U.S. patent application number 15/661346 was filed with the patent office on 2017-11-30 for use of microrna precursors as drugs for inducing cd34-positive adult stem cell expansion.
The applicant listed for this patent is Donald CHANG, Shi-Lung LIN, David TS WU. Invention is credited to Donald CHANG, Shi-Lung LIN, David TS WU.
Application Number | 20170342418 15/661346 |
Document ID | / |
Family ID | 56896284 |
Filed Date | 2017-11-30 |
United States Patent
Application |
20170342418 |
Kind Code |
A1 |
LIN; Shi-Lung ; et
al. |
November 30, 2017 |
USE OF MICRORNA PRECURSORS AS DRUGS FOR INDUCING CD34-POSITIVE
ADULT STEM CELL EXPANSION
Abstract
This invention generally relates to a composition and its
production method useful for developing drugs/vaccines and/or
therapies against a variety of degenerative diseases in humans.
Particularly, the present invention teaches the essential steps of
production and purification processes necessary for producing small
hairpin-like RNA (shRNA) compositions, such as microRNA precursors
(pre-miRNA) and short interfering RNAs (siRNA), which are useful
for treating human ageing related diseases, such as, but not
limited, Alzheimer's diseases, Parkinson's diseases, osteoporosis,
diabetes, and cancers. The novelty of the present invention is to
create an artificially enhanced adaptation environment for
prokaryotic cells to adopt eukaryotic pol-2 and/or pol-2-like
promoters for transcribing desired ncRNAs and/or their precursors
without going through error-prone prokaryotic promoters, so as to
improve the productive efficiency and reading fidelity of the shRNA
transcription in the prokaryotic cells. The resulting shRNAs,
preferably pre-miRNAs and siRNAs, are useful for developing
therapeutic drugs against human degenerative diseases, particularly
through a mechanism to induce CD34-positive stem cell expansion
and/or regeneration.
Inventors: |
LIN; Shi-Lung; (Arcadia,
CA) ; CHANG; Donald; (Cerritos, CA) ; WU;
David TS; (Arcadia, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIN; Shi-Lung
CHANG; Donald
WU; David TS |
Arcadia
Cerritos
Arcadia |
CA
CA
CA |
US
US
US |
|
|
Family ID: |
56896284 |
Appl. No.: |
15/661346 |
Filed: |
July 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15167226 |
May 27, 2016 |
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15661346 |
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14142512 |
Dec 27, 2013 |
9399773 |
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15167226 |
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13964705 |
Aug 12, 2013 |
9422559 |
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14142512 |
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13572263 |
Aug 10, 2012 |
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13964705 |
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14502608 |
Sep 30, 2014 |
9783811 |
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13572263 |
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13572263 |
Aug 10, 2012 |
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14502608 |
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14527439 |
Oct 29, 2014 |
9637747 |
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13572263 |
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13572263 |
Aug 10, 2012 |
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14527439 |
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61746786 |
Dec 28, 2012 |
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61761890 |
Feb 7, 2013 |
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61522843 |
Aug 12, 2011 |
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61522843 |
Aug 12, 2011 |
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62262280 |
Dec 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1135 20130101;
C12N 15/70 20130101; C12N 2310/141 20130101; C12N 2310/14 20130101;
C12N 2320/30 20130101; C12P 19/34 20130101; C12N 15/113 20130101;
C12N 2330/51 20130101; C12N 2330/50 20130101; C12N 15/111
20130101 |
International
Class: |
C12N 15/113 20100101
C12N015/113; C12P 19/34 20060101 C12P019/34; C12N 15/11 20060101
C12N015/11; C12N 15/70 20060101 C12N015/70 |
Claims
1. A method for inducing CD34-positive cell expansion or
regeneration using at least a microRNA or its precursor
(pre-miRNA), comprising: (a) providing a cell substrate containing
at least a CD34-positive cell, (b) providing at least a microRNA or
a pre-miRNA containing a shared seed sequence of SEQ.ID.NO:3, and
(c) contacting the microRNA or the pre-miRNA of (b) with the cell
substrate containing said at least a CD34-positive cell of (a), so
as to induce the expansion or regeneration of CD34-positive cell
population; wherein the concentration of the microRNA or the
pre-miRNA used for contacting is ranged from 50 to 500 micrograms
per milliliter (50-500 .mu.g/mL).
2. The method as defined in claim 1, wherein the microRNA or the
pre-miRNA is produced by eukaryotic promoter-driven RNA
transcription in prokaryotic cells.
3. The method as defined in claim 2, wherein said eukaryotic
promoter-driven RNA transcription is induced by contacting a
chemical agent containing 3-morpholinopropane-1-sulfonic acid
(MOPS) with at least a transformed prokaryotic cell carrying at
least an expression vector encoding a sequence of SEQ.ID.NO:6,
SEQ.ID.NO:7, SEQ.ID.NO:8, or SEQ.ID.NO:9.
4. The method as defined in claim 3, wherein said expression vector
is a recombinant plasmid encoding the sequence of SEQ.ID.NO:5.
5. The method as defined in claim 3, wherein said expression vector
is pLenti-EF1alpha-RGFP-miR302 containing either a cytomegalovirus
(CMV) or mammalian EF1alpha promoter, or both.
6. The method as defined in claim 2, wherein said prokaryotic cells
are E. coli competent cells.
7. The method as defined in claim 1, wherein the pre-miRNA contains
at least a hairpin-like sequence of SEQ.ID.NO:6, SEQ.ID.NO:7,
SEQ.ID.NO:8, or SEQ.ID.NO:9.
8. The method as defined in claim 1, wherein the pre-miRNA is a
prokaryote-produced miR-302 precursor (pro-miR-302).
9. The method as defined in claim 8, wherein said pro-miR-302
contains at least a sequence of miR-302a, miR-302b, miR-302c, or
miR-302d.
10. The method as defined in claim 8, wherein said pro-miR-302 is
used as a part of drug ingredients for pharmaceutical and
therapeutic applications.
11. The method as defined in claim 1, wherein the pre-miRNA is used
as a part of drug ingredients for pharmaceutical and therapeutic
applications.
12. The method as defined in claim 11, wherein the pre-miRNA is
used to treat aging-related diseases.
13. The method as defined in claim 1, wherein said induced
CD34-positive cells are used as a part of treatment components in
pharmaceutical or therapeutic applications.
14. The method as defined in claim 13, wherein said induced
CD34-positive cells are used to treat aging-related diseases.
15. The method as defined in claim 13, wherein said induced
CD34-positive cells reprogram the malignant properties of human
cancer cells into a low-grade benign or normal-like state in
vivo.
16. The method as defined in claim 13, wherein said induced
CD34-positive cells enhance scarless wound healing in vivo.
17. The method as defined in claim 1, wherein said CD34-positive
cells are adult stem cells and include skin, hair, muscle, blood
(hematopoietic), mesenchymal, and neural stem cells, or a
combination thereof.
Description
PRIORITY
[0001] This application is a Divisional of U.S. patent application
Ser. No. 15/167,226 filed on May 27, 2016, which claims priority to
the U.S. Provisional Application Ser. No. 62/262,280 filed on Dec.
2, 2015, which was entitled "miR-302 Attenuates A.beta.-induced
Neurotoxicity through Activation of Akt Signaling". The present
application also claims priority to the U.S. patent application
Ser. No. 14/502,608 filed on Sep. 30, 2014, and Ser. No. 14/527,439
filed on Oct. 29, 2014, all of which are entitled "Inducible Gene
Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven
Transcription in Prokaryotes and The Applications Thereof". Also,
the present application claims priority to the U.S. patent
application Ser. No. 13/964,705 filed on Aug. 12, 2013, entitled
"Production and Utilization of A Novel Anti-Cancer Drug in Therapy"
and the U.S. patent application Ser. No. 14/142,512 filed on Dec.
27, 2013, entitled "Production and Extraction of MicroRNA
Precursors as Drug for Cancer Therapy". The present application is
a continuation-in-part (CIP) application of the U.S. patent
application Ser. No. 14/502,608 filed on Sep. 30, 2014, and Ser.
No. 14/527,439 filed on Oct. 29, 2014, all of which are entitled
"Inducible Gene Expression Composition for Using Eukaryotic Pol-2
Promoter-Driven Transcription in Prokaryotes and The Applications
Thereof", as well as the U.S. patent application Ser. No.
13/964,705 filed on Aug. 12, 2013, entitled "Production and
Utilization of A Novel Anti-Cancer Drug in Therapy", and the U.S.
patent application Ser. No. 14/142,512 filed on Dec. 27, 2013,
entitled "Production and Extraction of MicroRNA Precursors as Drug
for Cancer Therapy", all of which are hereby incorporated by
reference as if fully set forth herein.
FIELD OF INVENTION
[0002] This invention generally relates to a composition and its
production method useful for developing drugs/vaccines and/or
therapies against a variety of degenerative diseases in humans.
Particularly, the present invention teaches the essential steps of
production and purification processes necessary for producing small
hairpin-like RNA (shRNA) compositions such as microRNA precursors
(pre-miRNA), and short interfering RNAs (siRNA), which are useful
for treating human ageing related diseases, such as, but not
limited, Alzheimer's diseases, Parkinson's diseases, osteoporosis,
diabetes, and cancers. The novelty of the present invention is to
create an artificially enhanced adaptation environment for
prokaryotic cells to adopt eukaryotic pol-2 and/or pol-2-like
promoters for transcribing desired ncRNAs and/or their precursors
without going through error-prone prokaryotic promoters, so as to
improve the productive efficiency and reading fidelity of the shRNA
transcription in the prokaryotic cells. The resulting shRNAs,
preferably pre-miRNAs and siRNAs, are useful for developing
therapeutic drugs against human degenerative diseases, particularly
through a mechanism to induce CD34-positive stem cell expansion
and/or regeneration. Furthermore, the present invention also
reveals a novel pre-miRNA-based drug composition that is able to
reprogram the malignant properties of high-grade liver cancers to a
low-grade benign or even relatively normal stage--a mechanism
called "Cancer Reversion". As cancer reversion is a totally new
concept in drug designs, the present invention devises the first
drug of its kinds using such a novel mechanism for cancer
therapy.
BACKGROUND
[0003] Stem cells are like a treasure box containing numerous
effective ingredients useful for stimulating new cell growth/tissue
regeneration, repairing and/or rejuvenating damaged/aged tissues,
treating degenerative diseases, and preventing tumor/cancer
formation/progression. Hence, it is conceivable that we can use
these stem cells as a tool for novel drug screening, identification
and production. As a result, the drugs so obtained will be useful
for developing pharmaceutical and therapeutic applications, such as
a biomedical utilization, device and/or apparatus for research,
diagnosis, and/or therapy, and a combination thereof.
[0004] MicroRNA (miRNA) is one of the main effective ingredients in
human embryonic stem cells (hESCs). Major hESC-specific miRNA
species include, but not limited, members of the miR-302 family,
miR-371.about.373 family, and miR-520 family. Among them, the
miR-302 family has been found to play a functional role in tumor
suppression (Lin et al., 2008 and 2010). MiR-302 contains eight (8)
familial members, including four (4) sense miR-302 (a, b, c, and d)
and four (4) antisense miR-302* (a*, b*, c*, and d*). These sense
and antisense members are partially matched and can form
double-stranded duplex, respectively. Precursors of miR-302 are
formed by miR-302a and a* (pre-miR-302a), miR-302b and b*
(pre-miR-302b), miR-302c and c* (miR-302c), and miR-302d and d*
(pre-miR-302d) with a link sequence in one end (stem loop),
respectively. In order to activate miR-302 function, miR-302
precursors (pre-miR-302s) are first processed into mature miR-302s
by cellular RNase III Dicers and further form RNA-induced silencing
complexes (RISCs) with certain argonaute proteins, subsequently
leading to either RNA interference (RNAi)-directed degradation or
translational suppression of targeted gene transcripts (mRNAs), in
particular oncogene mRNAs (Lin et al., 2008, 2010 and 2011).
[0005] MiR-302 is the most abundant ncRNA species found in hESCs
and induced pluripotent stem cells (iPSCs). Our previous studies
have shown that ectopic overexpression of miR-302 beyond the level
found in hESCs is able to reprogram both human normal and cancerous
cells to hESC-like iPSCs with a relatively slow cell cycle rate
(20-24 hours/cycle) similar to that of a morula-stage early human
zygote (Lin et al., 2008, 2010 and 2011; EP 2198025; U.S. Ser. No.
12/149,725; U.S. Ser. No. 12/318,806; U.S. Ser. No. 12/792,413).
Relative quiescence is a defined characteristic of these
miR-302-induced iPSCs, whereas hESCs and other previously reported
four-factor-induced (either Oct4-Sox2-Klf4-c-Myc or
Oct4-Sox2-Nanog-Lin28) iPSCs all showed a highly proliferative cell
cycle rate (12-15 hours/cycle) similar to that of a tumor/cancer
cell (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007;
Wang et al., 2008). To disclose this tumor suppression effect of
miR-302, we have identified the involvement of two miR-302-targeted
G1-checkpoint regulators, including cyclin-dependent kinase 2
(CDK2) and cyclin D (Lin et al., 2010; U.S. Ser. No. 12/792,413;
U.S. Ser. No. 13/964,705). It is known that cell cycle progression
is driven by activities of cyclin-dependent kinases (CDKs), which
forms functional complexes with positive regulatory subunits,
cyclins, as well as by negative regulators, CDK inhibitors (CKIs,
such as p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and
p27Kip1). In mammals, different cycling CDK complexes are involved
in regulating different cell cycle transitions, such as
cyclin-D-CDK4/6 for G1-phase progression, cyclin-E-CDK2 for G1-S
transition, cyclin-A-CDK2 for S-phase progression, and
cyclin-A/B-CDC2 (cyclin-AB-CDK1) for entry into M-phase. Hence, our
studies suggested that the tumor suppression function of miR-302
results from co-suppression of the cyclin-E-CDK2 and
cyclin-D-CDK4/6 pathways during G1-S transition.
[0006] Although miR-302 is useful for designing and developing
novel anti-cancer drugs/vaccines, its production is problematic
because natural miR-302 can only be found in human pluripotent stem
cells such as hESCs, of which the resource is very limited.
Alternatively, synthetic small interfering RNAs (siRNA) may be used
to mimic pre-miR-302; yet, since the structure of a pre-miR-302 is
formed by two mis-matched strands of miR-302 and miR-302*, those
perfectly matched siRNA mimics can not replace the function of
miR-302*, of which the sequence is totally different from the
antisense strand of siRNA. For example, the antisense strand of
siRNA-302a mimic is 5'-UCACCAAAAC AUGGAAGCAC UUA-3' (SEQ.ID.NO.1),
whereas native miR-302a* is 5'-ACUUAAACGU GGAUGUACUU GCU-3'
(SEQ.ID.NO.2). As miR-302 function results from both of its sense
miR-302 and antisense miR-302* strands, previous reports using
those siRNA mimics have shown different results from native miR-302
function. On the other hand, our recent discovery of iPSCs may
provide an alternative solution for pre-miR-302 production (EP
2198025; U.S. Ser. No. 12/149,725; U.S. Ser. No. 12/318,806).
Nevertheless, the cost of growing these iPSCs is still too high to
be used for industrial production.
[0007] Alternatively, the use of prokaryotic competent cells may be
a possible approach for producing human microRNAs and their
precursors. However, prokaryotic cells lack several essential
enzymes required for eukaryotic microRNA expression and processing,
such as Drosha and Dicer. Also, prokaryotic RNA polymerases do not
efficiently transcribe small RNAs with high secondary structures,
such as hairpin-like pre-miRNAs and shRNAs. In fact, there is no
true microRNA encoded in bacterial genomes and bacteria do not
naturally express microRNA. As a result, if we can force the
expression of human microRNAs in prokaryotes, the resulting
microRNAs will most likely remain in their precursor conformations
similar to pri-miRNA (a large primary cluster of multiple
pre-miRNAs) and/or pre-miRNA (one single hairpin RNA). Despite all
of the above problems, the real challenge is how to force the
expression of human microRNAs in prokaryotes. To overcome this
major problem, our priority application U.S. Ser. No. 13/572,263
has established a preliminary method; yet, it is currently not sure
whether these prokaryote-produced microRNAs (pro-miRNA) will
possess the same structures and functions as their human
counterparts. Also, the pro-miRNAs so obtained may be contaminated
with bacterial endotoxin, which is not suitable for direct use in
therapy.
[0008] As learning from current textbooks, every ordinary skill
person in the art knows very well that prokaryotic and eukaryotic
transcription machineries are different and hence not compatible to
each other. For example, based on current understandings,
eukaryotic RNA polymerases do not bind directly to a promoter
sequence and require additional accessory proteins (cofactors) to
initiate transcription, whereas prokaryotic RNA polymerases form a
holoenzyme that binds directly to a promoter sequence to start
transcription. It is also a common sense that eukaryotic messenger
RNA (mRNA) is synthesized in the nucleus by type-II RNA polymerases
(pol-2) and then processed and exported to the cytoplasm for
protein synthesis, whereas prokaryotic RNA transcription and
protein translation take place simultaneously off the same piece of
DNA in the same place. This is because prokaryotes such as bacteria
and archaea do not have any nucleus-like structure. Accordingly,
these differences make a prokaryotic cell difficult or even
impossible to produce eukaryotic RNAs using eukaryotic
promoters.
[0009] Prior arts attempt at producing mammalian peptides and/or
proteins in bacterial cells, such as U.S. Pat. No. 7,959,926 to
Buechler and U.S. Pat. No. 7,968,311 to Mehta, used bacterial or
bacteriophage promoters. For initiating expression, a desired gene
was cloned into a plasmid vector driven by a bacterial or
bacteriophage promoter. The gene must not contain any non-coding
intron because bacteria do not have any RNA splicing machinery to
process the intron. Then, the vector so obtained was introduced
into a competent strain of bacterial cells, such as Escherichia
coli (E. coli), for expressing the transcripts (mRNAs) of the gene
and subsequently translating the mRNAs into proteins. Nevertheless,
the bacterial and bacteriophage promoters, such as Tac, Lac, T3,
T7, and SP6 RNA promoters, are not pol-2 promoters and their
transcription activities tend to be an error-prone process which
causes mutations. In addition, Mehta further taught that
glycerol/glycerin might be used to increase the efficiency of
bacterial transformation; yet, no teaching was related to
enhancement of RNA transcription, in particular pol-2
promoter-driven prokaryotic RNA transcription. Due to lack of
possible compatibility between eukaryotic and prokaryotic
transcription systems, these prior arts were still limited by the
use of prokaryotic RNA promoters for gene expression in
prokaryotes.
[0010] Due to the problems of system incompatibility and possible
endotoxin contamination, there was previously no means for
producing human pre-miRNA/shRNA-like drugs in prokaryotes. Also, a
pre-miRNA/shRNA is sized about 70.about.85-nucleotides in length
which is too large and costly to be made by a RNA synthesis
machine. To overcome these problems, the present invention provides
a novel breakthrough--By adding some defined chemicals mimicking
certain transcriptional cofactors, we can create a novel adaptation
environment for prokaryotic cells to use eukaryotic pol-2 and/or
pol-2-like promoters for transcribing desired pre-miRNAs and shRNAs
without going through error-prone prokaryotic promoters. The
advantages are: first, cost-effective mass production due to the
fast growth of bacteria; second, easy handling because of no need
for growing dedicate hESCs or iPSCs; third, high fidelity
productivity in terms of pol-2 promoter-driven RNA transcription;
fourth, high purity of desired microRNAs due to lack of true
microRNA in prokaryotes; and last, no endotoxin, which can be
further removed by certain chemical treatments. Therefore, a method
for producing human pre-miRNAs and/or shRNAs in prokaryotic cells
without the problems of system incompatibility and endotoxin
contamination is highly desirable. Furthermore, the drugs so
obtained may present novel therapeutic effects other than the
currently known function of a microRNA.
SUMMARY OF THE INVENTION
[0011] The principle of the present invention is relied on the
different and incompatible properties between prokaryotic and
eukaryotic RNA transcription systems. Naturally, prokaryotic RNA
polymerases do not recognize eukaryotic promoters and vise versa.
However, the present invention has identified chemical agents that
can serve as transcriptional inducers to trigger and/or enhance
eukaryotic promoter-driven RNA transcription in prokaryotes. Hence,
the knowledge taught in the present invention is a totally novel
breakthrough beyond all current understandings regarding the
differences between prokaryotic and eukaryotic transcription
systems.
[0012] The present invention is related to an inducible gene
expression composition using certain chemical inducers to stimulate
and/or enhance eukaryotic promoter-driven RNA transcription in
prokaryotes. These chemical inducers have not been used in a cell
culture medium due to their bacteriostatic and/or bactericidal
properties, including 3-morpholinopropane-1-sulfonic acid [or named
3-(N-morpholino)propanesulfonic acid; MOPS], glycerin and ethanol,
as well as their functional analogs such as
2-(N-morpholino)ethanesulfonic acid (MES),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and
mannitol. Conceivably, chemicals with a similar structure like
these transcriptional inducers may share a similar function. For
example, MOPS is frequently used as a buffering agent in bacterial
cell lysis and hence is not suitable for growing bacteria. On the
other hand, ethanol is a well-known sanitizer and glycerin is
frequently used in bacterial transformation by destabilizing the
bacterial cell walls, indicating that glycerin is bacteriostatic
and ethanol is bactericidal, respectively. In view of these known
functionalities of MOPS, ethanol and glycerin, an ordinary skill in
the art would not anticipate the use of a trace amount (0.001% to
4% volume/volume concentration) of these chemicals for inducing
eukaryotic promoter-driven gene expression in prokaryotic cells
without first knowing the knowledge of the present invention.
[0013] Based on the above knowledge, the present invention is a
design and method for utilizing prokaryotic cells to produce human
microRNA precursors (pre-miRNAs) and/or shRNAs as therapeutical
drugs and/or vaccines for cancer therapy. More specifically, the
present invention is a design and method of utilizing prokaryotic
cells to produce a special kind of pre-miRNA-like agents, named
pro-miRNA, that are capable of reprogramming the malignant
properties of high-grade human cancer cells into a low-grade benign
or even relatively normal-like state. Preferably, these pro-miRNAs
are tumor suppressor microRNAs (TS-miRNA) similar to the precursors
of miR-302a, b, c, d, e, and/or f (pre-miR-302s) and their natural
familial cluster as well as their manually re-designed small
hairpin RNA (shRNA) homologues/derivatives, and/or a combination
thereof. The designs of pro-miRNA-like shRNA homologues/derivatives
include imperfectly and perfectly matched hairpin compositions of
the pro-miRNA and its homologous small interfering RNA (siRNA),
which may be formed in a single unit or in a multiple unit cluster.
These designs may improve the target specificity and/or reduce the
copy number of pro-miR-302 required for effective delivery and
therapy. The human cells suitable for such a drug treatment include
normal, tumor, and cancerous cells in vitro, ex vivo and/or in
vivo.
[0014] Preferably, the prokaryotic cells used for the present
invention are bacterial competent cells in particular, Escherichia
coli (E. coli), and the chemical inducer is MOPS, ethanol, or
glycerin, or a mixture thereof. Also preferably, the eukaryotic RNA
promoter used is either a eukaryotic pol-2 promoter (i.e. EF1alpha
promoter) or a pol-2 compatible (pol-2-like) viral promoter (i.e.
cytomegaloviral CMV promoter). The gene mediated by the eukaryotic
RNA promoter may code for either a non-coding or protein-coding
RNA, or both (such as an intron-containing gene transcript),
selected from the group consisted of microRNA (miRNA), small
hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA
(mRNA), their precursors and homologues, and a combination thereof.
For inducing gene expression, the prokaryotic cells are transfected
with the eukaryotic RNA promoter-mediated gene and then grown in a
culture medium similar to Luria-Bertani (LB) broth at 37.degree. C.
with addition of the chemical inducer(s) for >24 hours.
[0015] To demonstrate the inducibility of said chemical inducers
for human microRNA production in prokaryotes, we modified a
lentiviral vector pSpRNAi-RGFP-miR302 from our priority U.S. patent
application Ser. Nos. 12/149,725 and 12/318,806 to a new plasmid
vector pLenti-EF1a-RGFP-miR302, of which the SpRNAi-RGFP gene
expression is driven by a eukaryotic promoter such as EF1alpha or
CMV (FIG. 1A). After that, we transformed E. coli competent cells
with it and then used the production of red fluorescent protein
(RGFP) as a visible marker for measuring the transcription rate and
process of microRNA miR-302, as shown in FIG. 1B. Because the
miR-302 familial cluster was also modified to be encoded in the
5'-intron region [e.g. 5'-untranslated region (5'-UTR) or the first
intron] of the RGFP gene, the transcription of each RGFP mRNA led
to the production of one 4-hairpin miR-302 precursor cluster
(pri-miR-302) and/or four 1-hairpin miR-302 precursors
(pre-miR-302s), as shown in FIGS. 5 and 6. Due to lack of RNase III
Dicer in prokaryotes, the pri-miR-302 transcripts would be
eventually broke down (by certain single-strand RNases in E. coli)
into 1-hairpin pre-miR-302s, all of which could be extracted and
further used as therapeutic drugs in the present invention. Broadly
speaking, 5'-UTR and 3'-UTR are considered as a part of intron in
the present invention.
[0016] All miR-302 members share a totally identical sequence in
their first 5'-seventeen (17) nucleotides 5'-UAAGUGCUUC CAUGUUU-3'
(SEQ.ID.NO.3), and contain >82% homology in their full-length
23-nucleotides of a mature microRNA. Based on the results predicted
by online computing programs TARGETSCAN
(http://www.targetscan.org/) and PICTAR-VERT
(http://pictar.mdc-berlin.de/), these miR-302s concurrently target
against almost the same genes, including >600 human genes. In
addition, miR-302 also shares many overlapping target genes with
mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373,
mir-374, and mir-520 familial members, all of which may possess
similar functions. Most of these target genes are developmental
signals and transcriptional factors involved in initiating and/or
establishing certain lineage-specific cell differentiation during
early embryogenesis (Lin et al., 2008). Many of these target genes
are also well-known oncogenes; as a result, miR-302s likely
functions as a tumor suppressor to prevent the deviation of normal
hESC growth into tumor/cancer formation.
Induction of Eukaryotic Promoter-Driven Gene Expression in
Prokaryotes.
[0017] Escherichia coli (E. coli) competent cells were transformed
by the pLenti-EF1alpha-RGFP-miR302 plasmid (FIG. 1A) using a
z-competent E. coli transformation kit (Zymo Research, Irvine,
Calif.) and cultivated in Luria-Bertani (LB) broth supplemented
with a mixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerin
(inducers) at 37.degree. C. with frequent agitation at 170 rpm.
After overnight incubation, the transformed E. coli competent cells
expressed highly abundant red RGFP proteins that could be clearly
seen in the color of the LB broth, whereas the blank control E.
coli presented no RGFP, as shown in FIG. 2. The presence of
functional RGFP indicated that both of its encoded RNA and protein
are successfully produced and processed in the competent cells.
[0018] To further confirm the specificity of gene expression
induced by the chemical inducers, two transformed E. coli strains
were prepared: one carried a pLVX-Grn-miR302+367 plasmid vector
containing a CMV promoter-driven green fluorescent protein (GFP)
gene and the other carried the aforementioned
pLenti-EF1alpha-RGFP-miR302 vector. After overnight incubation with
only 0.1% (v/v) MOPS, the E. coli transformed with
pLVX-Grn-miR302+367 were changed to green color while the other
with pLenti-EF1alpha-RGFP-miR302 still showed red color, as shown
in FIG. 3. This result indicates that the chemical inducers like
MOPS can stimulate a specific RNA transcription and it related
protein production through either a eukaryotic pol-2 or a
pol-2-like viral promoter. Particularly it was noted that the RGFP
and GFP production is so abundant that even the E. coli cells are
visually stained by the respective red and green colors.
[0019] Among all chemicals tested in the present invention, the top
three most potent inducers are MOPS, glycerin and ethanol, as shown
in FIG. 4. The quantitative result of the induced RGFP production
was further confirmed by Western blot analysis, as shown in FIG. 5
and Example 3. Bacterial RuvB protein was served as a house-keeping
standard to normalize RGFP expression. The inducibility of these
identified inducers was also found to be dose-dependent in
proportional to their concentrations. Without any treatment,
negative control E. coli cells just showed their original color in
absence of any fluorescent stain. Therefore, according to all these
results, the present invention clearly provides a novel
chemical-inducible composition and its application for modulating
eukaryotic pol-2-driven or pol-2-like viral promoter-driven RNA
production in prokaryotic cells. In view of the above
demonstration, it is very obvious for an ordinary skill in the art
to use other genes or the related cDNAs in place of the RGFP gene
for producing functional RNAs and the related proteins in
prokaryotes.
Induction of Eukaryotic Promoter-Driven microRNA Expression in
Prokaryotes.
[0020] Accompanying the experiments of RGFP induction shown above,
we further measured the expression of pri-/pre-miR-302s and their
mature miR-302s in the pLenti-EF1alpha-RGFP-miR302-transformed
cells with or without chemical induction. As shown in FIG. 6 and
Example 4, the quantitative results of induced pri-/pre-miR-302
production have been confirmed by Northern blot analysis. Similar
to the results of the RGFP induction in FIGS. 4 and 5, the
pri-/pre-miR-302 expression was strongly detected in transformed
cells treated with MOPS, glycerin or ethanol, but not blank
control, indicating that these chemical inducers indeed stimulated
the expression of the encoded pri-/pre-miRNAs in prokaryotic cells
through a eukaryotic pol-2 promoter (FIG. 6). Due to the structural
similarity of pre-miRNAs and shRNAs, it is obvious for an ordinary
skill in the art to use the present invention to produce other
kinds of pri-/pre-miRNA species, such as but not limited miR-34,
miR-146, miR-371.about.373 and miR-520. For clarification, these
prokaryote-produced pri-/pre-miRNAs are called pro-miRNAs.
[0021] Since pLenti-EF1alpha-RGFP-miR302 contains a miR-302
familial cluster located in the 5'-UTR of the RGFP gene (FIGS. 1A
and 1B), the induced RGFP gene expression will also generate the
miR-302 cluster (pri-miR-302) and its derivative pre-miR-302a, b, c
and d (pre-miR-302s) as demonstrated in FIG. 1B. Due to lack of
RNase III Dicer in prokaryotes, the pri-miR-302 and pre-miR-302s so
obtained were found to remain as hairpin-like microRNA precursors,
which are useful for developing therapeutic drugs. In human cells,
these pre-miR-302s and pri-miR-302 can be processed into mature
miR-302 for eliciting its tumor suppression function. Similarly,
the present invention can also be used to produce other kinds of
TS-miRNA species and their precursors, such as the miR-34a,
miR-146a, miR-373 and miR-520 family.
[0022] The resulting pro-miRNAs can be easily extracted from
competent E. coli cells (Examples 5 and 6) and further purified by
high-performance liquid chromatography (HPLC) (FIGS. 10A and 10B).
Within the purified pro-miR-302s, we have identified all of the
miR-302 familial members (miR-302a, a*, b, b*, c, c*, d, and d*)
using analyses of microRNA microarrays (FIGS. 11B and 12) and RNA
sequencing [FIGS. 13A (pri-miR-302) and 13B (pre-miR-302s)].
Particularly, the sequencing results showed that these pro-miR-302s
all share exactly the same sequences as their natural pre-miR-302
counterparts (FIG. 13B). Furthermore, we have formulated these
pro-miR-302s into a soluble drug for IV/in-vivo injection in order
to test their therapeutic effects on human liver cancers in vivo
(Example 11). As shown in FIG. 14, after 3 injection treatments,
the pro-miR-302 drug successfully reduced >90% volume of the
engrafted human liver cancers in vivo, shirking the average cancer
size to <10% compared to the untreated cancers. Moreover,
histological examination with hematoxylin & eosin (H&E)
staining further demonstrated that this significantly therapeutic
effect was resulted from not only the reported tumor suppression
function of miR-302 (Lin et al., 2010) but also another novel
reprogramming function that has not yet been observed before. For
instance, FIG. 15 clearly showed that the pro-miR-302 drug can
reprogram the malignant properties of high-grade human liver
cancers in vivo to a much more benign stage almost similar to that
of normal liver tissues! These treated cancers can even form normal
liver-like structures, such as classical liver lobules, central
veins (CV) and portal triads (PT). Therefore, these evidences
strongly indicated that pro-miR-302 is able to not only inhibit
tumor/cancer cell growth but also reset the malignancy of human
cancers to a relatively benign or normal state in vivo, leading to
a totally novel therapeutic effect for cancer drug design.
[0023] In the present invention, both of the plasmid vector and its
encoded non-coding RNAs (i.e. pre-miRNA/shRNA) can be
simultaneously amplified in the prokaryotic cells, preferably E.
coli DH5alpha competent cells (Examples 1, 5 and 6). The method for
isolating the amplified pLenti-EF1alpha-RGFP-miR302 plasmid DNA and
the transcribed pri-/pre-miR-302s is described in Examples 5 and 6.
The technology for delivering plasmid vectors (i.e.
pLenti-EF1alpha-RGFP-miR302) into prokaryotic cells is called cell
transformation, while the method for delivering the amplified
non-coding RNAs (i.e. pro-/pri-/pre-miR-302s) into eukaryotic cells
can be selected from the group of endocytosis, chemical/glycerol
infusion, peptide/liposomal/chemical-mediated transfection,
electroporation, gene gun penetration, microinjection,
transposon/retrotransposon insertion and/or
adenoviral/retroviral/lentiviral infection.
Pre-miR-302-Induced Pluripotent Stem Cell Derivation.
[0024] MiR-302 has been reported to reprogram mammalian somatic
cells to human embryonic stem cell (hESC)-like induced pluripotent
stem cells (iPSCs) as demonstrated in our priority U.S. patent
application Ser. Nos. 12/149,725 and 12/318,806. Numerous stem cell
applications and therapies have been designed and developed using
these iPSCs. Nevertheless, since cultivating these iPSCs and hESCs
is very costly and laborious, it is difficult and inefficient to
collect miR-302 and its precursors from these pluripotent stem
cells. On the other hand, making synthetic shRNA mimics is another
possible alternative for pre-miR-302 production; yet, the cost is
still very expensive. Also, the similarity between synthetic shRNA
and natural pre-miR-302 is very questionable. To solve these
problems, the present invention provides a simple, cheap and
efficient method for mass production of pre-miR-302 in prokaryotes.
Moreover, the extraction and purification of these
prokaryote-produced pre-miR-302s (pro-miR-302s) is relatively easy
and cost-effective, as shown in FIG. 6 and Example 6 of the present
invention.
[0025] We have used the pLenti-EF1alpha-RGFP-miR302-transformed E.
coli cells to produce and isolate high quantity and quality of
pLenti-EF1alpha-RGFP-miR302 vector and pro-miR-302s, as shown in
Examples 5 and 6. Both pLenti-EF1alpha-RGFP-miR302 and pro-miR-302s
are useful for generating iPSCs. Following Example 2, when the
pro-miR-302s produced by the present invention were transduced into
human skin primary keratinocytes, the transfected keratinocytes
were reprogrammed to hESC-like iPSCs that expressed strong hESC
marker Oct4 (FIG. 7). In FIG. 8 and Example 8, we further performed
bisulfite DNA sequencing assays to show that global DNA
demethylation did occurr in the promoters of both Oct4 and Sox2
genes, two of the key reprogramming factors as well as hESC
markers. As global DNA demethylation and Oct4 expression are known
to be the first step of somatic cell reprogramming to form
hESC-like iPSCs (Simonsson and Gurdon, Nat Cell Biol. 6: 984-990,
2004), the pro-miR-302s isolated from the MOPS-induced E. coli cell
extracts is proven to be as effective as natural pre-miR-302s,
which are useful for iPSC derivation. Hence, pro-miR-302 and
pre-miR-302 likely possess the same function in stem cell
induction.
Pre-miR-302-Induced CD34-Positive Adult Stem Cell Expansion and/or
Regeneration.
[0026] MicroRNA miR-302 has been found to reprogram mammalian
somatic cells to embryonic stem cell (ESC)-like induced pluripotent
stem cells (iPSC) (Lin, 2008, 2010, 2011; U.S. patent application
Ser. Nos. 12/149,725 and 12/318,806 to Lin). Using these iPSCs,
many stem cell-associated applications and therapies have been
developed for advancing modern regenerative medicine. Yet, miR-302
is only abundantly found in human ESCs rather than differentiated
tissue cells. Also, isolation of miR-302 from human ESCs is highly
debatable, costly and tedious. To solve these problems, the present
invention provides a simple, cheap, fast and inducible composition
and method for mass production of hairpin-like miR-302 molecules
and/or their precursors/homologs in prokaryotes. Moreover, the
isolation of miR-302 and/or its precursors from prokaryotic cells
is relatively easy and cost-effective, as shown in FIG. 6 and
Example 6 of the present invention.
[0027] We have used the pLenti-EF1alpha-RGFP-miR302-transformed E.
coli cells to produce and isolate high quantity and quality of the
pLenti-EF1alpha-RGFP-miR302 vector and pre-miR-302s, as shown in
Examples 5 and 6. The use of pLenti-EF1alpha-RGFP-miR302 had been
shown to produce human ESC-like iPSCs in view of our previous U.S.
patent application Ser. Nos. 12/149,725 and 12/318,806. Also, the
iPSCs so obtained can be further differentiated into various
somatic tissue cells as demonstrated in our previous studies (Lin
et al, 2008, 2010 and 2011).
[0028] The applications of isolated miR-302 and/or pre-miR-302
molecules may further include the induction and expansion of
CD34-positive adult stem cells. As shown in FIGS. 17A and 17B, our
recent studies in wound healing therapy and cancer therapy using a
novel miR-302-formulated drug revealed that treatments of
relatively low concentrations (50.about.500 .mu.g/mL) of the
isolated miR-302/pre-miR-302 molecules not only greatly enhance
scar-less wound healing but also induce CD34-positive adult stem
cell expansion around the wounded area in pig skins in vivo. Based
on the miR-302-treated (miR-302s/pre-miR-302s+antibiotic ointment)
result of FIG. 17B in comparison with that of control (only
antibiotic ointment) result of FIG. 17A, it clearly showed a
.gtoreq.40-fold increase of CD34-positive adult stem cell
populations (labeled by green fluorescent antibodies) in vivo after
miR-302 treatments. The currently known CD34-positive adult stem
cell types include, but not limited, skin, hair, muscle, blood
(hematopoietic), mesenchymal, and neural stem cells. As a result,
since miR-302 can be used to induce CD34-positive adult stem cell
expansion and/or regeneration in vivo, this therapeutic effect may
also help to re-grow and/or revive functional adult stem cells for
treating degenerative diseases in humans, such as, but not limited,
Alzheimer's diseases, Parkinson's diseases, osteoporosis, diabetes,
and cancers.
Utilization of Pro-miR-302 for Tumor/Cancer Therapy In Vivo.
[0029] Our previous studies have demonstrated the feasibility of
this approach in treating human hepatocellular carcinoma HepG2
cells in vitro but not in vivo (Lin et al., 2010). As shown in FIG.
9, the treated tumor/cancer cells were reprogrammed to iPSCs
(labeled as mirPS-HepG2) and formed embryoid body-like cell
colonies. Moreover, miR-302 was also found to induce >95%
apoptosis in the treated cancer cell population. The top panels of
FIG. 9 further showed that flow cytometry analysis of the DNA
content in response to cell cycle stages revealed a significant
reduction in the mitotic cell populations after miR-302 treatments
(form 45.6% to 17.2%). These results indicated that miR-302 can
effectively attenuate the fast cell cycle rate of human liver
cancer cells and hence causes significant apoptosis in these cancer
cells.
[0030] The process of cancer progression was thought to be
irreversible due to accumulative gene mutations; yet, the present
invention discloses a novel pre-miRNA (pro-miR-302) function that
can reprogram high-grade malignant cancers back to a low-grade
benign or even normal-like stage in vivo, of which the mechanism
may be related to a very rare natural healing process called
spontaneous cancer regression. Spontaneous cancer regression occurs
rarely at a rate of less than 1 in 100,000 cancer patients. We
found that pro-mir-302 treatment is able to increase this rare
healing rate to >90% in human liver cancers. As shown in FIG.
14, the therapeutic results of using pro-miR-302s as a drug to
treat human liver cancer xenografts in SCID-beige nude mice (n=6)
demonstrated that this pro-mir-302 drug successfully reduced cancer
sizes from 728.+-.328 mm.sup.3 (untreated blank control, C) to
75.+-.15 mm.sup.3 (pro-mir-302-treated, T), indicating a .about.90%
reduction rate in the average cancer size, whereas treatments of
other synthetic siRNA mimics (siRNA-302) did not provide any
similar therapeutic effect.
[0031] Further histological examination (the most right panels of
FIG. 14) showed that normal liver lobule-like structures (circled
and pointed by a black arrow) were observed only in
pro-miR-302-treated cancer grafts but not other treatments or
controls, suggesting that a reprogramming mechanism has occurred to
reset the malignant cancer cell property back to a relatively
normal-like state (Cancer Reversion). This novel reprogramming
mechanism is likely resulted from the gene silencing effect of
miR-302 on human oncogenes in particular, those mutated oncogenes
involved in cancer progression. By silencing those mutated
oncogenes, pro-miR-302 is able to reset the cancerous gene
expression patterns back to a normal-like state, consequently
leading to the therapeutic result of cancer reversion.
Nevertheless, this in-vivo reprogramming mechanism is clearly
different from the previously reported somatic cell reprogramming
(Lin et al., 2008 and 2011) because no Oct4-positive pluripotent
stem cell has been identified.
[0032] More detailed histological examination (FIG. 15) further
confirmed that the pro-miR-302 drug did reprogram high-grade (Grade
IV) human liver cancer grafts to a more benign low-grade (less than
Grade II) state. As shown in FIG. 15, the treated cancer grafts
formed classical liver lobules containing central vein (CV)-like
and portal triad (PT)-like structures (indicated by black arrows),
highly similar to normal liver tissue structures (top).
Histological comparison among untreated, siRNA-treated,
pro-miR-302-treated human liver cancer grafts and normal liver
tissues in vivo (FIG. 16) also showed that the engrafted human
liver cancers without treatment (top) aggressively invaded into
surrounding normal tissues, such as muscles and blood vessels, and
formed massive cell-cell and cancer-tissue fusion structures,
demonstrating its high malignancy and metastasis. Treatment of
siRNA mimics (siRNA-302) did not significantly reduce the
malignancy of the engrafted liver cancers (upper middle), probably
due to the short half-life of siRNA in vivo. In contrast, treatment
of pro-miR-302 not only reprogrammed the engrafted cancer cells to
a normal liver cell-like morphology (no fusion) but also
successfully inhibited any cancer invasion into the surrounding
tissues (lower middle). Compared to normal liver tissues (bottom),
pro-miR-302-treated cancers clearly displayed similar lobule
structures, normal gland cell-like arrangements, and very clear
boundaries between cell-cell and cancer-tissue junctions (black
arrows), suggesting that these treated cancers have been greatly
downgraded to a very benign state. Further continuous treatments of
the pro-miR-302 drug over six to ten times could completely
eliminate the cancer xenografts in all six samples (n=6).
A. Definitions
[0033] To facilitate understanding of the invention, a number of
terms are defined below:
[0034] Nucleotide: a monomeric unit of DNA or RNA consisting of a
sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic
base. The base is linked to the sugar moiety via the glycosidic
carbon (1' carbon of the pentose) and that combination of base and
sugar is a nucleoside. A nucleoside containing at least one
phosphate group bonded to the 3' or 5' position of the pentose is a
nucleotide. DNA and RNA are consisted of different types of
nucleotide units called deoxyribonucleotide and ribonucleotide,
respectively.
[0035] Oligonucleotide: a molecule comprised of two or more DNAs
and/or RNAs, preferably more than three, and usually more than ten.
An oligonucleotide longer than 13 nucleotide monomers is also
called polynucleotiude. The exact size will depend on many factors,
which in turn depends on the ultimate function or use of the
oligonucleotide. The oligonucleotide may be generated in any
manner, including chemical synthesis, DNA replication, RNA
transcription, reverse transcription, or a combination thereof.
[0036] Nucleotide Analog: a purine or pyrimidine nucleotide that
differs structurally from adenine (A), thymine (T), guanine (G),
cytosine (C), or uracil (U), but is sufficiently similar to
substitute for the normal nucleotide in a nucleic acid
molecule.
[0037] Nucleic Acid Composition: a nucleic acid composition refers
to an oligonucleotide or polynucleotide such as a DNA or RNA
sequence, or a mixed DNA/RNA sequence, in either a single-stranded
or a double-stranded molecular structure.
[0038] Gene: a nucleic acid composition whose oligonucleotide or
polynucleotide sequence codes for an RNA and/or a polypeptide
(protein). A gene can be either RNA or DNA. A gene may encode a
non-coding RNA, such as small hairpin RNA (shRNA), microRNA
(miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors as
well as derivatives. Alternatively, a gene may encode a
protein-coding RNA essential for protein/peptide synthesis, such as
messenger RNA (mRNA) and its RNA precursors as well as derivatives.
In some cases, a gene may encode a protein-coding RNA that also
contains at least a microRNA or shRNA sequence.
[0039] Primary RNA Transcript: an RNA sequence that is directly
transcribed from a gene without any RNA processing or modification,
which may be selected from the group consisting of mRNA, hnRNA,
rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and their RNA
precursors as well as derivatives.
[0040] Precursor messenger RNA (pre-mRNA): primary RNA transcripts
of a protein-coding gene, which are produced by eukaryotic type-II
RNA polymerase (Pol-II) machineries in eukaryotes through an
intracellular mechanism termed transcription. A pre-mRNA sequence
contains a 5'-untranslated region (UTR), a 3'-UTR, exons and
introns.
[0041] Intron: a part or parts of a gene transcript sequence
encoding non-protein-reading frames, such as in-frame intron,
5'-UTR and 3'-UTR.
[0042] Exon: a part or parts of a gene transcript sequence encoding
protein-reading frames (cDNA), such as cDNA for cellular genes,
growth factors, insulin, antibodies and their analogs/homologs as
well as derivatives.
[0043] Messenger RNA (mRNA): assembly of pre-mRNA exons, which is
formed after intron removal by intracellular RNA splicing
machineries (spliceosomes) and served as a protein-coding RNA for
peptide/protein synthesis. The peptides/proteins encoded by mRNAs
include, but not limited, enzymes, growth factors, insulin,
antibodies and their analogs/homologs as well as derivatives.
[0044] Complementary DNA (cDNA): a single-stranded or
double-stranded DNA that contains a sequence complementary to an
mRNA sequence and does not contain any intronic sequence.
[0045] Sense: a nucleic acid molecule in the same sequence order
and composition as the homologous mRNA. The sense conformation is
indicated with a "+", "s" or "sense" symbol.
[0046] Antisense: a nucleic acid molecule complementary to the
respective mRNA molecule. The antisense conformation is indicated
as a "-" or "*" symbol or with an "a" or "antisense" in front of
the DNA or RNA, e.g., "aDNA" or "aRNA".
[0047] Base Pair (bp): a partnership of adenine (A) with thymine
(T), or of cytosine (C) with guanine (G) in a double stranded DNA
molecule. In RNA, uracil (U) is substituted for thymine. Generally
the partnership is achieved through hydrogen bonding.
[0048] Base Pair (bp): a partnership of adenine (A) with thymine
(T), or of cytosine (C) with guanine (G) in a double stranded DNA
molecule. In RNA, uracil (U) is substituted for thymine. Generally
the partnership is achieved through hydrogen bonding. For example,
a sense nucleotide sequence "5'-A-T-C-G-U-3" can form complete base
pairing with its antisense sequence "5'-A-C-G-A-T-3".
[0049] 5'-end: a terminus lacking a nucleotide at the 5' position
of successive nucleotides in which the 5'-hydroxyl group of one
nucleotide is joined to the 3'-hydroyl group of the next nucleotide
by a phosphodiester linkage. Other groups, such as one or more
phosphates, may be present on the terminus.
[0050] 3'-end: a terminus lacking a nucleotide at the 3' position
of successive nucleotides in which the 5'-hydroxyl group of one
nucleotide is joined to the 3'-hydroyl group of the next nucleotide
by a phosphodiester linkage. Other groups, most often a hydroxyl
group, may be present on the terminus.
[0051] Template: a nucleic acid molecule being copied by a nucleic
acid polymerase. A template can be single-stranded, double-stranded
or partially double-stranded, depending on the polymerase. The
synthesized copy is complementary to the template, or to at least
one strand of a double-stranded or partially double-stranded
template. Both RNA and DNA are synthesized in the 5' to 3'
direction. The two strands of a nucleic acid duplex are always
aligned so that the 5' ends of the two strands are at opposite ends
of the duplex (and, by necessity, so then are the 3' ends).
[0052] Nucleic Acid Template: a double-stranded DNA molecule,
double stranded RNA molecule, hybrid molecules such as DNA-RNA or
RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
[0053] Conserved: a nucleotide sequence is conserved with respect
to a pre-selected (referenced) sequence if it non-randomly
hybridizes to an exact complement of the pre-selected sequence.
[0054] Homologous or Homology: a term indicating the similarity
between a polynucleotide and a gene or mRNA sequence. A nucleic
acid sequence may be partially or completely homologous to a
particular gene or mRNA sequence, for example. Homology may be
expressed as a percentage determined by the number of similar
nucleotides over the total number of nucleotides.
[0055] Complementary or Complementarity or Complementation: a term
used in reference to matched base pairing between two
polynucleotides (i.e. sequences of an mRNA and a cDNA) related by
the aforementioned "base pair (bp)" rules. For example, the
sequence "5'-A-G-T-3" is complementary to the sequence
"5'-A-C-T-3", and also to "5'-A-C-U-3". Complementation can be
between two DNA strands, a DNA and an RNA strand, or between two
RNA strands. Complementarity may be "partial" or "complete" or
"total". Partial complementarity or complementation occurs when
only some of the nucleic acid bases are matched according to the
base pairing rules. Complete or total complementarity or
complementation occurs when the bases are completely or perfectly
matched between the nucleic acid strands. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as in detection methods that
depend on binding between nucleic acids. Percent complementarity or
complementation refers to the number of mismatch bases over the
total bases in one strand of the nucleic acid. Thus, a 50%
complementation means that half of the bases were mismatched and
half were matched. Two strands of nucleic acid can be complementary
even though the two strands differ in the number of bases. In this
situation, the complementation occurs between the portion of the
longer strand corresponding to the bases on that strand that pair
with the bases on the shorter strand.
[0056] Complementary Bases: nucleotides that normally pair up when
DNA or RNA adopts a double stranded configuration.
[0057] Complementary Nucleotide Sequence: a sequence of nucleotides
in a single-stranded molecule of DNA or RNA that is sufficiently
complementary to that on another single strand to specifically
hybridize between the two strands with consequent hydrogen
bonding.
[0058] Hybridize and Hybridization: the formation of duplexes
between nucleotide sequences which are sufficiently complementary
to form complexes via base pairing. Where a primer (or splice
template) "hybridizes" with target (template), such complexes (or
hybrids) are sufficiently stable to serve the priming function
required by a DNA polymerase to initiate DNA synthesis. There is a
specific, i.e. non-random, interaction between two complementary
polynucleotides that can be competitively inhibited.
[0059] Posttranscriptional Gene Silencing: a targeted gene knockout
or knockdown effect at the level of mRNA degradation or
translational suppression, which is usually triggered by either
foreign/viral DNA or RNA transgenes or small inhibitory RNAs.
[0060] RNA Interference (RNAi): a posttranscriptional gene
silencing mechanism in eukaryotes, which can be triggered by small
inhibitory RNA molecules such as microRNA (miRNA), small hairpin
RNA (shRNA) and small interfering RNA (siRNA). These small RNA
molecules usually function as gene silencers, interfering with
expression of intracellular genes containing either completely or
partially complementarity to the small RNAs.
[0061] Gene Silencing Effect: a cell response after a gene function
is suppressed, consisting but not limited of cell cycle
attenuation, G0/G1-checkpoint arrest, tumor suppression,
anti-tumorigenecity, cancer cell apoptosis, and a combination
thereof.
[0062] Non-coding RNA: an RNA transcript that cannot be used to
synthesize peptides or proteins through intracellular translation
machineries. Non-coding RNA includes long and short regulatory RNA
molecules such as microRNA (miRNA), small hairpin RNA (shRNA),
small interfering RNA (siRNA) and double strand RNA (dsRNA). These
regulatory RNA molecules usually function as gene silencers,
interfering with expression of intracellular genes containing
either completely or partially complementarity to the non-coding
RNAs.
[0063] MicroRNA (miRNA): single-stranded RNA capable of binding to
targeted gene transcripts (mRNAs) that have partial complementarity
to the sequence of microRNA. Mature microRNA is usually sized about
17-27 oligonucleotides in length and is able to either directly
degrade its intracellular mRNA target(s) or suppress the protein
translation of its targeted mRNA(s), depending on the
complementarity between the microRNA and its target mRNA(s). Native
microRNAs are found in almost all eukaryotes, functioning as a
defense against viral infections and allowing regulation of
specific gene expression during development of plants and animals.
In principle, one microRNA often target multiple target mRNAs to
fulfill its full functionality while on the other hand multiple
miRNAs may target the same gene transcripts to enhance the effect
of gene silencing.
[0064] MicroRNA Precursor (Pre-miRNA): hairpin-like single-stranded
RNA containing stem-arm and stem-loop regions for interacting with
intracellular RNase III Dicer endoribonucleases to produce one or
multiple mature microRNAs (miRNAs) capable of silencing a targeted
gene or a specific group of targeted genes that contain full or
partial complementarity to the mature microRNA sequence(s). The
stem-arm of a pre-miRNA can form either a perfectly (100%) or a
partially (mis-matched) hybrid duplexes, while the stem-loop
connects one end of the stem-arm duplex to form a circle or
hairpin-loop conformation required for being assembled into an
RNA-induced silencing complex (RISC) with some argonaute proteins
(AGO).
[0065] Prokaryote-produced MicroRNA Precursor (Pro-miRNA): small
hairpin-like RNA similar to natural microRNA precursor (pre-miRNA)
but transcribed from an artificially recombinant
microRNA-expressing plasmid driven by a eukaryotic promoter in
prokaryotic competent cells. For example, pro-miR-302 is
structurally as same as pre-miR-302 (FIGS. 13A and 13B) but
transcribed from either a pLVX-Grn-miR302+367 or
pLenti-EF1alpha-RGFP-miR302 vector in E. coli DH5alpha competent
cells (Example 1). As prokaryotic cells normally do not express
short RNAs with high secondary structures such as eukaryotic
pre-miRNA, the production of pro-miRNA in prokaryotes usually
requires the addition of chemical inducer(s) in order to stimulate
the eukaryotic promoter-driven pre-miRNA transcription (FIGS.
2-4).
[0066] Small interfering RNA (siRNA): short double-stranded RNA
sized about 18-27 perfectly base-paired ribonucleotide duplexes and
capable of degrading target gene transcripts with almost perfect
complementarity.
[0067] Small or short hairpin RNA (shRNA): single-stranded RNA that
contains a pair of partially or completely matched stem-arm
nucleotide sequences divided by an unmatched loop oligonucleotide
to form a hairpin-like structure. Many natural miRNAs are derived
from hairpin-like RNA precursors, namely precursor microRNA
(pre-miRNA).
[0068] Vector: a recombinant nucleic acid composition such as
recombinant DNA (rDNA) capable of movement and residence in
different genetic environments. Generally, another nucleic acid is
operatively linked therein. The vector can be capable of autonomous
replication in a cell in which case the vector and the attached
segment is replicated. One type of preferred vector is an episome,
i.e., a nucleic acid molecule capable of extrachromosomal
replication. Preferred vectors are those capable of autonomous
replication and expression of nucleic acids. Vectors capable of
directing the expression of genes encoding for one or more
polypeptides and/or non-coding RNAs are referred to herein as
"expression vectors" or "expression-competent vectors".
Particularly important vectors allow cloning of cDNA from mRNAs
produced using a reverse transcriptase. A vector may contain
components consisting of a viral or a type-II RNA polymerase
(Pol-II or pol-2) promoter, or both, a Kozak consensus translation
initiation site, polyadenylation signals, a plurality of
restriction/cloning sites, a pUC origin of replication, a SV40
early promoter for expressing at least an antibiotic resistance
gene in replication-competent prokaryotic cells, an optional SV40
origin for replication in mammalian cells, and/or a tetracycline
responsive element. The structure of a vector can be a linear or
circular form of single- or double-stranded DNA selected form the
group consisting of plasmid, viral vector, transposon,
retrotransposon, DNA transgene, jumping gene, and a combination
thereof.
[0069] Promoter: a nucleic acid to which a polymerase molecule
recognizes, or perhaps binds to, and initiates RNA transcription.
For the purposes of the instant invention, a promoter can be a
known polymerase or its cofector binding site, an enhancer and the
like, any sequence that can initiate synthesis of RNA transcripts
by a desired polymerase.
[0070] Eukaryotic Promoter: a sequence of nucleic acid motifs which
are required for RNA and/or gene transcription and can be
recognized by eukaryotic type II RNA polymerases (pol-2), pol-2
equivalent, and/or pol-2 compatible (pol-2-like) viral polymerases
for initiating the RNA/gene transcription.
[0071] Type-II RNA Polymerase (Pol-II or pol-2) Promoter: an RNA
promoter that can be recognized by eukaryotic type-II RNA
polymerases (Pol-II or pol-2) and hence is able to initiate the
transcription of eukaryotic messenger RNAs (mRNAs) and/or microRNAs
(miRNAs). For example, but not limited, a pol-2 promoter can be a
mammalian RNA promoter or a cytomegaloviral (CMV) promoter.
[0072] Type-II RNA Polymerase (Pol-II or pol-2) Equivalent: a
eukaryotic transcription machinery selected from the group
consisting of mammalian type-II RNA polymerases (Pol-II or pol-2)
and Pol-II compatible (pol-2-like) viral RNA polymerases.
[0073] Pol-II Compatible (pol-2-like) Viral Promoter: a viral RNA
promoter capable of using the eukaryotic pol-2 or pol-2 equivalent
transcription machineries for initiating gene and/or RNA
expression. For example, but not limited, a pol-2-like viral
promoter can be a cytomegaloviral (CMV) promoter or a retroviral
long terminal repeat (LTR) promoter.
[0074] Cistron: a sequence of nucleotides in a DNA molecule coding
for an amino acid residue sequence and including upstream and
downstream DNA expression control elements.
[0075] Intron Excision: a cellular mechanism responsible for RNA
processing, maturation and degradation, including RNA splicing,
exosome digestion, nonsense-mediated decay (NMD) processing, and a
combination thereof.
[0076] RNA Processing: a cellular mechanism responsible for RNA
maturation, modification and degradation, including RNA splicing,
intron excision, exosome digestion, nonsense-mediated decay (NMD),
RNA editing, RNA processing, and a combination thereof.
[0077] Targeted Cell: a single or a plurality of human cells
selected from the group consisting of a somatic cell, a tissue, a
stem cell, a germ-line cell, a teratoma cell, a tumor cell, a
cancer cell, and a combination thereof.
[0078] Cancerous Tissue: a neoplastic tissue derived from the group
consisting of skin cancer, prostate cancer, breast cancer, liver
cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a
combination thereof.
[0079] Expression-Competent Vector: a linear or circular form of
single- or double-stranded DNA selected form the group consisting
of plasmid, viral vector, transposon, retrotransposon, DNA
transgene, jumping gene, and a combination thereof.
[0080] Antibiotic Resistance Gene: a gene capable of degrading
antibiotics selected from the group consisted of penicillin G,
streptomycin, ampicillin (Amp), neomycin, G418, kanamycin,
erythromycin, paromycin, phophomycin, spectromycin, tetracycline
(Tet), doxycycline (Dox), rifapicin, amphotericin B, gentamycin,
chloramphenicol, cephalothin, tylosin, and a combination
thereof.
[0081] Restriction/Cloning Site: a DNA motif for restriction enzyme
cleavage including but not limited AatII, AccI, AflII/III, AgeI,
ApaI/LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I,
BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I,
DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III/RV, EheI, FspI,
HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI,
MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI,
NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI,
Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI
cleavage site.
[0082] Gene Delivery: a genetic engineering method selected from
the group consisting of polysomal transfection, liposomal
transfection, chemical transfection, electroporation, viral
infection, DNA recombination, transposon insertion, jumping gene
insertion, microinjection, gene-gun penetration, and a combination
thereof.
[0083] Genetic Engineering: a DNA recombination method selected
from the group consisting of DNA restriction and ligation,
homologous recombination, transgene incorporation, transposon
insertion, jumping gene integration, retroviral infection, and a
combination thereof.
[0084] Cell Cycle Regulator: a cellular gene involved in
controlling cell division and proliferation rates, consisting but
not limited of CDK2, CDK4, CDK6, cyclins, BMI-1, p14/p19Arf,
p15Ink4b, p16Ink4a, p18Ink4c, p21 Cip1/Waf1, and p27Kip1, and a
combination thereof.
[0085] Tumor Suppression Effect: a cellular anti-tumor and/or
anti-cancer mechanism and response consisting of, but not limited,
cell cycle attenuation, cell cycle arrest, inhibition of tumor cell
growth, inhibition of cell tumorigenecity, inhibition of
tumor/cancer cell transformation, induction of tumor/cancer cell
apoptosis, induction of normal cell recovery, reprogramming
high-grade malignant cancer cells to a more benign low-grade state
(tumor regression), and a combination thereof.
[0086] Cancer Therapy Effect: a cell response and/or cellular
mechanism resulted from a drug treatment, including, but not
limited, inhibition of oncogene expression, inhibition of cancer
cell proliferation, inhibition of cancer cell invasion and/or
migration, inhibition of cancer metastasis, induction of cancer
cell death, prevention of tumor/cancer formation, prevention of
cancer relapse, suppression of cancer progression, repairing
damaged tissue cells, reprogramming high-grade malignant cancers to
a more benign low-grade state (cancer regression/remission), and a
combination thereof.
[0087] Gene Silencing Effect: a cell response after a gene function
is suppressed, consisting of, but not limited, inhibition of
oncogene expression, inhibition of cell proliferation, cell cycle
arrest, tumor suppression, cancer regression, cancer prevention,
cell apoptosis, cell repairing and/or rejuvenation, cell
reprogramming, reprogramming diseased cells to a relatively normal
state (spontaneous healing), and a combination thereof.
[0088] Cancer Reversion: a reprogramming mechanism that resets the
malignant properties of high-grade cancers back to a relatively
normal-like low-grade state in vitro, ex vivo or in vivo.
[0089] Targeted Cell: a single or a plurality of human cells
selected from the group consisting of a somatic cell, a tissue, a
stem cell, a germ-line cell, a teratoma cell, a tumor cell, a
cancer cell, and a combination thereof.
[0090] Cancerous Tissue: a neoplastic tissue derived from the group
consisting of skin cancer, prostate cancer, breast cancer, liver
cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a
combination thereof.
[0091] Transcriptional Inducer: a chemical agent that can induce
and/or enhance eukaryotic RNA and/or gene transcription from a
pol-2 or pol-2-like promoter in prokaryotic cells. For example, a
transcription inducer contains, but not limited, a chemical
structure similar to MOPS, ethanol, glycerin, as well as their
functional analogs such as 2-(N-morpholino)ethanesulfonic acid
(MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
and mannitol, or a mixture thereof.
[0092] Antibody: a peptide or protein molecule having a
pre-selected conserved domain structure coding for a receptor
capable of binding a pre-selected ligand.
[0093] Pharmaceutical and/or Therapeutic Application: a biomedical
utilization, device and/or apparatus useful for diagnosis, stem
cell generation, stem cell research and/or therapy development,
tissue/organ repair and/or rejuvenation, wound healing treatment,
tumor suppression, cancer therapy and/or prevention, disease
treatment, drug production, and a combination thereof.
B. Compositions and Applications
[0094] A composition and method for producing a new kind of
prokaryote-produced microRNA precursors (pro-miRNAs) capable of
reprogramming the malignant properties of human cancers into a
low-grade benign or normal-like state in vitro, ex vivo and in
vivo, comprising: (a) at least a chemical inducer agent containing
a structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS),
ethanol, or glycerin, or a mixture thereof and (b) a plurality of
prokaryotic cells that contain at least a pre-miRNA-encoding gene
mediated by eukaryotic pol-2 and/or pol-2-like promoter-driven
transcription; wherein said (a) and (b) are mixed together under a
condition to induce the expression of said gene, so as to generate
the encoded pre-miRNA in the prokaryotic cells. Notably, the
chemical inducer is able to stimulate eukaryotic promoter-driven
RNA transcription in prokaryotes!
[0095] In principle, the present invention provides a novel
composition design and its applicable strategy for inducing a quick
adaptation of prokaryotes to use eukaryotic pol-2 and pol-2-like
promoters for directly expressing certain desired microRNA
precursors (pre-miRNA) without the need of using error-prone
prokaryotic promoters or growing laborious and costly hybridomas or
mammalian cells.
[0096] Preferably, said prokaryote is a bacterial cell strain in
particular, Escherichia coli (E. coli), and said chemical inducer
is 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, or
glycerin, or a mixture thereof. Also preferably, said eukaryotic
promoter is either a eukaryotic pol-2 promoter, such as EF1alpha,
or a pol-2 compatible (pol-2-like) viral promoter, such as
cytomegaloviral (CMV) promoter or retroviral long terminal repeat
(LTR) promoter. The pre-miRNA-encoding gene mediated by said
eukaryotic promoter is coded for either a non-coding or a
protein-coding RNA transcript, or both (such as an
intron-containing gene transcript), selected from the group
consisted of microRNA (miRNA), small hairpin RNA (shRNA), small
interfering RNA (siRNA), messenger RNA (mRNA) and their precursors
as well as shRNA/siRNA homologues, and a combination thereof. The
protein-coding RNA may be selected from, but not limited to, the
group consisted of a gene encoding enzyme, growth factor, antibody,
insulin, botulinum toxin (botox), a functional protein and/or its
analogs, and a combination thereof. Preferably, said condition for
inducing the expression of said pre-miRNA-encoding gene is a
bacterial culturing condition such as Luria-Bertani (LB) broth at
37.degree. C. with the addition of said chemical inducer(s).
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] The patent or application file contains at least one color
drawing. Copies of this patent or patent application publication
with color drawing will be provided by the USPTO upon request and
payment of the necessary fee.
[0098] Referring particularly to the drawings for the purpose of
illustration only and not limitation, there is illustrated:
[0099] FIGS. 1A and 1B show a eukaryotic promoter-driven expression
vector composition (1A) and its expression mechanism (1B) for RNA
transcript and/or protein production in prokaryotes. For
demonstrating the present invention, a new
pLenti-EF1alpha-RGFP-miR302 vector (FIG. 1A) is served as an
example composition for transforming E. coli DH5alpha competent
cells to produce RGFP proteins as well as miR-302s and their
precursors (pre-miR-302s) under the stimulation of MOPS, glycerin
and/or ethanol. pLenti-EF1alpha-RGFP-miR302 is a lentiviral plasmid
vector that is designed by the inventors to expresses various
microRNAs/shRNAs, mRNAs and/or proteins/peptides in both
prokaryotes and eukaryotes. According to the disclosed mechanism
(1B), it is easy for an ordinary skill in the art to use any
microRNA/shRNA in place miR-302 or any mRNA/protein in place of
RGFP as described in the present invention. Black arrows indicate
the pathways occurring in both prokaryotic and eukaryotic cells,
while blank arrows indicate the steps only occurring in the
eukaryotic cells.
[0100] FIG. 2 depicts the results of bacterial culture broths
treated with (left) or without (right) the mixture of 0.1% (v/v)
MOPS and 0.05% (v/v) glycerin. The E. coli competent cells have
been transformed by pLenti-EF1alpha-RGFP-miR302 before the
treatment of chemical inducers.
[0101] FIG. 3 shows the results of different bacterial pellets
after treated with 0.1% (v/v) MOPS. The E. coli competent cells
have been transformed by either pLVX-Grn-miR302+367 (green) or
pLenti-EF1alpha-RGFP-miR302 (red) before the MOPS treatment.
[0102] FIG. 4 shows the inducibility of various chemical inducers
for inducing pol-2 promoter-driven gene expression in E. coli
competent cells. Among all chemicals tested, the top three most
potent inducers are MOPS, glycerin and ethanol. The chemical
concentration used can be ranged from about 0.001% to 4%, most
preferably, from 0.01% to 1%.
[0103] FIG. 5 shows the Western blotting results of red RGFP
protein expression induced by MOPS, glycerin and ethanol,
respectively. Bacterial RuvB protein was used as a house-keeping
standard to normalize the detected RGFP expression. Proteins
extracted from blank E. coli cells, i.e. transformed with no
vector, were used as a negative control.
[0104] FIG. 6 shows the Northern blotting results of the expression
of the miR-302 familial cluster (.about.700 nt) and its derivative
precursors (pre-miR-302s with 1 to 4 hairpins) induced by MOPS,
glycerin and ethanol, respectively. RNAs extracted from blank E.
coli cells were used as a negative control.
[0105] FIG. 7 shows iPSC generation using miR-302 and/or
pre-miR-302 isolated from bacterial competent cell extracts (BE),
which is confirmed by Northern blot analysis as shown in FIG. 6. As
reported, miR-302-reprogrammed iPSCs (or called mirPSCs) form
sphere-like cell colonies and express strong Oct4 as a standard
hESC marker.
[0106] FIG. 8 shows the global DNA demethylation of Oct4 and Sox2
gene promoters induced by the miR-302 and/or pre-miR-302 isolated
from bacterial competent cell extracts (BE), which is confirmed by
Northern blot analysis as shown in FIG. 6. As demonstrated by
Simonsson and Gurdon (Nat Cell Biol. 6, 984-990, 2004), both signs
of global DNA demethylation and Oct4 expression are required for
somatic cell reprogramming to form iPSCs.
[0107] FIG. 9 shows the in vitro tumorigenicity assays of human
liver cancer cell line HepG2 in response to miR-302 transfection.
The cells obtained after miR-302 transfection are labeled as
mirPS-HepG2, indicating the change of their cancer cell properties
into an induced pluripotent stem cell (iPSC)-like state. Changes of
morphology and cell cycle rate before and after miR-302
transfection were compared. Each cell DNA content respective to
cell cycle stages was shown by a peak chart of flow cytometry
analysis above the cell morphology (n=3, p<0.01).
[0108] FIGS. 10A and 10B show the results of HPLC purification and
analysis using a synthetic standard uDNA (by Sigma-Genosys) and
freshly extracted pro-miR-302s isolated from
pLenti-EF1alpha-RGFP-miR302-transformed E. coli cells. The standard
uDNA was designed to be equal to a natural pre-miR-302a as:
5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC
UUCCAUGUUU UGGUGAUGG-3' (SEQ.ID.NO.4).
[0109] FIGS. 11A and 11B show the results of microRNA (miRNA)
microarray analyses using small RNAs extracted from either blank E.
coli competent cells or pLenti-EF1alpha-RGFP-miR302
(RGFP-miR302)-transfected cells. The extracted small RNAs were
further purified by HPLC as shown in the green-labeled area of FIG.
10B. FIG. 11A shows that RNAs from blank E. coli cells present
almost no microRNA (green dots mean non-statistically significant
whereas red dots indicate positive results). This is because
prokaryotes lack several essential enzymes required for microRNA
expression and processing, such as Pol-2, Drosha and RNase III
Dicer. Also, prokaryotic RNA polymerases do not efficiently
transcribe small RNAs with high secondary structures, such as
hairpin-like pre-miRNAs and shRNAs. As a result, only using the
present invention we can stimulate the expression of specific
microRNAs, such as miR-302a, a*, b, b*, c, c*, d and d* as shown in
FIG. 11B, in prokaryotic cells. Since prokaryotic cells possess no
Dicer, all microRNAs remain in their precursor conformations, such
as pri-miRNA (4-hairpin cluster) and/or pre-miRNA (1 hairpin
precursor). Taken together, the results of FIGS. 10B and 11B have
established two facts as: (1) small RNAs extracted from the
RGFP-miR302-transfected cells contain mostly pure miR-302
precursors, and (2) there is almost no other kind of microRNA
contamination in the E. coli competent cells.
[0110] FIG. 12 shows the lists of expressed microRNAs extracted
from either blank E. coli competent cells (Group 1 as shown in FIG.
11A) or pLenti-EF1alpha-RGFP-miR302-transfected cells (Group 2 as
shown in FIG. 11B). Signals less than 500 are not statistically
significant (as shown in green in FIGS. 11A and 11B), which may be
caused by either low copy number expression or high background.
[0111] FIGS. 13A and 13B show the sequencing results of the miR-302
familial cluster (13A) and the individual pro-miR-302a,
pro-miR-302b, pro-miR-302c, and pro-miR-302d sequences (13B). The
result of the miR-302 familial cluster (=pri-miR-302) is
5'-AAUUUUUUUC UUCUAAAGUU AUGCCAUUUU GUUUUCUUUC UCCUCAGCUC
UAAAUACUCU GAAGUCCAAA GAAGUUGUAU GUUGGGUGGG CUCCCUUCAA CUUUAACAUG
GAAGUGCUUU CUGUGACUUU AAAAGUAAGU GCUUCCAUGU UUUAGUAGGA GUGAAUCCAA
UUUACUUCUC CAAAAUAGAA CACGCUAACC UCAUUUGAAG GGAUCCCCUU UGCUUUAACA
UGGGGGUACC UGCUGUGUGA AACAAAAGUA AGUGCUUCCA UGUUUCAGUG GAGGUGUCUC
CAAGCCAGCA CACCUUUUGU UACAAAAUUU UUUUGUUAUU GUGUUUUAAG GUUACUAAGC
UUGUUACAGG UUAAAGGAUU CUAACUUUUU CCAAGACUGG GCUCCCCACC ACUUAAACGU
GGAUGUACUU GCUUUGAAAC UAAAGAAGUA AGUGCUUCCA UGUUUUGGUG AUGGUAAGUC
UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA AAAUAACUUU AUUGUAUUGA CCGCAGCUCA
UAUAUUUAAG CUUUAUUUUG UAUUUUUACA UCUGUUAAGG GGCCCCCUCU ACUUUAACAU
GGAGGCACUU GCUGUGACAU GACAAAAAUA AGUGCUUCCA UGUUUGAGUG UGGUGGUUCC
UACCUAAUCA GCAAUUGAGU UAACGCCCAC ACUGUGUGCA GUUCUUGGCU ACAGGCCAUU
ACUGUUGCUA-3' (SEQ.ID.NO.5), while the individual sequences of
pro-miR-302a, pro-miR-302b, pro-miR-302c, and pro-miR-302d are as
follows: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC
UUCCAUGUUU UGGUGAUGG-3' (SEQ.ID.NO.6), 5'-GCUCCCUUCA ACUUUAACAU
GGAAGUGCUU UCUGUGACUU UAAAAGUAAG UGCUUCCAUG UUUUAGUAGG AGU-3'
(SEQ.ID.NO.7), 5'-CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA
AGUAAGUGCU UCCAUGUUUC AGUGGAGG-3' (SEQ.ID.NO.8), and 5'-CCUCUACUUU
AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU GAGUGUGG-3'
(SEQ.ID.NO.9), respectively.
[0112] FIG. 14 shows the in vivo therapeutic results of a
pre-investigational new drug (pre-IND) trial using pro-miR-302 as
an injection drug to treat human liver cancer xenografts in
SCID-beige nude mice. Following three treatments (once per week),
the pro-miR-302 drug (=pre-miR-302) successfully reduced cancer
sizes from 728.+-.328 mm.sup.3 (untreated blank control, C) to
75.+-.15 mm.sup.3 (pro-mir-302-treated, T), indicating a .about.90%
reduction rate in the average cancer size! No significant
therapeutic effect was found in the treatments of synthetic siRNA
mimics (siRNA-302). Further histological examination (most right)
found that normal liver lobule-like structures (circles pointed by
a black arrow) were formed only in pro-miR-302-treated cancers but
not other treatments or controls, suggesting that a reprogramming
mechanism may occur to reset the malignant cancer cell properties
back to a relatively normal-like state, called "Cancer
Reversion".
[0113] FIG. 15 shows the histological similarity between normal
liver tissues and pro-mir-302-treated human liver cancer xenografts
in vivo. After three treatments (once per week), the pro-mir-302
drug successfully reprogrammed high-grade (grade IV) human liver
cancer grafts to a more benign low-grade (less than grade II)
state. Similar to normal liver tissues (top), the treated cancer
grafts could form classical liver lobules, containing central vein
(CV)-like and portal triad (PT)-like structures (indicated by black
arrows). As cancer cells are generally more acidic than normal
liver cells, the result of hematoxylin & eosin (H&E)
staining shows more purple in cancer cells whereas more red in
normal liver cells.
[0114] FIG. 16 shows the patho-histological comparison among
untreated, siRNA-treated, pro-mir-302-treated human liver cancer
grafts and normal liver tissues in SCID-beige nude mice. Without
treatment (top), the engrafted human liver cancer aggressively
invaded into normal tissues, such as muscles and blood vessels, and
formed massive cell-cell and cancer-tissue fusion structures,
indicating its malignancy and high metastasis. Treatment of siRNA
mimics (siRNA-302) did not significantly reduce the malignancy of
the engrafted cancer (upper middle), probably due to the short
half-life of siRNA. In contrast, pro-miR-302 treatment not only
reprogrammed the engrafted cancer to a relatively normal-like
morphology (no fusion) but also greatly inhibited cancer invasion
into the surrounding tissues (lower middle). Compared to normal
liver tissues (bottom), pro-miR-302-treated cancers formed
normal-like lobule structures, gland-like cell arrangements, and
clear boundaries between cell-cell and cancer-tissue junctions
(black arrows), indicating that these treated cancers have been
downgraded to a very benign state.
[0115] FIGS. 17A and 17B show comparison of the healing results
between untreated (17A) and miR-302-treated (17B) wounds in vivo.
The isolated miR-302 molecules (20.about.400 .mu.g/mL) were
formulated with di-/tri-glycylglycerins, a delivery reagent, and
antibiotic ointment to form candidate drugs for testing topic
treatments of large 2 cm.times.2 cm open wounds on pig back skins
in vivo (n=6 for each group). After about two-week treatments (one
treatment per day), the healed wounds were dissected and further
made into tissue sections for histological examination under a
microscope. The data showed that no or very little scar (scarless)
could be seen in the miR-302-treated wounds (17B top, n=6/6),
whereas almost all untreated (treated with only antibiotic
ointment) wounds contained large scars (17A top, n=5/6). Also, a
significantly large amount of CD34-positive adult stem cell
clusters (labeled by green fluorescent antibodies) were found in
the miR-302-treated wounds (17B bottom, n=6/6), but not in
untreated control wounds (17A bottom, n=0/6). These results
indicate that pre-miR-302 is able to induce CD34-positive adult
stem cell expansion and/or regeneration, so as to enhance tissue
repairing and regeneration, leading to a very beneficial
therapeutic effect on lesions caused by human degenerative
diseases, such as Alzheimer's diseases, Parkinson's diseases,
osteoporosis, diabetes, and cancers. Such therapeutic effect may
also help to reprogram high-grade malignant cancers into low-grade
benign or even normal-like tissues, a novel mechanism called Cancer
Reversion or Cancer Regression.
EXAMPLES
[0116] In the experimental disclosure which follows, the following
abbreviations apply: M (molar); mM (millimolar); .mu.m
(micromolar); mol (moles); pmol (picomoles); gm (grams); mg
(milligrams) .mu.g (micrograms); ng (nanograms); L (liters); ml
(milliliters); .mu.l (microliters); .degree. C. (degrees
Centigrade); RNA (ribonucleic acid); DNA (deoxyribonucleic acid);
dNTP (deoxyribonucleotide triphosphate); PBS (phosphate buffered
saline); NaCl (sodium chloride); HEPES
(N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPES
buffered saline); SDS (sodium dodecyl sulfate); Tris-HCl
(tris-hydroxymethylaminomethane-hydrochloride); ATCC (American Type
Culture Collection, Rockville, Md.); hESC (human embryonic stem
cells); and iPSC (induced pluripotent stem cells).
1. Bacterial Cell Culture and Chemical Treatments
[0117] E. coli DH5alpha competent cells were acquired as a part
from the z-competent E. coli transformation kit (Zymo Research,
Irvine, Calif.) and then transformed by mixing with 5 .mu.g of a
pre-made plasmid vector such as pLenti-EF1alpha-RGFP-miR302 or
pLVX-Grn-miR302+367. Non-transformed cells were normally grown in
Luria-Bertani (LB) broth supplemented with 10 mM MgSO.sub.4 and 0.2
mM glucose at 37.degree. C. with frequent agitation at 170 rpm,
whereas the transformed cells are cultivated in the above LB broth
further supplemented with additional 100 .mu.g/ml ampicillin. For
chemical induction, 0.5 to 2 ml of MOPS, glycerin and/or ethanol,
respectively or in combination, was added into 1 litter LB broth
supplemented with 10 mM MgSO.sub.4 and 0.2 mM glucose in the
presence of 100 .mu.g/ml ampicillin. For negative control, the
transformed cells were cultivated in the above
ampicillin-supplemented LB broth but without adding any chemical
inducer. The results are shown in FIGS. 2-4.
2. Human Cell Culture and MicroRNA Transfection
[0118] Human liver cancer cell line HepG2 was obtained from ATCC
and maintained according to manufacturer's suggestions. For
transfection, 15 .mu.g of pre-miR-302 was dissolved in 1 ml of
fresh RPMI medium and mixed with 50 .mu.l of X-tremeGENE HP DNA
transfection reagent (Roche, Indianapolis, Ind.). After 10 min
incubation, the mixture is added into a 100-mm cell culture dish
containing 50%-60% confluency of HepG2. The medium was refreshed 12
to 18 hours later. After these transfected cells formed sphere-like
iPSC colonies, the medium was changed to a knockout DMEM/F-12
medium (Invitrogen) supplemented with 20% knockout serum, 1% MEM
nonessential amino acids, 100 .mu.M .beta.-mercaptoethanol, 1 mM
GlutaMax, 1 mM sodium pyruvate, 10 ng/ml bFGF, 10 ng/ml FGF-4, 5
ng/ml LIF, 100 IU/ml penicillin/100 .mu.g/ml streptomycin, 0.1
.mu.M A83-01, and 0.1 .mu.M valproic acid (Stemgent, San Diego,
Calif.), and the cells were cultivated at 37.degree. C. under 5%
CO.sub.2. The result is shown in FIG. 9.
3. Protein Extraction and Western Blot Analysis
[0119] Cells (10.sup.6) were lysed with a CelLytic-M
lysis/extraction reagent (Sigma) supplemented with protease
inhibitors, Leupeptin, TLCK, TAME and PMSF, following the
manufacturer's suggestion. Lysates were centrifuged at 12,000 rpm
for 20 min at 4.degree. C. and the supernatant was recovered.
Protein concentrations were measured using an improved SOFTmax
protein assay package on an E-max microplate reader (Molecular
Devices, CA). Each 30 .mu.g of cell lysate was added to SDS-PAGE
sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT)
conditions, and boiled for 3 min before loading onto a 6-8%
polyacylamide gel. Proteins were resolved by SDS-polyacrylamide gel
electrophoresis (PAGE), electroblotted onto a nitrocellulose
membrane and incubated in Odyssey blocking reagent (Li-Cor
Biosciences, Lincoln, NB) for 2 hours at room temperature. Then, a
primary antibody was applied to the reagent and incubated the
mixture at 4.degree. C. Primary antibodies included Oct3/4 (Santa
Cruz Biotechnology, Santa Cruz, Calif.), RuvB (Santa Cruz) and RGFP
(Clontech). After overnight, the membrane was rinsed three times
with TBS-T and then exposed to goat anti-mouse IgG conjugated
secondary antibody to Alexa Fluor 680 reactive dye (1:2,000;
Invitrogen-Molecular Probes), for 1 hour at the room temperature.
After three additional TBS-T rinses, fluorescent scanning of the
immunoblot and image analysis was conducted using Li-Cor Odyssey
Infrared Imager and Odyssey Software v.10 (Li-Cor). The results are
shown in FIG. 5.
4. RNA Extraction and Northern Blot Analysis
[0120] Total RNAs (10 .mu.g) were isolated with a mirVana.TM. miRNA
isolation kit (Ambion, Austin, Tex.), fractionated by either 15%
TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel
electrophoresis, and electroblotted onto a nylon membrane.
Detection of miR-302s and the related pre-miR-302s was performed
with a [LNA]-DNA probe (5'-[TCACTGAAAC] ATGGAAGCAC TTA-3')
(SEQ.ID.NO.10) probe. The probe has been purified by
high-performance liquid chromatography (HPLC) and tail-labeled with
terminal transferase (20 units) for 20 min in the presence of
[.sup.32P]-dATP (>3000 Ci/mM, Amersham International, Arlington
Heights, Ill.). The results are shown in FIG. 6.
5. Plasmid Amplification and Plasmid DNA/Total RNA Extraction
[0121] E. coli DH5alpha competent cells after transformation (from
Example 1) were cultivated in LB broth supplemented with 10 mM
MgSO.sub.4 and 0.2 mM glucose at 37.degree. C. with frequent
agitation at 170 rpm. For inducing eukaryotic promoter-driven RNA
transcription, 0.5 to 2 ml of MOPS, glycerin, and/or ethanol was
added into every 1 litter of LB broth for propagating the
transformed cells overnight. The amplified plasmid DNAs and
expressed mRNAs/microRNAs in the transformed cells were isolated
using a HiSpeed plasmid purification kit (Qiagen, Valencia,
Calif.), following the manufacturer's protocol but with a minor
modification that RNase A was not added into the P1 buffer. After
that, the final extracted products containing both plasmids and
mRNAs/microRNAs were dissolved in DEPC-treated ddH.sub.2O and
stored at -80.degree. C. before use. For purifying only the
amplified plasmid vectors, RNase A was added into the P1 buffer and
the extraction procedure was performed following the manufacturer's
protocol.
6. MicroRNA and Pre-miRNA Isolation/Purification
[0122] For purifying microRNAs and pre-miRNAs, the total RNAs
isolated from Example 5 were further extracted using a mirVana.TM.
miRNA isolation kit (Ambion, Austin, Tex.), following the
manufacturer's protocol. The final products so obtained were
dissolved in DEPC-treated ddH.sub.2O and stored at -80.degree. C.
before use. Because bacterial RNAs are naturally degraded very fast
(within a few hours) whereas eukaryotic hairpin-like microRNA
precursors (pre-miRNAs and pri-miRNAs) remain largely stable at
4.degree. C. (half-life up to 3-4 days), we can use this half-life
difference to acquire relatively pure pri-/pre-miRNAs for other
applications. For example, the pre-miR-302s so obtained can be used
to reprogram somatic cells to hESC-like iPSCs, as shown in FIG.
9.
7. Immunostaining Assay
[0123] Embedding, sectioning and immunostaining tissue samples were
performed as previously reported (Lin et al., 2008). Primary
antibodies include Oct4 (Santa Cruz) and RGFP (Clontech, Palo Alto,
Calif.). Fluorescent dye-labeled goat anti-rabbit or horse
anti-mouse antibody was used as the secondary antibody
(Invitrogen-Molecular Probes, Carlsbad, Calif.). Positive results
were examined and analyzed at 100.times. or 200.times.
magnification under a fluorescent 80i microscopic quantitation
system with a Metamorph imaging program (Nikon). The result is
shown in FIG. 7.
8. Bisulfite DNA Sequencing
[0124] Genomic DNAs were isolated from 2,000,000 cells using a DNA
isolation kit (Roche) and 1 .mu.g of the isolated DNAs was further
treated with bisulfite (CpGenome DNA modification kit, Chemicon,
Temecula, Calif.), following the manufacturers' suggestion. The
bisulfite treatment converted all unmethylated cytosine to uracil,
while methylated cytosine remained as cytosine. For bisulfite DNA
sequencing, we amplified the promoter region of the Oct4 gene with
PCR primers: 5'-GAGGCTGGAG CAGAAGGATT GCTTTGG-3' (SEQ.ID.NO.11) and
5'-CCCTCCTGAC CCATCACCTC CACCACC-3' (SEQ.ID.NO.12). For PCR, the
bisulfite-modified DNAs (50 ng) were mixed with the primers (total
100 pmol) in 1.times.PCR buffer, heated to 94.degree. C. for 2 min,
and immediately cooled on ice. Next, 25 cycles of PCR were
performed as follows: 94.degree. C. for 1 min and 70.degree. C. for
3 min, using an Expand High Fidelity PCR kit (Roche). The PCR
product with a correct size was further fractionized by 3% agarose
gel electrophoresis, purified by a gel extraction filter (Qiagen),
and then used in DNA sequencing. After that, a detailed profile of
DNA methylation sites was generated by comparing the unchanged
cytosine in the converted DNA sequence to the unconverted one, as
shown in FIG. 8.
9. DNA-Density Flow Cytometry
[0125] Cells were trypsinized, pelleted and fixed by re-suspension
in 1 ml of pre-chilled 70% methanol in PBS for 1 hour at
-20.degree. C. The cells were pelleted and washed once with 1 ml of
PBS and then pelleted again and resuspended in 1 ml of 1 mg/ml
propidium iodide, 0.5 .mu.g/ml RNase in PBS for 30 min at
37.degree. C. After that, about 15,000 cells were analyzed on a BD
FACSCalibur (San Jose, Calif.). Cell doublets were excluded by
plotting pulse width versus pulse area and gating on the single
cells. The collected data were analyzed using the software package
Flowjo using the "Watson Pragmatic" algorithm. The result was shown
in the top panels of FIG. 9.
10. MicroRNA (miRNA) Microarray Analysis
[0126] At about 70% confluency, small RNAs from each cell culture
were isolated, using the mirVana.TM. miRNA isolation kit (Ambion).
The purity and quantity of the isolated small RNAs were assessed,
using 1% formaldehyde-agarose gel electrophoresis and
spectrophotometer measurement (Bio-Rad), and then immediately
frozen in dry ice and submitted to LC Sciences (San Diego, Calif.)
for miRNA microarray analyses. Each microarray chip was hybridized
a single sample labeled with either Cy3 or Cy5 or a pair of samples
labeled with Cy3 and Cy5, respectively. Background subtraction and
normalization were performed as manufacturer's suggestions. For a
dual sample assay, a p-value calculation was performed and a list
of differentially expressed transcripts more than 3-fold
(yellow-red signals) was produced. The final microarray results
were shown in FIGS. 11A and 11B, and the list of differentially
expressed microRNAs was shown in FIG. 12, which compared the small
RNAs extracted from blank E. coli cell lysates (Group 1) to those
extracted from pLenti-EF1alpha-RGFP-miR302-transformed cell lysates
(Group 2).
11. In Vivo In Vivo Liver Cancer Therapy Trials
[0127] Xenografting human liver cancers into immunocompromised
SCID-beige mice is a valid animal model for studying liver cancer
metastasis and therapy. To establish this model, we mixed 5 million
human hepatocarcinoma (HepG2) cells with 100 .mu.L of matrix gel
and subcutaneously engrafted the mixture into each flank of the
mouse hind limbs, respectively. As a result, both sides of the
mouse hind limbs were subjected to approximately the same amount of
cancer cell engraftment. Cancers were observed about two weeks
post-engraftment and sized about 15.6.+-.8 mm.sup.3 in average
(starting cancer size before treatment). For each mouse, we
selected the side with a larger cancer as the treatment group and
the other smaller one as the control group. Since the same mouse
was treated with a blank formulation reagent (negative control) in
one side and the formulated drug (pro-mir-302) in the other side,
the results so obtained can minimize any possible variation due to
individual differences.
[0128] To deliver pro-mir-302 into the targeted cancer regions in
vivo, we contracted a professional formulation company, Latitude
(San Diego, Calif.), to liposomally encapsulate pro-miR-302s into
160.about.200 nm-diameter nanoparticles. These
pro-miR-302-containing nanoparticles have been tested to be almost
100% stable at room temperature for over two weeks and at 4.degree.
C. for over one month, whereas other synthetic siRNA mimics
(siRNA-302) were all quickly degraded over 50% within 3 to 5 days
under the same conditions, indicating that pro-miRNA rather than
siRNA is stable enough to be used as a drug for therapy. For
toxicity assay, we have further injected maximally 300 .mu.L of the
formulated pro-miR-302 (1 mg/mL) into the mouse tail vein (n=8),
respectively, and observed no detectable side effect in all tested
mice over six months. In general, non-modified ribonucleic acids
are relatively not immunogenic and can be easily metabolized by
tissue cells, rendering a safe tool for in vivo therapy.
[0129] For testing drug potency, we subcutaneously injected 200
.mu.L of the formulated pro-mir-302 in one side and 200 .mu.L of
the blank formulation reagent in the other side of the mice,
respectively, and continued the same injection pattern for three
times (one injection per week). The drug and reagent were applied
to the surrounding region of the cancer site and absorbed by the
cancer and its surrounding tissues within 18 hours. Samples were
collected one week after the third injection. Hearts, livers,
kidneys and the engrafted cancers were removed for further
histological examination. Tumor formation was monitored by
palpation and tumor volume was calculated using the formula (length
x width)/2. Tumor lesions were counted, dissected, weighed, and
subjected to histological examination using H&E and
immunostaining assays. Histological examination showed no
detectable tissue lesions in heart, liver, and kidney. The results
were shown in FIGS. 14, 15 and 16.
12. Statistic Analysis
[0130] Any change over 75% of signal intensity in the analyses of
immunostaining, western blotting and northern blotting was
considered as a positive result, which in turn is analyzed and
presented as mean.+-.SE. Statistical analysis of data was performed
by one-way ANOVA. When main effects were significant, the Dunnett's
post-hoc test was used to identify the groups that differed
significantly from the controls. For pairwise comparison between
two treatment groups, the two-tailed student t test was used. For
experiments involving more than two treatment groups, ANOVA was
performed followed by a post-hoc multiple range test. Probability
values of p<0.05 was considered significant. All p values were
determined from two-tailed tests.
REFERENCES
[0131] 1. Lin S L and Ying S Y. (2006) Gene silencing in vitro and
in vivo using intronic microRNAs. Ying S Y. (Ed.) MicroRNA
protocols. Humana press, Totowa, N.J., pp 295-312. [0132] 2. Lin S
L, Chang D and Ying S Y. (2006) Transgene-like animal models using
intronic microRNAs. Ying S Y. (Ed.) MicroRNA protocols. Humana
press, Totowa, N.J., pp 321-334. [0133] 3. Lin S L, Chang D,
Chang-Lin S, Lin C H, Wu D T S, Chen D T, and Ying S Y. (2008)
Mir-302 reprograms human skin cancer cells into a pluripotent
ES-cell-like state. RNA 14, 2115-2124. [0134] 4. Lin S L and Ying S
Y. (2008) Role of mir-302 microRNA family in stem cell pluripotency
and renewal. Ying S Y. (Ed.) Current Perspectives in MicroRNAs.
Springer Publishers press, New York, pp 167-185. [0135] 5. Lin S L,
Chang D, Ying S Y, Leu D and Wu D T S. (2010) MicroRNA miR-302
inhibits the tumorigenecity of human pluripotent stem cells by
coordinate suppression of CDK2 and CDK4/6 cell cycle pathways.
Cancer Res. 70, 9473-9482. [0136] 6. Lin S L, Chang D, Lin C H,
Ying S Y, Leu D and Wu D T S. (2011) Regulation of somatic cell
reprogramming through inducible mir-302 expression. Nucleic Acids
Res. 39, 1054-1065. [0137] 7. Simonsson S and Gurdon J. (2004) DNA
demethylation is necessary for the epigenetic reprogramming of
somatic cell nuclei. Nat Cell Biol. 6, 984-990. [0138] 8. Takahashi
et al. (2006). Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell
126, 663-676. [0139] 9. Wang et al. (2008). Embryonic stem
cell-specific microRNAs regulate the G1-S transition and promote
rapid proliferation. Nat. Genet. 40, 1478-1483. [0140] 10. Wernig
et al. (2007). In vitro reprogramming of fibroblasts into a
pluripotent ES-cell-like state. Nature 448, 318-324. [0141] 11. Yu
et al. (2007). Induced pluripotent stem cell lines derived from
human somatic cells. Science 318, 1917-1920. [0142] 12. U.S. Pat.
No. 7,959,926 to Buechler. [0143] 13. U.S. Pat. No. 7,968,311 to
Mehta. [0144] 14. European Patent No. EP 2198025 to Lin. [0145] 15.
U.S. patent application Ser. No. 12/149,725 to Lin. [0146] 16. U.S.
patent application Ser. No. 12/318,806 to Lin. [0147] 17. U.S.
patent application Ser. No. 12/792,413 to Lin. [0148] 18. U.S.
patent application Ser. No. 13/572,263 to Lin. [0149] 19. U.S.
patent application Ser. No. 13/964,705 to Lin.
Sequence CWU 1
1
12123RNAArtificial SequenceSynthetic oligonucleotides 1ucaccaaaac
auggaagcac uua 23223RNAArtificial SequenceSynthetic
oligonucleotides 2acuuaaacgu ggauguacuu gcu 23317RNAArtificial
SequenceSynthetic oligonucleotides 3uaagugcuuc cauguuu 17469RNAHomo
sapiens 4ccaccacuua aacguggaug uacuugcuuu gaaacuaaag aaguaagugc
uuccauguuu 60uggugaugg 695720RNAArtificial SequenceSynthetic
oligonucleotides 5aauuuuuuuc uucuaaaguu augccauuuu guuuucuuuc
uccucagcuc uaaauacucu 60gaaguccaaa gaaguuguau guuggguggg cucccuucaa
cuuuaacaug gaagugcuuu 120cugugacuuu aaaaguaagu gcuuccaugu
uuuaguagga gugaauccaa uuuacuucuc 180caaaauagaa cacgcuaacc
ucauuugaag ggauccccuu ugcuuuaaca uggggguacc 240ugcuguguga
aacaaaagua agugcuucca uguuucagug gaggugucuc caagccagca
300caccuuuugu uacaaaauuu uuuuguuauu guguuuuaag guuacuaagc
uuguuacagg 360uuaaaggauu cuaacuuuuu ccaagacugg gcuccccacc
acuuaaacgu ggauguacuu 420gcuuugaaac uaaagaagua agugcuucca
uguuuuggug augguaaguc uucuuuuuac 480auuuuuauua uuuuuuuaga
aaauaacuuu auuguauuga ccgcagcuca uauauuuaag 540cuuuauuuug
uauuuuuaca ucuguuaagg ggcccccucu acuuuaacau ggaggcacuu
600gcugugacau gacaaaaaua agugcuucca uguuugagug uggugguucc
uaccuaauca 660gcaauugagu uaacgcccac acugugugca guucuuggcu
acaggccauu acuguugcua 720669RNAArtificial SequenceSynthetic
oligonucleotides 6ccaccacuua aacguggaug uacuugcuuu gaaacuaaag
aaguaagugc uuccauguuu 60uggugaugg 69773RNAArtificial
SequenceSynthetic oligonucleotides 7gcucccuuca acuuuaacau
ggaagugcuu ucugugacuu uaaaaguaag ugcuuccaug 60uuuuaguagg agu
73868RNAArtificial SequenceSynthetic oligonucleotides 8ccuuugcuuu
aacauggggg uaccugcugu gugaaacaaa aguaagugcu uccauguuuc 60aguggagg
68968RNAArtificial SequenceSynthetic oligonucleotides 9ccucuacuuu
aacauggagg cacuugcugu gacaugacaa aaauaagugc uuccauguuu 60gagugugg
681023DNAArtificial SequenceSynthetic oligonucleotides 10tcactgaaac
atggaagcac tta 231127DNAArtificial SequenceSynthetic
oligonucleotides 11gaggctggag cagaaggatt gctttgg
271227DNAArtificial SequenceSynthetic oligonucleotides 12ccctcctgac
ccatcacctc caccacc 27
* * * * *
References