,
Applicants compared two vectors based on the murine stem cell virus
(MSCV) and the self-inactivating (SIN) retroviral vector,
respectively. The 5'-long terminal repeat (5'-LTR) promoter of the
SIN provirus is transcriptionally inactive, thus using the SIN
5'-UTR promoter in this construct (e.g., USP-p53C, see below)
serves as a control for the construct using the functional MSCV Pol
II promoter (e.g., LUSP-p53C, see below).
[0171] Into each vector, Applicants cloned the U6 RNA polymerase
III promoter upstream of a sequence encoding p53C, a short hairpin
RNA (shRNA) that targets murine p53. The shRNA p53C is predicted to
fold into a simple, symmetrical shRNA with a 29-nucleotide stem and
an eight nt loop (FIG. 1A, left). Also in each vector, a
puroR-IRES-GFP (PIG) cassette under the control of the PGK promoter
was operably linked downstream of the U6-shRNA cassette (FIG.
1B).
[0172] Primary murine embryonic fibroblasts (MEFs) were infected
with either the SIN-U6shRNA-PIG (UP)-p53C construct (USP-p53C), the
MSCV-U6shRNA-PIG (LUP)-p53C construct (LUSP-p53C), or a control
virus, and subject to puromycin selection to establish
stably-infected cell lines.
[0173] After treatment with the DNA-damaging agent adriamycin to
induce p53 expression, cells stably-integrated by the above
constructs were harvested, and their p53 expression levels were
assessed by Western blotting. Interestingly, Applicants found that
p53 knockdown was far more effective in cells transduced with the
SIN retrovirus (FIG. 1C), indicating that the internal U6 Pol III
promoter is sufficient for expression of the p53C shRNA in
mammalian cells (since the SIN Pol II promoter is inactive in the
USP-p53C construct). Also surprisingly, our observations suggest
that transcription from the upstream MSCV LTR promoter, a strong
RNA polymerase II promoter, inhibited shRNA function and p53
knockdown in this context. This effect may be due to promoter
interference between the LTR Pol II and U6 Pol III promoters.
[0174] Similar results were obtained for several other shRNAs with
simple stem/loop folds similar to p53C (data not shown), verifying
the general applicability of using RNA polymerase III promoters
alone for expression of this style (the simple stem-loop style) of
shRNA.
Example II
LTR Pol II Promoter is More Effective than RNA Polymerase III
Promoter in Directing Integrated miR30-Design shRNAs Suppression of
Target Genes
[0175] This example demonstrates that synthetic shRNAs with folds
designed to mimic endogenous microRNA (mRNA) precursors can
effectively inhibit target gene expression. To illustrate,
Applicants used the exemplary miR30-design shRNAs (designated
microRNA-based shRNAs, or mishRNAs) to demonstrate stable
suppression of gene expression in mammalian cells, which strategy
can be generaly applied to other microRNA (mRNA) precursors.
Specifically, Applicants recovered a mishRNA referred to as
p53.1224 (so named because the predicted siRNA begins at nucleotide
1224 of the p53 cDNA) from the mishRNA library (a genome wide
miR30-based shRNA library).
[0176] As shown above, standard stem-loop shRNAs are most
effectively expressed from RNA polymerase III (Pol III) promoters
such as the U6 promoter. Applicants sub-cloned a U6
promoter-p53.1224 cassette into a murine stem cell virus (MSCV) and
a self-inactivating (SIN) retroviral vector, thus generating two
vectors designed to express miR-based shRNAs (as opposed to the
stem-loop shRNA): MSCV/LTR-U6miR30-PIG (LUMP)-p53.1224 and
SIN-U6miR30-PIG (UMP)-p53.1224 (FIG. 1B). One difference between
the mishRNA and the standard stem-loop shRNA is that the miR30
precursor RNA is approximately 300 nt in length and is predicted to
fold into an extensive secondary structure (FIG. 1A, right).
[0177] Applicants have previously constructed similar vectors
expressing a standard stem-loop shRNA targeting p53 (p53C),
producing MSCV/LTR-U6shRNA-PIG (LUSP)-p53C or SIN-U6shRNA-PIG
(USP)-p53C (see above and FIG. 1B). All four constructs were
introduced into early passage murine embryonic fibroblasts (MEFs).
The resulting cell populations were assessed for p53 knockdown
after adriamycin treatment (a DNA damaging agent that stabilizes
p53), and the ability to form colonies when plated at low density
(a functional readout of p53 loss).
[0178] Contrast to what was observed for the simple stem-loop
shRNA, the MSCV-based p53.1224 mishRNA (LUMP-p53.1224) driven by a
functional Pol II promoter was more effective at suppressing p53
than its SIN-based counterpart (UMP-p53.1224) devoid of a
functional Pol II promoter, producing nearly undetectable p53
levels as assessed by immunoblotting (FIG. 1C, compare lanes 6, 7,
and 8). As shown above, for the standard stem-loop shRNA, the
SIN-based p53C shRNA (USP-p53C) was more effective at suppressing
p53 than its MSCV-derived counterpart (LUSP-p53C), verifying that
the U6 promoter is sufficient for stem/loop shRNA expression (FIG.
1C). The ability of each vector to suppress p53 correlated
precisely with its ability to stimulate colony formation at low
density, with cells expressing the MSCV-based p53.1224 vector
producing as many colonies as p53-null cells (FIG. 1D).
[0179] Southern blotting using a GFP probe verified that these
differences were not due to variation in retroviral copy number
(data nor shown).
[0180] This vector preference was also observed for several other
mishRNAs and stem-loop shRNAs targeting diverse gene products (data
not shown). Thus, in general, mishRNAs can be remarkably potent
when stably expressed from retroviral vectors, particularly those
with a functional 5'-LTR (with a Pol II promoter). In the examples
shown herein, this system achieved near-complete (if not complete)
target gene knockdown.
Example III
Pol II Promoter Contributes to Functional shRNA Production
[0181] The more potent knockdown produced by mishRNAs expressed
from the MSCV vector compared with the SIN vector implies that the
5'-LTR contributes to optimal mishRNA expression. To determine
whether the 5'-LTR promoter, a strong Pol II promoter, is
sufficient for effective gene knockdown using mishRNAs,
Applicants-introduced the p53.1224 shRNA into an MSCV vector
lacking a U6 promoter (MSCV/LTRmiR30-PIG (LMP) (FIG. 1B). To
facilitate comparison, Applicants introduced this vector and its
LUMP and UMP counterparts into NIH3T3 cells at a low multiplicity
of infection (<5% efficiency) such that the vast majority of
transduced cells should contain single proviral integrations.
Remarkably, both vectors harboring the MSCV LTR (LUMP-p53.1224 and
LMP-p53.1224) suppressed p53 expression extremely efficiently, and
were far superior to UMP-p53.1224, which expresses p53.1224 from
the U6 promoter alone (FIG. 1E). Similar results were obtained in
other cell types including wild type and p19ARF-null MEFs (data not
shown).
[0182] Thus, transcription of mishRNAs from Pol II promoters (such
as the retroviral LTR in this example) is sufficient for highly
effective target gene knockdown, even when expressed at single
copy, and even in the absence of any Pol III promoters. Such
features are extremely valuable for knockdown screens using complex
libraries, where infected cells are unlikely to contain multiple
copies of a given shRNA vector.
[0183] The fact that the 5'-LTR Pol II promoter produced results
similar to those of the 5'-LTR+U6 promoters (Pol II and Pol III
promoters), suggested that in this case, U6 may be mainly acting as
a "spacer." As the combined effects of the 5'-LTR and U6 promoters
appeared to be more effective than U6 alone, promoter interference
is unlikely, and rather, it suggests dominance of the LTR
promoter.
[0184] Interestingly, GFP is less abundant in cells with LTR-miR30
transcript. While not wishing to be bound by any particular theory,
this is likely due to degradation of this transcript after nuclear
processing by Drosha. Since microRNA clusters, much GFP appeared to
be translated from the IRES on the long LTR transcript.
Example IV
In Vivo Loss-of-Function Phenotypes can be Recapitulated using
miR30-Design shRNAs Expressed from Pol II Promoters
[0185] Stable expression of stem/loop shRNAs can produce loss of
function phenotypes in mice. To determine whether miR30-derived
shRNAs expressed from pol II promoters can efficiently modulate
gene expression in vivo, Applicants targeted genes for which the
null phenotype was known. For example, inactivation of the BH3-only
protein Bim (a pro-apoptotic member of the Bcl-2 family)
accelerates lymphomagenesis in E.mu.-myc transgenic mice. To this
end, Applicants have demonstrated that miR30-design shRNAs
targeting Bim would also cooperate with myc during lymphomagenesis.
Indeed, mice reconstituted with E.mu.-myc hematopoetic stem cells
(HSCs) transduced with two independent miR30-design shRNAs
targeting Bim (collectively referred to as shBim, and expressed
from the LTR of MSCV/LTRmiR30-SV40-GFP (LMS), a derivative of LMP
that lacks a Pol III promoter) formed tumors much more rapidly than
animals reconstituted with stem cells expressing a control vector
(FIG. 2A, P<0.05). Importantly, lymphomas arising in animals
transduced with shBim were GFP-positive, expressed low levels of
Bim (FIG. 2B), and displayed a mature (IgM.sup.+) B cell phenotype
uniquely characteristic of Bim null lymphomas (data not shown).
Thus, in vivo loss of function phenotypes can be recapitulated
using miR30-design shRNAs expressed from Pol II promoters.
Example V
Identification and Characterization of Genes that Modify Drug
Responses In Vivo
[0186] siRNAs have been used to identify modulators of drug action,
but are not suitable for long-term assays or in vivo systems. The
miR30-based vectors described above enable the identification and
characterization of genes that modify drug responses in vivo.
[0187] As an illustrative example, Applicants examined the ability
of a miR30-design p53 shRNA to promote chemoresistance in E.mu.-myc
lymphomas, which respond poorly to therapy in the absence of p53.
Applicants introduced LMS-p53.1224 (co-expressing GFP) into
chemosensitive E.mu.-myc lymphoma cells at .about.10% infection
efficiency and transplanted the mixed cell populations into
syngeneic recipient mice. Upon lymphoma manifestation, animals were
treated with the chemotherapeutic drug adriamycin and monitored for
tumor response. Strikingly, mice harboring lymphomas expressing
LMS-p53.1224 did not regress following adriamycin treatment and
showed significantly reduced overall survival relative to control
tumor-bearing mice (FIG. 2C). This indicates that the LMS-p53.1224
construct effectively knocked-down p53 expression in tumor cells,
resulting in their poor response (or chemoresistance) to therapy.
Furthermore, the percentage of GFP positive cells dramatically
increased in lymphomas harboring p53.1224 but not the control
vector, indicating a selective advantage for p53.1224 expressing
cells (FIG. 2D).
[0188] These results demonstrated that sufficient p53 knockdown may
promote in vivo chemoresistance. Such an animal mode (or tumor
cells therein) may also be used to screen (in vivo or in vitro) for
compounds that can overcome chemoresistance in p53 negative
cells.
[0189] Together, these data indicate that mishRNAs expressed from
Pol II promoters are suitable for a variety of in vivo
applications, with strong potential for transgenic animals, tissue
specific gene knockdowns and in vivo forward genetic screens.
Example VI
Pol II Promoter-Driven Inducible and Reversible shRNA Production
from Low-Copy Stable Integration
[0190] RNAi inhibits gene function without altering DNA sequence,
therefore its effects are potentially reversible. Given our
findings that low copy Pol II promoters can effectively drive
mishRNAs from a single integrated construct, Applicants adapted the
traditional inducible protein expression systems, such as the
tetracycline (tet)-regulated Pol II promoter TRE-CMV, to achieve
inducible stable expression of mishRNAs.
[0191] Many inducible promoters are known in the art in the context
of protein expression. These inducible systems can all be adpated
to express the mishRNAs of the subject invention. In one
illustrative example, the TRE-CMV promoter consists of a tandem
array of tet transactivator binding sites fused to a minimal CMV
promoter. Transactivator protein tTA transactivates the TRE-CMV
promoter in the absence of the tetracycline analog doxycycline
(Dox). This promoter system has been shown to be highly effective
for conditional expression of protein-coding cDNAs both in vitro
and in vivo. Thus when adapted for use in the subject invention,
shRNA expression can mediate target gene knockdown in the absence
of Dox both in vitro and in vivo.
[0192] Using a SIN vector backbone, Applicants cloned a mishRNA
targeting human Rb (Rb.670) downstream of the TRE-CMV promoter,
producing SIN-TREmiR30-PIG, or TMP-Rb.670; FIG. 3A). HeLa cells
stably expressing the tet transactivator protein tTA (tet-off) were
infected with TMP-Rb.670 at single copy in the absence of Dox. Rb
levels in these cell populations were slightly decreased compared
with uninfected controls, indicating potential shRNA production
from the TRE-CMV promoter (FIG. 3B). Indeed, when single cell
clones were generated from this population, 6 of 13 showed
excellent Rb knockdown (FIG. 3C and data not shown), demonstrating
that the TRE-CMV promoter can effectively drive shRNA expression at
low copy number.
[0193] To examine inducible regulation of shRNA expression, Rb.670C
cells, which showed significant Rb knockdown in Dox-free medium
(FIG. 3C), were cultured in various Dox concentrations for many
days. Cell growth and viability were indistinguishable at all Dox
concentrations. However, Applicants observed a clear
dose-dependency of Rb expression, with maximum Rb knockdown
achieved in low Dox concentrations and vice versa (FIG. 3D). At Dox
concentrations of less than 0.005 ng/mL, Dox produced minimal Rb
expression. However, cells grown in 0.008 ng/mL Dox showed slight
de-repression of Rb. Normal Rb expression was restored in cells
cultured in approximately 0.05 ng/mL Dox and higher, suggesting
that shRNA expression is not leaky at these Dox concentrations.
[0194] Thus, Dox concentration can tightly control the extent of
stable gene knockdown. This effect was also observed in time-course
studies, where Applicants observed normalization of Rb expression
upon Dox addition, and rapid Rb knockdown upon Dox removal (FIG.
3E), demonstrating the reversibility of the induced mishRNA
expression. Remarkably, in all cases GFP and Rb levels were
inversely correlated (FIGS. 3D and 3E), with intermediate GFP
expression observed between 0.002 and 0.008 ng/mL Dox. As GFP and
shRNA are produced from the same transcript, GFP expression may be
regarded as a surrogate marker of shRNA production.
[0195] A great advantage of tet-regulated systems is that
expression from the TRE-CMV promoter can be either induced (tet-ON)
or repressed (tet-OFF) by Dox, depending on which tet
transactivator protein is used. To test a tet-ON shRNA expression
system, Applicants utilized U2OS cells stably expressing the
reverse tTA (rtTA) protein, which in contrast to tTA, requires Dox
to activate transcription.
[0196] Applicants also isolated a clone (Rb.670R5; FIG. 3F) of U2OS
cells infected with TMP-Rb.670 and stably expressing the reverse
tTA (rtTA; tet-on) protein. As predicted, Dox concentration and Rb
knockdown were positively correlated in these cells (FIGS. 3G and
3H).
[0197] At Dox concentrations of less than 0.005 ng/mL, Dox produced
minimal Rb expression. However, cells grown in 0.008 ng/mL Dox
showed slight de-repression of Rb. Normal Rb expression was
restored in cells cultured in approximately 0.05 ng/mL Dox and
higher, suggesting that shRNA expression is not leaky at these Dox
concentrations. As GFP protein is translated from an IRES, it can
be produced from transcripts originating from both the PGK and
TRE-CMV promoters. As GFP is not detected in cells grown in high
Dox concentrations, it appears that GFP production from the PGK
promoter transcript is very weak. Our results suggest that the
majority of GFP in untreated Rb.670C cells arises from the CMV-TRE
transcript, production of which is blocked by Dox in a
dose-dependent manner. As the TRE-CMV transcript also carries the
miR30-based shRNA fold, GFP expression may be regarded as a
surrogate marker of shRNA production.
[0198] Using the same Tet-responsive system, good protein
expression regulation was also achieved in several other cell
clones, including those expressing a PTEN-miR30 construct.
[0199] These observation verifies that low copy delivery of the TMP
vector (also lacking a Pol III promoter) allows regulated mishRNA
expression in either tet-on or tet-off configurations, and altering
Dox concentration in this system allows tight control of the extent
of stable gene knockdown.
Example VII
Reversible Induction of Pol II-Driven Tet-Responsive p53 shRNA
Production in Primary Cells
[0200] The instant regulatable shRNA expression is not only
operable in immortalized cell lines, but also functional to
regulate suppression of genes (e.g., the tumor suppressor gene p53)
in primary cells.
[0201] For example, inactivation of the tumor suppressor p53
immortalizes wild type MEFs, and transforms MEFs over-expressing
oncogenic Ras. Early passage MEFs were co-transduced with
TMP-p53.1224 and a retrovirus expressing the tTA (tet-off) protein.
Many doubly infected MEFs (designated wild type/tTA/TMP-p53.1224,
or WtT) showed stable p53 knockdown when cultured in Dox-free
medium. WtT cells plated at low density formed colonies comparable
in size and number to those formed by p53-null MEFs (FIG. 4A),
suggesting that p53 was functionally inactivated in most cells.
Colony formation of WtT cells cultured in Dox in parallel was
similar to that of control cells (FIG. 4A), suggesting normal p53
expression. p53-null MEF growth was unaffected by Dox, ruling out
non-specific effects (FIG. 4A).
[0202] Applicants also isolated several WtT clones and examined
their p53 regulation in response to Dox. p53 expression in WtT
cells increased rapidly and GFP expression was lost upon Dox
treatment (FIGS. 4B and 4C). WtT clones cultured in Dox failed to
form colonies when plated at low density. Instead, by day 8 of Dox
treatment, all cells had a flattened morphology characteristic of
senescent cells, and many were positive for senescence-associated
.beta.-galactosidase (SA-.beta.-gal; FIG. 4C). This dormant
phenotype was stable for weeks of continuous culture in Dox.
Therefore, p53.1224 shRNA expression can be tightly regulated by
Dox treatment in wild type MEFs doubly infected with tTA and
TMP-p53.1224.
[0203] The rapid and coordinated senescence response observed when
endogenous p53 expression was restored in MEFs immortalized by p53
knockdown was reversed upon Dox removal (FIG. 4D, left panel, upper
well), in agreement with previous observations in other MEF culture
systems. Control cells continually cultured in Dox remained dormant
(FIG. 4D, left panel, lower well). Newly proliferating cells (FIG.
4D, left panel, upper well) remained responsive to p53
re-expression, as they lost GFP expression and failed to form
colonies when re-plated in Dox (FIG. 4D, right panel, lower
well).
[0204] These results suggest that wild type MEFs can be reversibly
switched between cycling and senescent states simply by regulating
p53 knockdown. WtT cells transformed by infection with activated
Ras (FIG. 4E, upper panels) also became morphologically senescent
and SA-.beta.-gal positive when treated with Dox (FIG. 4E, lower
panels), with p53 and GFP expression changes similar to that of
parental WtT cells (FIG. 4F).
[0205] Furthermore, Applicants conclude that restoration of p53
expression in wild type MEFs immortalized by stable p53 knockdown
causes a rapid and coordinated senescence response. This
demonstrates that at least in cancers with p53 loss-of-function
mutations, cancers can be treated by restoring p53 expression to
induce senescence. This technique can also be extended to test any
potential target genes whose functions are lost in diseases, such
as in cancer. Specifically, the system of the instant invention may
be used to test whether loss-of-function of a candidate gene causes
certain disease state, and whether restoring such target gene
function in diseased tissues can reverse the disease status, or at
least slow down disease progression.
Example VIII
Reversible In Vivo Gene Knockdown Using Tet-Responsive Promoter
[0206] Tet-regulated over-expression systems have revolutionized
the study of the role of oncogenes in tumor survival in vivo.
Tet-regulated RNAi holds similar promise for regulated knockdown of
tumor suppressor genes. To illustrate this concept, Applicants
injected WtT-Ras MEFs subcutaneously into the flanks of nude mice
formed visible, rapidly growing and strongly GFP positive tumors
after approximately 2 weeks, verifying that these cells were
functionally transformed (FIG. 5A; upper panels). To inactivate
p53.1224 shRNA in established tumors, mice were treated with Dox
via their drinking water. After only 2 days of Dox treatment, tumor
GFP intensity was markedly diminished compared with untreated mice,
and after 4 days tumors were almost GFP negative (FIG. 5A).
Remarkably, tumor growth slowed upon Dox treatment, and tumors
began shrinking after approximately 4 to 6 days (FIG. 5B). Animals
treated with Dox for 10 days often showed continued tumor
regression and became tumor-free (FIG. 5B). This regression was
p53-dependent, as tumors derived from p53-null MEFs infected with
tTA, TMP-53.1224 and Ras lost GFP expression but continued to grow
when treated with Dox (data not shown). Similar results were
obtained for several WtT-Ras clones and WtT clones infected with
E1A/Ras, with variable tumor growth rates and regression kinetics
(data not shown).
[0207] Dox-treated animals with regressing tumors were taken off
Dox treatment after various times. In many cases, usually when
initial tumor size was less, mice became tumor-free and remained so
for weeks. However, removing Dox from animals with larger
regressing tumors or after a briefer Dox treatment often allowed
renewed GFP expression and tumor growth (FIG. 5C). Interestingly,
tumors isolated from Dox-treated animals contained cells with
unusually compact nuclei, and widespread apoptosis was seen
compared with untreated controls (FIG. 5D), suggesting that tumor
regression was at least in part due to p53-dependent apoptosis.
Indeed, as predicted, p53 expression was dramatically elevated in
tumors from Dox-treated animals (FIG. 5E).
[0208] In summary, by adapting a standard Pol II promoter-driven
tet-responsive promoter normally used for inducible protein
expression, Applicants for the first time have demonstrated
inducible and reversible target gene knockdown in vivo. p53
re-expression in tumors caused regression associated with
widespread apoptosis, in contrast to the senescence observed when
p53 was re-expressed in the same cells in culture. These findings
highlight the ability of this technology for the study of many
aspects of biology, including identification and/or validation of
potential drug targets in animal models. The tet system has obvious
advantages over unidirectional Cre-lox strategies, and many key
reagents, such as tissue-specific tet transactivator mice, are
readily available.
[0209] In summary, expression of mRNA-design short hairpin RNAs
(shRNAs) allows stable, post-transcriptional suppression of gene
activity, which is optionally reversible. Applicants have developed
a new retroviral vector system that uses RNA polymerase II
promoters to express shRNAs based on the human miR30 precursor.
Single copy expression of shRNAs from this vector yields potent and
stable gene knockdown in cultured cells and in vivo. Expression of
an shRNA targeting p53 using this system mimics complete p53 loss
and renders tumor cells chemoresistant in vivo. By improving
standard tet-inducible promoters for shRNA expression, we show
stable, incremental, and reversible gene knockdown of various
target genes in tet-on or tet-off configurations. Interestingly,
cultured wild type mouse fibroblasts can be switched from
proliferative to senescent states simply through regulated
knockdown of p53. We find that tumors derived from wild type mouse
fibroblasts transformed by Ras overexpression and p53 knockdown
regress upon p53 re-activation in vivo, suggesting that ongoing
suppression of p53 is essential for tumor maintenance in this
context. This system proves useful for studying potential
therapeutic targets in cancer, and in most other biological
systems.
[0210] All vectors described in these experiments are compatible
with genome-wide, sequence verified banks of miR30 shRNAs (or any
other similar banks of miR shRNAs) targeting human and mouse genes,
creating a formidable resource for diverse, large scale RNAi
studies in mammalian systems.
Methods
[0211] The following methods and reagents were used in the Examples
above. These are merely for illustrative purpose, and are by no
means limiting. Other comparable minor variations can be readily
made without undue experimentation for adapting to specific
problems.
Vector Construction.
[0212] The retroviral vector MSCV-PIG has an EcoRI site in the
polylinker and another between the Puro.sup.R cassette and the IRES
sequence. To facilitate cloning into the polylinker, the second
site was destroyed using a PCR-based strategy: a PCR product was
generated using MSCV-PIG template, forward primer
5'-TCTAGGCGCCGGAATTAGATCTCTCG-3' (SEQ ID NO: 1), and reverse primer
5'-CCTGCAATTGGATGCATGGGGTCGTGC-3' (SEQ ID NO: 2), and digested with
BglII and MfeI. This fragment was cloned into MSCV-PIG digested
with BglII/EcoRI, yielding MSCV-PIGdRI. MSCV-U6miR30-PIG was made
by ligating the 762 bp BamHI-MfeI "U6-miR30 context" cassette from
pSM2 into MSCV-PIGdRI digested with BglII/EcoRI. MSCV-LTRmiR30-PIG
was made by ligating the 256 bp SalI-MfeI "miR30 context" cassette
from pSM2 into MSCV-PIGdRI digested with XhoI/EcoRI.
MSCV-LTRmiR30-SV40GFP (LMS) was made in two steps. Firstly, a 1.2
kb EcoRI-ClaI SV40GFP fragment from pBabeGFP was ligated into
MSCV-puro (Clontech) digested with EcoRI/ClaI, forming
MSCV-SV40GFP. This was digested with XhoI/EcoRI, and the 256 bp
SalI-MfeI "miR30 context" cassette from pSM2 was inserted, forming
MSCV-LTRmiR30-SV40GFP. SIN-PIGdRI was made by ligating the 2524 bp
EcoRI-SalI fragment from MSCV-PIGdRI into pQCXIX (Clontech)
digested with EcoRI/XhoI. SIN-TREmiR30-PIG was constructed in two
steps. Firstly, a PCR product spanning the TRE-CMV promoter was
generated using template plasmid PQTXIX (a kind gift from Abba
Malina, generated by cloning the XbaI-EcoRI TRE-CMV promoter
fragment from pUHD10.3 into pQCXIX (Clontech) digested with
XbaI/EcoRI), using the primers 5'-GAATTGAAGATCT GGGGGATCGATC-3'
(SEQ ID NO: 3) and 5'-CATCAATTGCTAGAATTCTGGTTGCT
CGAGAGGCTGGATCGGTCCCGGTGTCTTC-3' (SEQ ID NO:4). This PCR product
was digested with BglII/MfeI and ligated into SIN-PIGdRI digested
with BglII/EcoRI (removing its CMV promoter), forming SIN-TRE-PIG.
SIN-TREmiR30-PIG was completed by ligating the 256 bp SalI-MfeI
"miR30 context" cassette from pSM2 into SIN-TRE-PIG digested with
XhoI/EcoRI. DNA fragments encoding various mishRNA folds were
generated using oligonucleotide template PCR as described
previously, or subcloned as 110 bp XhoI/EcoRI fragments from the
pSM2 mishRNA library. Oligonucleotides were designed at
katahdin.cshl.org:9331/siRNA/RNAi.cgi?type=shRNA (incorporated
herein by reference). PCR products were digested with XhoI/EcoRI
and ligated into the unique XhoI/EcoRI sites within the "miR30
context" in the vectors described above.
Cell Culture and Expression Analysis
[0213] Cells were grown in DMEM containing 10% fetal bovine serum
at 37.degree. C. with 7.5% CO.sub.2. Doxycycline (Clontech) was
dissolved in water and generally used at a final concentration of
100 ng/mL. Medium containing Dox was refreshed every two days.
Infections and colony formation assays were carried out as
previously described. SA-.beta.-gal activity was detected as
previously described, with sample equilibration and X-gal staining
done at pH 5.5. For western blotting analysis, Laemmli buffer
protein lysates were run on SDS-PAGE, and transferred to Immobilon
PVDF membrane (Millipore). Antibodies were anti-p53 (1:1000 IMX25,
Vector Laboratories), anti-PTEN (1:1000 486, a kind gift from
Michael Myers), anti-GFP (1:5000, Clontech), anti-tubulin (1:5000
B-5-1-2, Sigma), anti-actin (1:5000, Sigma), and anti-Rb (1:1000
G3-245, Pharmingen with 1:100 XZ-55 and C36 hybridoma
supernatants).
Lymphoma Studies.
[0214] E.mu.-myc lymphomagenesis and drug treatment studies were
performed as previously described (Schmitt, 2000; Hemann, 2003).
Chemosensitive lymphoma cells were isolated from tumors arising in
mice transplanted with E.mu.-myc; p19.sup.ARF+/-HSCs, which
invariably lose the wild type p19.sup.ARF allele while retaining
wild type p53.
Nude Mouse Studies
[0215] Approximately 10.sup.6 transformed cells were injected
subcutaneously into the two rear flanks of nude mice. Mice were
treated with 0.2 mg/mL Dox in a 0.5% sucrose solution in
light-proof bottles, refreshed every four days. Tumor volume (mm 3)
was calculated as (length.times.width.sup.2.times..pi./6). For
analysis of protein expression, tumors were snap-frozen and
pulverised in liquid nitrogen using a mortar and pestle. Powdered
tumor was lysed in Laemmli buffer and western analysis was
performed as above. For histology, tumor tissue was fixed for 24
hours in 4% formaldehyde in PBS prior to embedding and sectioning.
Apoptosis was measured by TUNEL assay (In situ Cell Death Detection
Kit, POD; Roche).
[0216] Results described herein above are published in Nat Genet.
37(11): 1289-95, 2005 (Dickins et al., 2005). Other related work is
published in Nat Genet. 37(11): 1281-88, 2005 (Silva et al., 2005).
The entire contents of these publications, including the related
online (supplemental) information and contents of the publications
cited therein are incorporated herein by reference. The subject
system can also be used in lentiviral, pre-miR-30based siRNA
expression vectors, such as those including a
tetracyclin-responsive Pol II promoter and thus can be used to
tightly regulate the expression of target genes in transduced
cells. See Stegmeier et al., Proc. Natl. Acad. Sci. U.S.A. 102:
13212-17, 2005 (incorporated herein in its entirety).
Example IX
In vivo Transgenic Animal Model for Tissue-Specific and Inducible
Terget Gene Knockdown
[0217] This example demonstrates knockdown of a target gene, e.g.,
p53, in a tissue-specific, inducible and/or reversible manner, in a
germline (transgenic) animal model.
[0218] To achieve regulated transgene expression in germline
transgenic mice, two lines of transgenic mice were generated: one
expressing a tet transactivator protein (either tTA/tet-off or
rtTA/tet-on), optionally in a tissue-specific manner using
tissue-specific promoters; and another harboring a
tetracycline-responsive (TRE) promoter driving the transgene of
interest. Crossing these two lines yielded double transgenic mice
that expressed the transgene, in a Dox-regulatable manner (either
tet-on or tet-off), in cells where the transactivator (tTA or rtTA)
was expressed.
[0219] Alternatively, the tTA or rtTA construct may be introduced
(e.g. via infection or transfection, etc.) into cells of a
transgenic animal bearing TRE-mishRNA-expression cassette.
[0220] For example, to demonstrate that tet-regulated miR30-based
shRNA expression can be achieved in animals, Applicants generated
several transgenic founder lines harboring a TRE-p53.1224 shRNA
cassette (using standard pronuclear injection protocols). To test
shRNA activity in these animals, Applicants isolated MEFs (mouse
embryonic fibroblasts) from F2 transgenic embryos and wild-type
controls, infected them with a retrovirus expressing the tTA
(tet-off) protein, and assessed p53 knockdown after p53 induction
by adriamycin. Specifically, primary MEFs derived from embryos from
a cross between wild-type B6 females mated to TRE-p53.1224 founder
lines A and B, were infected with either tTA-IRES-Neo or
tTA-IRES-GFP retrovirus. Then tTA-IRES-Neo MEFs were selected for 7
days in G418 prior to harvesting in order to eliminate uninfected
cells. The tTA-IRES-GFP MEFs were unselected, though the MEFs were
infected at high percentage. All cells were adriamycin treated
prior to harvesting.
[0221] Of the two founder lines tested so far, one (line A) showed
striking p53 knockdown in transgene-positive cells (results not
shown). This knockdown was similar to that seen when the p53.1224
shRNA was expressed from a retroviral LTR promoter (supra; Dickins
et al., Nat Genet. 37(11): 1289-95, 2005). Importantly, p53
induction was normal in uninfected transgene-positive cells (e.g.,
by comparing founder line A MEFs either uninfected or infected with
tTA-IRES-Neo. All cells were adriamycin treated prior to
harvesting. Results not shown). This demonstrates that shRNA
expression and p53 knockdown is tTA-dependent and not leaky.
[0222] Moreover, as expected, these MEF lines showed a rapid
re-expression of p53 upon Doxycycline treatment, indicating that
shRNA expression was tightly controlled by Doxycycline (results not
shown).
[0223] To our knowledge, the above experiments for the first time
demonstrated that tetracycline effectively regulated shRNA
expression in a germline transgenic setting. This enables one to
reversibly switch any endogenous gene on or off, simply by
administering a reversible activator or inhibitor of a
transcriptional regulator, such as Doxycycline (or other Tet
homolog), preferably via drinking water. This technology is
especially powerful in examining gene function in vivo, for
example, during embryonic or postnatal development, tumorigenesis,
or after treatment of tumors with chemotherapeutic drugs.
[0224] As indicated above, Applicants have also crossed the
TRE-p53.1224 transgenic mice to established transgenic lines that
express the tTA (tet-off) protein in a tissue-specific manner. As
expected, Applicants detected p53.1224 siRNA in the liver of
LAP-tTA; TRE-p53.1224 double trangenic mice, where tTA expression
was restricted to the liver (lane 2 of FIG. 6). After 4 days of
doxycycline administration, some attenuation of siRNA production
was observed (lane 3 of FIG. 6). Applicans have also been assessing
p53 knockdown in the liver of these mice, in order to determine
whether longer term doxycycline administration will further or
completely block siRNA production. Note that the spleens of these
mice were devoid of siRNA (see lanes 4-6 of FIG. 6), consistent
with liver-specific expression of the siRNA.
[0225] The system can also be used to generate animal models for
studying the effect of turning on/off certain target genes in the
progression of certain diseases, such as cancer.
[0226] For example, the E1-myc mouse is prone to developing
lymphoma, which is accelerated further by loss of p53 function. To
model this process using tet-regulated p53 knockdown in vivo,
Applicants crossed E.mu.-myc mice to TRE-p53.1224 mice and
E.mu.-tTA mice, which expressed tTA specifically in the B cell
compartment. As myc and tTA should be expressed coordinately in B
cells of the E1-myc; E.mu.-tTA; TRE-p53.1224 triple transgenic
mice, Applicants expected reversible p53 knockdown in
oncogene-expressing cells.
[0227] Consistent with this prediction, in a spleen (a tissue
enriched for lymphoma cells) isolated from lymphoma-laden triple
transgenic mice, Applicants observed highly abundant p53.1224
siRNA, at levels known to promote p53 knockdown and tumor
progression (lanes 9-11 of FIG. 6). In contrast, spleens from
E1-myc; TRE-1224 double transgenic mice do not express the siRNA,
indicating that the TRE-1224 transgene requires tTA for
expression.
[0228] Lymphoma cells isolated from these triple trangenic mice
were then transplanted into several recipient nude mice to allow
controlled p53 re-activation. Specifically, tumor-bearing recipient
mice were treated with Doxycycline. Survival of heavily
tumor-bearing transplant recipients was extended by many days when
doxycycline was administered via the drinking water. Furthermore,
p53.1224 siRNA expression was completely suppressed in the lymph
nodes and spleen of these treated mice, indicating effective
switching of shRNA expression in vivo.
[0229] These results demonstrated that Applicants can produce
transgenic mice where miR30-based shRNA production was
tissue-specific, and can be inducibly and reversibly regulated
simply by administering or omitting doxycycline in the drinking
water.
[0230] The practice of aspects of the present invention may employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986). All patents, patent applications and references cited
herein are incorporated in their entirety by reference.
EQUIVALENTS
[0231] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *
References