U.S. patent application number 16/578893 was filed with the patent office on 2020-02-27 for methods, systems, and compositions for neuronal differentiation of multipotent stromal cells.
The applicant listed for this patent is EXOSTEM BIOTEC LTD.. Invention is credited to Chaya BRODIE, Shimon SLAVIN.
Application Number | 20200063135 16/578893 |
Document ID | / |
Family ID | 43309468 |
Filed Date | 2020-02-27 |
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United States Patent
Application |
20200063135 |
Kind Code |
A1 |
BRODIE; Chaya ; et
al. |
February 27, 2020 |
METHODS, SYSTEMS, AND COMPOSITIONS FOR NEURONAL DIFFERENTIATION OF
MULTIPOTENT STROMAL CELLS
Abstract
Some embodiments of the invention comprise methods, systems, and
compositions to selectively induce, whether in vitro or in vivo,
the neuronal differentiation of multipotent stromal cells through
the application of microRNAs, including but not limited to
miRNA-124, miRNA-137 and/or miRNA-9* expression products of those
miRNAs, and molecules and compositions containing functional
elements of those miRNAs. Some embodiments of the invention also
comprise the therapeutic administration and use of such induced
cells to treat mammalian injuries and diseases, including but not
limited to, nervous system injuries or diseases that may otherwise
result in decreased cell or system function.
Inventors: |
BRODIE; Chaya; (Southfield,
MI) ; SLAVIN; Shimon; (Tel-Aviv, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EXOSTEM BIOTEC LTD. |
Tel Aviv |
|
IL |
|
|
Family ID: |
43309468 |
Appl. No.: |
16/578893 |
Filed: |
September 23, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13377558 |
Feb 23, 2012 |
10421961 |
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PCT/US2010/038168 |
Jun 10, 2010 |
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16578893 |
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61185773 |
Jun 10, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/1353 20130101;
C12N 2510/00 20130101; A61P 43/00 20180101; C12N 2330/10 20130101;
A61K 31/7105 20130101; C12N 5/0619 20130101; C12N 2501/65 20130101;
C12N 2310/141 20130101; C12N 2501/13 20130101; A61P 25/28 20180101;
A61P 25/00 20180101; C12N 15/113 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7105 20060101 A61K031/7105; C12N 5/0793
20060101 C12N005/0793 |
Claims
1. A method of trans-differentiating mammalian multipotent stromal
cells into neuronal stem cells, the method comprising the steps of:
a) providing a population of mammalian multipotent stromal cells;
and b) expressing microRNA-124 in the population of mammalian
multipotent stromal cells so as to generate a population of
neuronal stem cells expressing nestin, thereby
trans-differentiating mammalian multipotent stromal cells into
neuronal stem cells.
2. The method of claim 1, wherein the mammal is a human.
3. The method of claim 1, further comprising step (c) comprising
analyzing a marker in said population of neuronal stem cells,
wherein said marker comprises a neuronal morphology.
4. The method of claim 1, wherein said mammalian multipotent
stromal cells are derived from a tissue selected from the group
consisting of adipose tissue, umbilical cord tissue, bone marrow
tissue and placenta tissue.
5. The method of claim 1, further comprising expressing in said
mammalian multipotent stromal cells glial derived neurotrophic
factor (GDNF).
6. The method of claim 1, further comprising expressing in said
mammalian multipotent stromal cells microRNA-137, microRNA-9* or
both.
7. The method of claim 1, wherein said expressing comprises
transfection, overexpression of exogenous miRNA or transduction of
pre-miRNA.
8. A method of treating a mammal suffering from nervous system
injury or disease, comprising: a. performing the method of claim 1;
and b. administering said trans-differentiated mammalian
multipotent stromal cells, secreted factors therefrom or both to
said mammal; thereby treating a mammal suffering from nervous
system injury or disease.
9. The method of claim 8, wherein said mammal is a human.
10. The method of claim 8, further comprising expressing in said
mammalian multipotent stromal cells microRNA-137, microRNA-9* or
both.
11. The method of claim 8, wherein said nervous system injury is a
spinal injury.
12. The method of claim 8, wherein said nervous system disease is a
neurodegenerative disorder.
13. The method of claim 11, wherein said neurodegenerative disorder
is selected from amyotrophic lateral sclerosis, and multiple
sclerosis.
14. The method of claim 11, wherein said neurodegenerative disorder
is a motor neuron disease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 13/377,558, filed on Feb. 23, 2012, which is a
national phase of PCT Patent Application No. PCT/US2010/038168,
filed on Jun. 10, 2010, which claims the benefit of priority of
U.S. Provisional Patent Application No. 61/185,773, filed on Jun.
10, 2009. The contents of the above applications are all hereby
expressly incorporated by reference, in their entirety.
TECHNICAL FIELD
[0002] Without limitation, certain embodiments of the invention
relate to induction and application of cell types for the treatment
of mammalian nervous system injuries and diseases.
BACKGROUND
[0003] Certain nervous system injuries, autoimmune diseases
affecting the central or peripheral nervous system, and
neurodegenerative diseases are characterized by loss of specific
cells, or abnormal functions of existing nerve cells, which cause
the patient to present with different neurological signs and
symptoms and potentially irreversible loss of neurological
functions. As one example only, some patients suffering stroke,
spinal cord injury, or other neural injury and degeneration
experience loss of functioning cell types, or neurological
conditions like Parkinson's disease and Alzheimer's disease which
in turn results in loss of or abnormal function of system function.
Currently therapeutic options for treating and restoring such cell
and system functions are limited. Thus, a need remains for methods,
systems, and compositions to promote additional therapies,
including therapies addressed to replacement of missing or damaged
nervous system cells, tissues, and functions.
BRIEF SUMMARY
[0004] Without limitation to only those embodiments described
herein and without disclaimer, some embodiments of the invention
comprise methods, systems, and compositions to selectively induce,
whether in vitro or in vivo, the neuronal differentiation of
multipotent stromal cells through the application of microRNAs,
including but not limited to miRNA-124, miRNA-137 and/or miRNA-9*
expression products of those miRNAs, and molecules and compositions
containing functional elements of those miRNAs. Some embodiments of
the invention also comprise the therapeutic administration and use
of such induced cells to treat mammalian injuries and diseases,
including but not limited to, nervous system injuries or diseases
that may otherwise result in decreased cell or system function.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] Some embodiments of the present invention will now be
described, by way of example only and without disclaimer of other
embodiments, with reference to the accompanying drawings, in
which:
[0006] FIG. 1 shows bright field images of MSCs treated with growth
factors or transfected with control miRNA and miRNA-124 or
miRNA-137 for 3, 5 and 9 days.
[0007] FIG. 2 is a data representation showing that miRNA-124,
miRNA-137 and miRNA-9* induce neuronal markers in MSCs.
[0008] FIG. 3 shows Western Blot results following transfection of
cells with tested miRNAs or treatment with DMEM.
[0009] FIG. 4 shows results of transfecting adipose and cord
derived MSCs with control miRNA, miRNA-124, or miRNA-137.
DETAILED DESCRIPTION
[0010] Without limitation to only those embodiments expressly
disclosed herein and without disclaiming any embodiments, some
embodiments of the invention comprise methods, systems, and
composition to selectively induce, whether in vitro or in vivo, the
neuronal differentiation of multipotent stromal cells ("MSCs")
through the application of microRNAs ("miRNA(s)" or "miR(s)"),
including but not limited to, miRNA-124 and/or miRNA-137, and/or
miRNA-9*, expression products of those miRNAs, and molecules and
compositions containing functional elements of those miRNAs. Some
embodiments of the invention also comprise the therapeutic
administration and use of such induced cells to treat mammalian
injuries and diseases, including but not limited to, nervous system
injuries or diseases that may otherwise result in decreased cell or
system function. In some embodiments, such induction of
differentiated MSCs, and/or the resulting cells, may be used to
treat cell, tissue, or organ damage in a patient by administering
to said patient a therapeutically effective amount of an miRNA of
interest, or of differentiated MSCs induced by such miRNAs.
[0011] We have discovered unexpectedly that certain miRNAs are
capable of inducing long-term neuronal differentiation of MSCs for
the use of cell-based therapies in subjects presenting with nervous
system injury and disease, including but not limited to,
neurodegenerative disorders and spinal injury. Such subjects may
include mammals, including but not limited to, humans. Thus, we
have discovered novel applications for such miRNAs and resulting
induced MSCs which, among other possible uses, can reduce or
alleviate the effects of certain nervous system injuries or
diseases in mammals.
[0012] Without limitation, some embodiments of the invention
comprise methods, systems, and/or compositions for inducing
neuronal differentiation of MSCs through the use and expression of
miRNA-124, miRNA-137, and/or miRNA-9*. MSCs are mesoderm-derived
cells that typically reside in adult bone marrow, typically at very
low concentration (about 1 in 10,000 nucleated cells). MSCs can
differentiate to generate cells such as bone marrow stroma, blood
vessels, fat, bone and cartilage. These cells may also have the
potential to differentiate into neurons\ or glia-like cells
depending on the environmental signals. Moreover, these cells may
be further induced to express or maintained specific neuronal or
glial phenotypes by incubation with different combinations of
growth factors and hormones.
[0013] MSCs have been shown to exert therapeutic effects in a
variety of neurological diseases and dysfunctions in experimental
animal models and more recently in pilot clinical trials. Their
effects have been mainly attributed to immunosuppressive and
neuroprotective functions. In experimental autoimmune encephalitis
("EAE"), an animal model of multiple sclerosis ("MS"), treatment of
mice with bone marrow derived MSCs resulted in significant
suppression of disease manifestations. Some studies demonstrated
that in addition to down regulation of autoimmunity neural
differentiation of these cells increased their therapeutic effect
in various instances such as the ischemic brain.
[0014] In our work, we tested the effect of three
neuronal-associated miRNAs, miRNA-124, miRNA-137, and miRNA-9*, on
the differentiation of human MSCs. These miRNAs are not normally
expressed in MSCs. We discovered that the expression of miRNA-124,
miRNA137, or miRNA-9* induced neuronal differentiation of MSCs, as
indicated by the morphology of the cells and by the increased
expression of .beta.III-tubulin and MAP2. miRNA-124, miRNA-137 and
miRNA-9* induced an increase in tyrosine hydroxylase, suggesting
differentiation of the MSCs to dopaminergic phenotype. One of the
targets of pre-miRNA124 is the transcription factor REST that
represses a large number of neuronal genes. Our results indicate
that neuronal-associated miRNAs may induce long-term neuronal
differentiation of MSCs for the use of cell-based therapy in
neurodegenerative and neuroinflammatory disorders and spinal
injury. One advantage of the use of miRNAs over the existing
methods is that one can stably express pre-miRNAs in MSCs that will
result in long-term neuronal differentiation, as compared with
transient differentiation that is induced by treatment with growth
factors. As such, easy access to patient's own bone marrow derived
MSCs and the feasibility to enrich and expand MSCs in large numbers
indicates that neuronal differentiation of such cells can serve as
autologous neuronal stem cells that can be available for treatment
of a large number of acquired or congenital neurological disorders
associated with lack of or damaged neurons. As one example only
without limitation, MSCs can be prepared from fat removed by
liposuction and from cord blood or the placenta. Reduced
immunogenicity of MSCs may facilitate the use of allogeneic neurons
off the shelf or from matched or partially mismatched family member
for treatment of conditions caused, as nonlimiting examples only,
by congenital deficiencies of essential enzymes or other essential
products.
[0015] Without limitation to only embodiments expressly disclosed
herein, and without disclaiming any embodiments, some embodiments
of the invention comprise:
[0016] 1. the neuronal differentiation of MSCs through culture or
other exposure to miRNAs, including but not limited to, miRNA124
and/or miRNA 137, and/or miRNA 9*.
[0017] 2. transfection of MSCs with such miRNAs;
[0018] 3. administration of MSCs induced in vitro into neuronal
differentiation to a subject suffering from nervous system injury
or disease; and/or
[0019] 4. administration of MSCs transfected with such miRNAs to a
subject suffering from nervous system injury or disease.
[0020] In some embodiments, without limitation, with reproducible
transdifferentiation of MSCs to neurons, the therapeutic use of
MSCs can be obtained and expanded, whether in vitro or in vivo, to
include, as some examples only, treatment of cerebrovascular
disease, spinal cord injury, treatment of neurodegenerative
disorders such as amyotrophic lateral sclerosis ("ALS"), multiple
sclerosis ("MS"), and related motor neuron diseases. Ongoing
clinical studies already indicate that infusion of MSCs
intrathecally and intravenously can improve partially the clinical
manifestation of the disease in patients with MS and to a lesser
extent in patients with ALS. Such clinical studies provide evidence
that both intrathecal and intravenous infusions of MSCs are safe
procedures since none of the treated patients has developed any
severe side effect. Thus, cell therapy with MSCs represents
prophetically an important approach for the treatment of a large
number of neurological disorders, especially where MSCs can be
induced into neurons or oligodendrocytes and/or secrete factors
that can induce neurogenesis of locally residing stem cells.
Examples
[0021] The following examples of some embodiments of the invention
are provided without limiting the invention to only those
embodiments described herein and without disclaiming any
embodiments.
[0022] microRNAs
[0023] microRNAs ("miRNAs") represent a family of endogenous, small
(as some nonlimited examples, 19-23 nucleotides) non-coding RNAs
that function through the RNA interference ("RNAi") pathway to
effect post-transcriptional gene silencing. miRNAs target the mRNAs
of specific genes based on complementarity, and mediate either mRNA
cleavage (perfect complementarity) or translation repression
(partial complementarity). miRNAs have been demonstrated to play
important roles in development and may function as fundamental
genetic regulatory elements that serve to establish or maintain
specific expression profiles determining cell fate.
[0024] Manipulating neuronal differentiation of MSCs may involve
regulatory pathways that orchestrate the program of gene expression
during the differentiation process. Differentiation often requires
shifts in the mRNA and protein constitution of cells. One class of
gene regulatory molecules are the microRNAs, a subclass of small
RNAs, that are thought to use the elements of the RNA-interference
pathway to post transcriptionally down-regulate the expression of
protein-coding genes. miRNAs may play an important role in cell
differentiation since they are predicted to individually regulate
hundreds of target genes simultaneously.
[0025] Methods
[0026] To determine the effect of miRNA-124, miRNA-137, and
miRNA-9* on the differentiation of MSCs, we employed three
different preparations of these cells in passages 4-12. MSC cells
were plated in DMEM+10% FCS for 24 hr and were then transfected
with double-stranded RNA oligonucleotides of the mature sequence of
the three miRNAs and with a negative control oligonucleotide. The
miRNAs used were as follows:
TABLE-US-00001 Dharmacon mimic products:
MI0000443/MIMAT0000422-Human Selected Precursor/Mature Mature:
hsa-miR-124 [MIMAT0000422] Precursor: hsa-miR-124-1 [MI0000443]
Organism: Human Mature Sequence: (SEQ ID NO. 1)
UAAGGCACGCGGUGAAUGCC MI0000454/MIMAT0000429-Human Selected
Precursor/Mature Mature: hsa-miR-137 [MIMAT0000429] Precursor:
hsa-miR-137 [MI0000454] Organism: Human Mature Sequence: (SEQ ID
NO. 2) UUAUUGCUUAAGAAUACGCGUAG miRNA 9* sequence: (SEQ ID NO. 3)
AUAAAGCUAGAUAACCGAAAGU miRNA 9 mimic: (SEQ ID NO. 4)
UCUUUGGUUAUCUAGCUGUAUGA
[0027] Following 3 days, cells were transferred to Neurobasal
Medium (NB) supplemented with B27. Cell morphology was monitored
every 24 hr and analysis of neuronal markers by either
immunofluorescence staining, Western blot analysis or real-time PCR
was performed following 5, 7 and 9 days post-transfection. As a
positive control for the induction of neuronal differentiation, we
used cells stimulated with combination of Shh, FGF8 and bFGF.
[0028] Results
[0029] miRNA-124, miRNA-137, and miRNA-9* promote neuronal
differentiation of MSCs. The pictures of FIG. 1 are representative
of six separate experiments that gave similar results. As presented
in FIG. 1, transfection of the cells with miRNA-137, miRNA-124 or
miRNA-9* decreased cell proliferation and induced morphological
differentiation in the cells already after 72 hr of transfection.
Transfection of the MSCs with miRNA-137 induced rapid and robust
morphological changes and the cells acquired a typical neuronal
phenotype with compact cell bodies and elongated processes with
varicosities. miRNA-124-transfected cells exhibited a strong
decrease in cell proliferation followed by the generation of a
number of cell types; elongated cells with long processes, small
cells with multiple shorter processes and flat star-like cells.
Cells transfected with the control miRNA resembled the control
untreated cells. Interestingly, the effect of miRNA-137 was more
rapid and stronger than that of the GF. About 90% of the miRNA-137
transfected cells exhibited neuronal morphology.
[0030] miRNA-124, miRNA-137, and miRNA-9* increase the expression
of neuronal markers in MSCs. To further examine the effect of
miRNA-124, miRNA-137 and miRNA-9* on neuronal differentiation, we
examined the expression of the neural stem cell marker, nestin, the
astrocytic marker GFAP and the neuronal markers beta III-tubulin
and tyrosine hydroxylase. Cells were transfected with the
appropriate miRNA or treated with DMEM or with neurobasal
medium+B27. Following 5 days, the expression of nestin mRNA was
determined using real-time PCR and the expression of nestin, GFAP,
beta III-tubulin and tyrosine hydroxylase was examined after 9 days
of treatment by Western blot analysis. The results represent five
different experiments that gave similar results. FIG. 2 shows that
after 5 days of transfection, there was a large increase in nestin
mRNA as determined by real-time PCR. In contrast, after 9 days of
transfection with the different miRNAs we found an increase in the
expression of beta III-tubulin, whereas no expression of nestin or
GFAP was observed. In addition, we found that miRNA-137 and
miRNA-9* induced a large increase in the expression of tyrosine
hydroxylase, whereas a smaller increase was observed in miRNA-124
transfected cells. The expression of all these markers in the
control miRNA transfected cells was absent or negligible.
[0031] miR-9* induced the dopaminergic marker, tyrosine
hydroxylase, in MSCs. FIG. 3 shows Western Blot results following
MSC transfection with the appropriate miRNAs or treatment with
DMEM. Following 9 days, the expression tyrosine hydroxylase was
examined by Western blot analysis. The results represent five
different experiments that gave similar results. miRNA-9* induced
the dopaminergic marker, tyrosine hydroxylase, in MSCs.
[0032] Our results demonstrate that miRNA-124, miRNA-137 and
miRNA-9* induce neuronal differentiation of MSCs, albeit to
respectively different degrees in our test model. miRNA-137 induces
a more rapid and robust effect resulting in a homogenous population
of neuronal cells. The high level of tyrosine hydroxylase expressed
in these cells suggests that these cells display a dopaminergic
phenotype.
[0033] miRNA-124 also induces neuronal differentiation as
determined by the high level of .beta.III-tubulin compared to the
control miRNA-treated cells. In our work, this treatment resulted
in a mixed population of cells which expressed lower level of
tyrosine hydroxylase. None of the treatments induced astrocytic
differentiation as determined by the lack of GFAP expression.
[0034] Moreover, following 5 days of treatment, both miRNAs induced
a large transient increase in nestin expression, indicating
generation of neural stem cell-like or neuronal progenitor-like
cells. A controlled differentiation of MSCs to NSC or NPC-like
cells may be further exploited to differentiate these cells to
different neuronal lineages or to neurons with different phenotypes
using specific transcription factors or specific combination of
growth factors.
[0035] miRNA-124 and miRNA-9* have been reported to be involved in
neuronal differentiation and neurite outgrowth. Similarly, there is
one report demonstrating the effect of miRNA137 on neuronal
differentiation of glioma stem cells and NSCs. However, no effects
of miRNAs have been reported on the neuronal differentiation of
MSCs and no effect of miRNA124 and miRNA137 has been shown on the
generation of neurons with a specific phenotype. Moreover, none of
these miRNAs has been reported to induce cells with a NSC/NPC
phenotype.
[0036] miRNA-124 and miRNA-137 induced neuronal differentiation in
the Adipose and cord derived MSCs. Adipose and cord derived MSCs
were transfected with control miRNA, miRNA-124 and miRNA-137
similar to the bone marrow MSCs (FIG. 4).
[0037] Preparation of adipose-derived MSCs: Adipose-derived MSCs
were obtained from liposuction from the thighs or abdominal walls.
100-200 ml aspirates were processed in a special designed Cytori
separator that separates the MSCs from the fats cells and debris.
The cells were further processed and maintained as described for
the bone-marrow derived MSCs.
[0038] Preparation of human umbilical (cord) MSCs: Fresh human
umbilical cords were obtained after birth (with parental consent)
and collected in DMEM at 4.degree. C. The umbilical cord vessels
were removed and the mesenchymal tissue (Wharton's jelly) was
minced into small pieces. Following centrifugation, at 250.times.g
for 5 min the tissue was washed with serum-free DMEM was treated
with collagenase at 37.degree. C. for 18 h followed by digestion
with 2.5% trypsin at 37.degree. C. for 30 min. The dissociated MSCs
were further dispersed and maintained in conditions similar to
those described for bone marrow-derived MSCs.
[0039] After 12 days, mRNA was extracted and the levels of
b3-tubulin and the house keeping gene S12 were determined using
real-time PCR.
[0040] Our results (FIG. 4) demonstrate that miRNA-124 and
miRNA-137 induce neuronal differentiation not only in bone-marrow
derived MSCs but also in adipose-tissue and cord blood-derived
cells. The neuronal marker beta III tubulin was induced in these
cells following miRNA treatment. Each of these cell sources has its
own advantages. Bone-marrow derived MSCs are very well
characterized and have been used for over 20 years successfully
with no oncogenic potential. Adipose-derived MSCs are less
characterized but can be obtained in larger numbers and cord blood
cells can be easily obtained in a non-invasive manner they do not
require complete genetic compatibility between the donor and the
patient and therefore are more accessible.
[0041] Construction of a plasmid containing pre-miRNA and GDNF.
Since GDNF has been implicated in the survival of dopaminergic
neurons, we constructed plasmids that co-express pre-miRNA-124 or
pre-miRNA-137 together with GDNF under separate promoters.
[0042] Without limitation to only embodiments described herein, and
without disclaiming any embodiments, steps for sequence and
procedure of cloning the pre-miRNA GDNF vectors and that of the
miRNAs are described with respect to step by step cloning of GDNF
into premir vectors (CD-511_1 or PCDH-CMV-MCS-EF1-copGFP from
System Biosciences).
[0043] cDNA of Homo sapiens glial cell derived neurotrophic factor
("GDNF") template was obtained from Origene. For cloning GDNF into
premir 124 and 137 vectors (System Biosciences), primers with Xho1
and Sal1 restriction enzyme digestion sites for GDNF ORF were
designed as follows:
TABLE-US-00002 Forward: (SEQ ID NO. 5) cacc ctcgag(XhoI) atg aag
tta tgg gat gtc gtg gct gtc tgc Reverse: (SEQ ID NO. 6) aaa
gtcgac(SalI) tca gat aca tcc aca cct ttt agc gga atg
[0044] After PCR, the GDNF DNA product was cleaned, then digested
with Xho1 and Sal1, and the DNA was cleaned again, resulting in DNA
of GDNF now ready for cloning.
[0045] Xho1 restriction site was added into the vector of premir
124 and premir 137 by using primers:
TABLE-US-00003 Forward: (SEQ ID NO. 7) gac gcc acc atg gag agc ctc
gag (XhoI) agc ggc ctg ccc gcc Reverse: (SEQ ID NO. 8) ggc ggg cag
gcc gct ctc gag (XhoI) gct ctc cat ggt ggc gtc
[0046] The GFP gene was removed from premir 124 and 137 vector by
using restriction enzymes Xho1 and Sal1, then the vector was
cleaned.
[0047] Ligation of GDNF and premir 124 and 137 vectors. The ligated
plasmids were transformed into One shot Top10 chemical competent
cell. Plasmids with premir 124 and 137 were selected by culturing
the clones, following by processing with mini prep.
[0048] The plasmids were digested by using Xho1 and Sal1 to detect
the insert of GDNF. The plasmids were then sequenced. The sequence
of miR-124 and 137 and the backbone of the premir vector:
TABLE-US-00004 MiR-124: (SEQ ID NO. 9)
GAACAAAGAGCCTTTGGAAGACGTCGCTGTTATCTCATTGTCTGTG
TGATTGGGGGAGCTGCGGCGGGGAGGATGCTGTGGTCCCTTCCTCCGGCG
TTCCCCACCCCCATCCCTCTCCCCGCTGTCAGTGCGCACGCACACGCGCC
GCTTTTTATTTCTTTTTCCTGGTTTTCTTATTCCATCTTCTACCCACCCC
TCTTCCTTTCTTTCACCTTTCCTTCCTTCCTTCCTCCTTTCCTTCCTCAG
GAGAAAGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCC
ATACAATTAAGGCACGCGGTGAATGCCAAGAATGGGGCTGGCTGAGCACC
GTGGGTCGGCGAGGGCCCGCCAAGGAAGGAGCGACCGACCGAGCCAGGCG
CCCTCCGCAGACCTCCGCGCAGCGGCCGCGGGCGCGAGGGGAGGGGTCTG
GAGCTCCCTCCGGCTGCCTGTCCCGCACCGGAGCCCGTGGGGTGGGGAGG
TGTGCAGCCTGTGACAGACAGGGGCTTAGAGATGC MiR-137: (SEQ ID NO. 10)
CAGCACTCTTCTGTGTTAAGTATTTGATTTTGTGATTTGTCTTTCAG
AATTGGAAATAGAGCGGCCATTTGGATTTGGGCAGGAAGCAGCCGAGCAC
AGCTTTGGATCCTTCTTTAGGGAAATCGAGTTATGGATTTATGGTCCCGG
TCAAGCTCAGCCCATCCCCAGGCAGGGGCGGGCTCAGCGAGCAGCAAGAG
TTCTGGTGGCGGCGGCGGCGGCAGTAGCAGCGGCAGCGGTAGCAGCGGCA
GCGGTAGCAGCGGCAGCGGCAGCTTGGTCCTCTGACTCTCTTCGGTGACG
GGTATTCTTGGGTGGATAATACGGATTACGTTGTTATTGCTTAAGAATAC
GCGTAGTCGAGGAGAGTACCAGCGGCAGGGGGGCAGCGGCCGCCCTCCCC
AGCCCACCAGCTGGCCACTAAACGCCCGTGGTTGCCAAGGTAGCACTTTC
TTGTTCTTTTCATTTCCTCGGGTGTTTTCGCACTGGTTCCACCGGAAAGG
CTGTGCGCTGCGCCTCTGGTGACCAGGACTGGA
[0049] The sequence of backbone vector (CD-511_1) was attached:
[0050] LOCUS CD511B_1_pCDH_CMV_7544 bp ds-DNA circular 16 Dec.
2008:
TABLE-US-00005 DEFINITION ACCESSION VERSION SOURCE ORGANISM COMMENT
COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers
misc_feature 2315..2764 /label=EF1 promoter /ApEinfo_fwdcolor=cyan
/ApEinfo_revcolor=green misc_feature 2765..2789 /label=EF1
promoter(1) /ApEinfo_label=EF1 promoter /ApEinfo_fwdcolor=cyan
/ApEinfo_revcolor=green misc_feature 2874..3629 /label=copGFP
/ApEinfo_fwdcolor=#00ff00 /ApEinfo_revcolor=green misc_feature
3639..4229 /label=WPRE /ApEinfo_fwdcolor=cyan
/ApEinfo_revcolor=green misc_feature 2790..2860 /label=EF1
promoter(2) /ApEinfo_label=EF1 promoter /ApEinfo_fwdcolor=cyan
/ApEinfo_revcolor=green misc_feature 2765..2789 /label=EFfwd primer
/ApEinfo_fwdcolor=#ff80ff /ApEinfo_revcolor=green misc_feature
1922..2183 /label=CMV /ApEinfo_fwdcolor=#ff80ff
/ApEinfo_revcolor=green misc_feature 2272..2314 /label=MCS
/ApEinfo_fwdcolor=#80ff00 /ApEinfo_revcolor=green misc_feature
2205..2271 /label=CMV(1) /ApEinfo_label=CMV
/ApEinfo_fwdcolor=#ff80ff /ApEinfo_revcolor=green misc_feature
2184..2204 /label=DAB 90 primer forward /ApEinfo_fwdcolor=cyan
/ApEinfo_revcolor=green ORIGIN 1 acgcgtgtag tcttatgcaa tactcttgta
gtcttgcaac atggtaacga tgagttagca 61 acatgcctta caaggagaga
aaaagcaccg tgcatgccga ttggtggaag taaggtggta 121 cgatcgtgcc
ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 181
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
ccactgctta 301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
tgcccgtctg ttgtgtgact 361 ctggtaacta gagatccctc agaccctttt
agtcagtgtg gaaaatctct agcagtggcg 421 cccgaacagg gacctgaaag
cgaaagggaa accagagctc tctcgacgca ggactcggct 481 tgctgaagcg
cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 541
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
aatataaatt 661 aaaacatata gtatgggcaa gcagggagct agaacgattc
gcagttaatc ctggcctgtt 721 agaaacatca gaaggctgta gacaaatact
gggacagcta caaccatccc ttcagacagg 781 atcagaagaa cttagatcat
tatataatac agtagcaacc ctctattgtg tgcatcaaag 841 gatagagata
aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 901
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
ttaggagtag 1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
aagagcagtg ggaataggag 1081 ctttgttcct tgggttcttg ggagcagcag
gaagcactat gggcgcagcc tcaatgacgc 1141 tgacggtaca ggccagacaa
ttattgtctg gtatagtgca gcagcagaac aatttgctga 1201 gggctattga
ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1261
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
tggagtaata 1381 aatctctgga acagattgga atcacacgac ctggatggag
tgggacagag aaattaacaa 1441 ttacacaagc ttaatacact ccttaattga
agaatcgcaa aaccagcaag aaaagaatga 1501 acaagaatta ttggaattag
ataaatgggc aagtttgtgg aattggttta acataacaaa 1561 ttggctgtgg
tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1621
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
aagaagaagg 1741 tggagagaga gacagagaca gatccattcg attagtgaac
ggatctcgac ggttaacttt 1801 taaaagaaaa ggggggattg gggggtacag
tgcaggggaa agaatagtag acataatagc 1861 aacagacata caaactaaag
aattacaaaa acaaattaca aaaattcaaa attttatcga 1921 tactagtatt
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1981
gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
2041 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg 2101 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg 2161 caaatgggcg gtaggcgtgt acggtgggag
gtctatataa gcagagctcg tttagtgaac 2221 cgtcagatcg cctggagacg
ccatccacgc tgttttgacc tccatagaag attctagagc 2281 tagcgaattc
gaatttaaat ggatccgcgg ccgcaaggat ctgcgatcgc tccggtgccc 2341
gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
2401 attgaacggg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
gtcgtgtact 2461 ggctccgcct ttttcccgag ggtgggggag aaccgtatat
aagtgcagta gtcgccgtga 2521 acgttctttt tcgcaacggg tttgccgcca
gaacacagct gaagcttcga ggggctcgca 2581 tctctccttc acgcgcccgc
cgccctacct gaggccgcca tccacgccgg ttgagtcgcg 2641 ttctgccgcc
tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa 2701
agctcaggtc gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc
2761 ggctctccac gctttgcctg accctgcttg ctcaactcta cgtctttgtt
tcgttttctg 2821 ttctgcgccg ttacagatcc aagctgtgac cggcgcctac
gctagacgcc accatggaga 2881 gcgacgagag cggcctgccc gccatggaga
tcgagtgccg catcaccggc accctgaacg 2941 gcgtggagtt cgagctggtg
ggcggcggag agggcacccc caagcagggc cgcatgacca 3001 acaagatgaa
gagcaccaaa ggcgccctga ccttcagccc ctacctgctg agccacgtga 3061
tgggctacgg cttctaccac ttcggcacct accccagcgg ctacgagaac cccttcctgc
3121 acgccatcaa caacggcggc tacaccaaca cccgcatcga gaagtacgag
gacggcggcg 3181 tgctgcacgt gagcttcagc taccgctacg aggccggccg
cgtgatcggc gacttcaagg 3241 tggtgggcac cggcttcccc gaggacagcg
tgatcttcac cgacaagatc atccgcagca 3301 acgccaccgt ggagcacctg
caccccatgg gcgataacgt gctggtgggc agcttcgccc 3361 gcaccttcag
cctgcgcgac ggcggctact acagcttcgt ggtggacagc cacatgcact 3421
tcaagagcgc catccacccc agcatcctgc agaacggggg ccccatgttc gccttccgcc
3481 gcgtggagga gctgcacagc aacaccgagc tgggcatcgt ggagtaccag
cacgccttca 3541 agacccccat cgccttcgcc agatcccgcg ctcagtcgtc
caattctgcc gtggacggca 3601 ccgccggacc cggctccacc ggatctcgct
aagtcgacaa tcaacctctg gattacaaaa 3661 tttgtgaaag attgactggt
attcttaact atgttgctcc ttttacgcta tgtggatacg 3721 ctgctttaat
gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 3781
tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
3841 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
gccaccacct 3901 gtcagctcct ttccgggact ttcgctttcc ccctccctat
tgccacggcg gaactcatcg 3961 ccgcctgcct tgcccgctgc tggacagggg
ctcggctgtt gggcactgac aattccgtgg 4021 tgttgtcggg gaaatcatcg
tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 4081 tgcgcgggac
gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 4141
gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
4201 ggatctccct ttgggccgcc tccccgcctg gtacctttaa gaccaatgac
ttacaaggca 4261 gctgtagatc ttagccactt tttaaaagaa aaggggggac
tggaagggct aattcactcc 4321 caacgaaaat aagatctgct ttttgcttgt
actgggtctc tctggttaga ccagatctga 4381 gcctgggagc tctctggcta
actagggaac ccactgctta agcctcaata aagcttgcct 4441 tgagtgcttc
aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 4501
agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
4561 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
gtttattgca 4621 gcttataatg gttacaaata aagcaatagc atcacaaatt
tcacaaataa agcatttttt 4681 tcactgcatt ctagttgtgg tttgtccaaa
ctcatcaatg tatcttatca tgtctggctc 4741 tagctatccc gcccctaact
ccgcccagtt ccgcccattc tccgccccat ggctgactaa 4801 ttttttttat
ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 4861
gaggaggctt ttttggaggc ctagactttt gcagagacgg cccaaattcg taatcatggt
4921 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
atacgagccg 4981 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
ctaactcaca ttaattgcgt 5041 tgcgctcact gcccgctttc cagtcgggaa
acctgtcgtg ccagctgcat taatgaatcg 5101 gccaacgcgc ggggagaggc
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 5161 actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5221
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
5281 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
ctccgccccc 5341 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg acaggactat
5401 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
ccgaccctgc 5461 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
cgtggcgctt tctcatagct 5521 cacgctgtag gtatctcagt tcggtgtagg
tcgttcgctc caagctgggc tgtgtgcacg 5581 aaccccccgt tcagcccgac
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5641 cggtaagaca
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5701
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
5761 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
agagttggta 5821 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt tgcaagcagc 5881 agattacgcg cagaaaaaaa ggatctcaag
aagatccttt gatcttttct acggggtctg 5941 acgctcagtg gaacgaaaac
tcacgttaag ggattttggt catgagatta tcaaaaagga 6001 tcttcaccta
gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 6061
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
6121 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
acgatacggg 6181 agggcttacc atctggcccc agtgctgcaa tgataccgcg
agacccacgc tcaccggctc 6241 cagatttatc agcaataaac cagccagccg
gaagggccga gcgcagaagt ggtcctgcaa 6301 ctttatccgc ctccatccag
tctattaatt gttgccggga agctagagta agtagttcgc 6361 cagttaatag
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 6421
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
6481 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
agaagtaagt 6541 tggccgcagt gttatcactc atggttatgg cagcactgca
taattctctt actgtcatgc 6601 catccgtaag atgcttttct gtgactggtg
agtactcaac caagtcattc tgagaatagt 6661 gtatgcggcg accgagttgc
tcttgcccgg cgtcaatacg ggataatacc gcgccacata 6721 gcagaacttt
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 6781
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
6841 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
aatgccgcaa 6901 aaaagggaat aagggcgaca cggaaatgtt gaatactcat
actcttcctt tttcaatatt 6961 attgaagcat ttatcagggt tattgtctca
tgagcggata catatttgaa tgtatttaga 7021 aaaataaaca aataggggtt
ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7081 aaaccattat
tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7141
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
7201 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 7261 ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 7321 accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 7381 attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 7441 tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 7501
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctg (SEQ ID NO.
11)
[0051] We found that MSCs transfected with these plasmids secrete
GDNF and express the respective miRNAs. Thus, the GDNF secreted by
the differentiated dopaminergic neurons is expected to provide
survival signals to the differentiated cells and to endogenous
dopaminergic neurons.
[0052] Construction of inducible miRNAs. Implanted MSCs have been
reported to migrate to damaged tissues in the central nervous
systems and to exert neurotrophic and immunomodulatory effects.
Specifically, in Parkinson's animal models, implanted MSCs have
been shown to engraft in the lesioned striatum. In some
embodiments, without limitation, inducible pre-miRNA expression
vectors might be used that will allow the induction of the specific
pre-miRNA expression at desired time points. Thus, MSCs would be
transfected with the specific pre-miRNA and its expression would be
induced at different time points prior or following the engraftment
of the MSCs in the lesioned striatum. For such studies we have
employed the inducible miRNA and living color, fluorescent protein
reporters using the Tet-on system (Clontech). This system allows
the induction of the specific miRNA by the addition of a promoter,
as one example, only, by doxycyline, and the identification of
cells in which the miRNA is produced.
[0053] In summary, we have demonstrated the ability of miRNA124,
miRNA137 and miRNA-9* to induce transdifferentiation of MSCs to
NSC/NPC and neurons with a specific neuronal phenotype (miRNA137).
Additional neuronal miRNAs such as miRNA-9 and miR218 may also
effect transdifferentiation of MSCs and induce neuronal
differentiation.
[0054] An advantage of using miRNAs over the existing methods is
that one can stably express pre-miRNAs in the MSCs which will
result in long-term neuronal differentiation as compared with
transient differentiation that is induced by treatment with growth
factors.
[0055] Our work indicates that neuronal-associated miRNAs may be
employed to induce long-term neuronal differentiation of MSCs for
the use of cell-based therapy in neurodegenerative disorders and
spinal injury, as some examples only, as shown by:
[0056] 1. Neuronal differentiation of MSCs by microRNAs (miRNA-124,
miRNA-137, miRNA-9*);
[0057] 2. Specific dopaminergic differentiation of MSCs by
miRNA-137, miRNA-124 and miRNA-9*; and
[0058] 3. Induction by microRNAs of transient differentiation of
MSCs to neural stem cell like- or neural progenitor-like cells.
Transfection with the miRNAs provides a window of opportunity where
cells can be differentiated to the different lineages of the
central nervous system (neurons, astrocytes and oligodendrocytes)
or to a specific neuronal phenotype using transcription factors or
a specific combination of growth factors. This window can be
controlled by level of miRNA expression or by a specific time point
post-transfection.
[0059] The ability of miRNAs to transdifferentiate MSCs to
uncommitted progenitor cells and to different neuronal cell subsets
makes it possible to use these cells for treatment of a large
variety of neurological diseases, including spinal cord and
peripheral nerve injuries, damage to the central nervous system
caused by hemorrhage or obstructive lesions ("CVA") or to traumatic
central or peripheral nerve injury. In addition,
transdifferentiated MSCs may be employed in the case of
neurodegenerative diseases caused by idiopathic autoimmune diseases
("EAE") or diseases such as Parkinson's disease or Alzheimer/s
disease or diseases with unknown etiology such as ALS. Moreover,
improvement of neurological functions by transdifferentiated MSCs
may also be used in various degenerative disorders caused by
drug-induced neuronal damage and/or toxicity.
[0060] Thus, in our work, miRNA-124, miRNA-137 and miRNA-9* promote
neural differentiation of MSC's, with accompanying morphological
changes and expression of phenotypic markers.
[0061] The inducing miRNA(s) of some embodiments would be
administered and dosed in accordance with good medical practice,
taking into account the techniques of use to accomplish the desired
effect of target MSCs, the clinical condition of the individual
patient, the site and method of administration, scheduling of
administration, patient age, sex, body weight and other factors
known to medical practitioners. The "pharmaceutically effective
amount" for purposes herein is thus determined by such
considerations as are known in the art. The amount must be
effective to achieve improvement, including but not limited to, the
desired differentiation of MSCs in vivo and/or in vitro, decreased
damage or injury, or improvement or elimination of symptoms and
other indicators as are selected as appropriate measures by those
skilled in the art.
[0062] Embodiments of the invention may expand the therapeutic
window for treatment of nervous system injury and diseases and
could be applied to treatment of a large patient population which
suffers such injury and diseases each year in the United States.
Thus, in some embodiments, the invention comprises novel methods to
prevent, control, or alleviate mammalian nervous system injury and
disease, including without limitation, brain damage, neural
degeneration, or spinal cord injury, through the selective
application of inducing miRNAs comprising embodiments of the
invention. In accordance with some embodiments, without limitation,
one may effect such therapeutic intervention through the use and/or
administration of one or more such miRNAs to induce differentiation
in target cells in vivo or in vitro for use in treatment to limit
the effects of such injury or disease. Thus, without limitation and
without disclaimer of subject matter, some embodiments comprise
novel compositions and methods to prevent, control, or alleviate
mammalian injury, including without limitation, brain damage,
through the selective application and/or induction of
transdifferentiated MSCs.
[0063] This application may reference various publications by
author, citation, and/or by patent number, including without
limitation, articles, presentations, and United States patents. The
disclosures of each of any such references in their entireties are
hereby incorporated by reference into this application.
[0064] While the present invention has been particularly shown and
described with reference to the foregoing preferred and alternative
embodiments, it should be understood by those skilled in the art
that various alternatives to the embodiments of the invention
described herein may be employed in practicing the invention
without departing from the spirit and scope of the invention as
defined in the following claims. It is intended that the following
claims define the scope of the invention and that the method and
apparatus within the scope of these claims and their equivalents be
covered thereby. This description of the invention should be
understood to include all novel and non-obvious combinations of
elements described herein, and claims may be presented in this or a
later application to any novel and non-obvious combination of these
elements. The foregoing embodiments are illustrative, and no single
feature or element is essential to all possible combinations that
may be claimed in this or a later application. Where the claims
recite "a" or "a first" element of the equivalent thereof, such
claims should be understood to include incorporation of one or more
such elements, neither requiring nor excluding two or more such
elements.
Sequence CWU 1
1
11120RNAHomo sapiens 1uaaggcacgc ggugaaugcc 20223RNAHomo sapiens
2uuauugcuua agaauacgcg uag 23322RNAHomo sapiens 3auaaagcuag
auaaccgaaa gu 22423RNAArtificial sequencemiRNA 9 mimic 4ucuuugguua
ucuagcugua uga 23540DNAArtificial sequencePCR
primermisc_feature(5)..(10)Xho1 restriction site 5caccctcgag
atgaagttat gggatgtcgt ggctgtctgc 40640DNAArtificial sequencePCR
primermisc_feature(4)..(9)Sal1 restriction site 6aaagtcgaca
tcagatacat ccacaccttt tagcggaatg 40739DNAArtificial sequencePCR
primermisc_feature(19)..(24)Xho1 restriction site 7gacgccacca
tggagagcct cgagagcggc ctgcccgcc 39839DNAArtificial sequencePCR
primermisc_feature(16)..(21)Xho1 restriction site 8ggcgggcagg
ccgctctcga ggctctccat ggtggcgtc 399531DNAArtificial
sequenceRecombinant miR-124 9gaacaaagag cctttggaag acgtcgctgt
tatctcattg tctgtgtgat tgggggagct 60gcggcgggga ggatgctgtg gtcccttcct
ccggcgttcc ccacccccat ccctctcccc 120gctgtcagtg cgcacgcaca
cgcgccgctt tttatttctt tttcctggtt ttcttattcc 180atcttctacc
cacccctctt cctttctttc acctttcctt ccttccttcc tcctttcctt
240cctcaggaga aaggcctctc tctccgtgtt cacagcggac cttgatttaa
atgtccatac 300aattaaggca cgcggtgaat gccaagaatg gggctggctg
agcaccgtgg gtcggcgagg 360gcccgccaag gaaggagcga ccgaccgagc
caggcgccct ccgcagacct ccgcgcagcg 420gccgcgggcg cgaggggagg
ggtctggagc tccctccggc tgcctgtccc gcaccggagc 480ccgtggggtg
gggaggtgtg cagcctgtga cagacagggg cttagagatg c 53110530DNAArtificial
sequenceRecombinant miR-137 10cagcactctt ctgtgttaag tatttgattt
tgtgatttgt ctttcagaat tggaaataga 60gcggccattt ggatttgggc aggaagcagc
cgagcacagc tttggatcct tctttaggga 120aatcgagtta tggatttatg
gtcccggtca agctcagccc atccccaggc aggggcgggc 180tcagcgagca
gcaagagttc tggtggcggc ggcggcggca gtagcagcgg cagcggtagc
240agcggcagcg gtagcagcgg cagcggcagc ttggtcctct gactctcttc
ggtgacgggt 300attcttgggt ggataatacg gattacgttg ttattgctta
agaatacgcg tagtcgagga 360gagtaccagc ggcagggggg cagcggccgc
cctccccagc ccaccagctg gccactaaac 420gcccgtggtt gccaaggtag
cactttcttg ttcttttcat ttcctcgggt gttttcgcac 480tggttccacc
ggaaaggctg tgcgctgcgc ctctggtgac caggactgga 530117544DNAArtificial
sequenceCD-511_1 vector DNA 11acgcgtgtag tcttatgcaa tactcttgta
gtcttgcaac atggtaacga tgagttagca 60acatgcctta caaggagaga aaaagcaccg
tgcatgccga ttggtggaag taaggtggta 120cgatcgtgcc ttattaggaa
ggcaacagac gggtctgaca tggattggac gaaccactga 180attgccgcat
tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
240tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
ccactgctta 300agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
tgcccgtctg ttgtgtgact 360ctggtaacta gagatccctc agaccctttt
agtcagtgtg gaaaatctct agcagtggcg 420cccgaacagg gacctgaaag
cgaaagggaa accagagctc tctcgacgca ggactcggct 480tgctgaagcg
cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
540gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
agcgggggag 600aattagatcg cgatgggaaa aaattcggtt aaggccaggg
ggaaagaaaa aatataaatt 660aaaacatata gtatgggcaa gcagggagct
agaacgattc gcagttaatc ctggcctgtt 720agaaacatca gaaggctgta
gacaaatact gggacagcta caaccatccc ttcagacagg 780atcagaagaa
cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
840gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
aaaacaaaag 900taagaccacc gcacagcaag cggccactga tcttcagacc
tggaggagga gatatgaggg 960acaattggag aagtgaatta tataaatata
aagtagtaaa aattgaacca ttaggagtag 1020cacccaccaa ggcaaagaga
agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080ctttgttcct
tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
1140tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
aatttgctga 1200gggctattga ggcgcaacag catctgttgc aactcacagt
ctggggcatc aagcagctcc 1260aggcaagaat cctggctgtg gaaagatacc
taaaggatca acagctcctg gggatttggg 1320gttgctctgg aaaactcatt
tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380aatctctgga
acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg
aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat
gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta
tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac
ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
ggttaacttt 1800taaaagaaaa ggggggattg gggggtacag tgcaggggaa
agaatagtag acataatagc 1860aacagacata caaactaaag aattacaaaa
acaaattaca aaaattcaaa attttatcga 1920tactagtatt atgcccagta
catgacctta tgggactttc ctacttggca gtacatctac 1980gtattagtca
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
2040tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg 2100ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg 2160caaatgggcg gtaggcgtgt acggtgggag
gtctatataa gcagagctcg tttagtgaac 2220cgtcagatcg cctggagacg
ccatccacgc tgttttgacc tccatagaag attctagagc 2280tagcgaattc
gaatttaaat ggatccgcgg ccgcaaggat ctgcgatcgc tccggtgccc
2340gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
ggggtcggca 2400attgaacggg tgcctagaga aggtggcgcg gggtaaactg
ggaaagtgat gtcgtgtact 2460ggctccgcct ttttcccgag ggtgggggag
aaccgtatat aagtgcagta gtcgccgtga 2520acgttctttt tcgcaacggg
tttgccgcca gaacacagct gaagcttcga ggggctcgca 2580tctctccttc
acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg
2640ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag
gtaagtttaa 2700agctcaggtc gagaccgggc ctttgtccgg cgctcccttg
gagcctacct agactcagcc 2760ggctctccac gctttgcctg accctgcttg
ctcaactcta cgtctttgtt tcgttttctg 2820ttctgcgccg ttacagatcc
aagctgtgac cggcgcctac gctagacgcc accatggaga 2880gcgacgagag
cggcctgccc gccatggaga tcgagtgccg catcaccggc accctgaacg
2940gcgtggagtt cgagctggtg ggcggcggag agggcacccc caagcagggc
cgcatgacca 3000acaagatgaa gagcaccaaa ggcgccctga ccttcagccc
ctacctgctg agccacgtga 3060tgggctacgg cttctaccac ttcggcacct
accccagcgg ctacgagaac cccttcctgc 3120acgccatcaa caacggcggc
tacaccaaca cccgcatcga gaagtacgag gacggcggcg 3180tgctgcacgt
gagcttcagc taccgctacg aggccggccg cgtgatcggc gacttcaagg
3240tggtgggcac cggcttcccc gaggacagcg tgatcttcac cgacaagatc
atccgcagca 3300acgccaccgt ggagcacctg caccccatgg gcgataacgt
gctggtgggc agcttcgccc 3360gcaccttcag cctgcgcgac ggcggctact
acagcttcgt ggtggacagc cacatgcact 3420tcaagagcgc catccacccc
agcatcctgc agaacggggg ccccatgttc gccttccgcc 3480gcgtggagga
gctgcacagc aacaccgagc tgggcatcgt ggagtaccag cacgccttca
3540agacccccat cgccttcgcc agatcccgcg ctcagtcgtc caattctgcc
gtggacggca 3600ccgccggacc cggctccacc ggatctcgct aagtcgacaa
tcaacctctg gattacaaaa 3660tttgtgaaag attgactggt attcttaact
atgttgctcc ttttacgcta tgtggatacg 3720ctgctttaat gcctttgtat
catgctattg cttcccgtat ggctttcatt ttctcctcct 3780tgtataaatc
ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
3840gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
gccaccacct 3900gtcagctcct ttccgggact ttcgctttcc ccctccctat
tgccacggcg gaactcatcg 3960ccgcctgcct tgcccgctgc tggacagggg
ctcggctgtt gggcactgac aattccgtgg 4020tgttgtcggg gaaatcatcg
tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 4080tgcgcgggac
gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
4140gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
cagacgagtc 4200ggatctccct ttgggccgcc tccccgcctg gtacctttaa
gaccaatgac ttacaaggca 4260gctgtagatc ttagccactt tttaaaagaa
aaggggggac tggaagggct aattcactcc 4320caacgaaaat aagatctgct
ttttgcttgt actgggtctc tctggttaga ccagatctga 4380gcctgggagc
tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
4440tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
gagatccctc 4500agaccctttt agtcagtgtg gaaaatctct agcagtagta
gttcatgtca tcttattatt 4560cagtatttat aacttgcaaa gaaatgaata
tcagagagtg agaggaactt gtttattgca 4620gcttataatg gttacaaata
aagcaatagc atcacaaatt tcacaaataa agcatttttt 4680tcactgcatt
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
4740tagctatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
ggctgactaa 4800ttttttttat ttatgcagag gccgaggccg cctcggcctc
tgagctattc cagaagtagt 4860gaggaggctt ttttggaggc ctagactttt
gcagagacgg cccaaattcg taatcatggt 4920catagctgtt tcctgtgtga
aattgttatc cgctcacaat tccacacaac atacgagccg 4980gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
5040tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
taatgaatcg 5100gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
ttccgcttcc tcgctcactg 5160actcgctgcg ctcggtcgtt cggctgcggc
gagcggtatc agctcactca aaggcggtaa 5220tacggttatc cacagaatca
ggggataacg caggaaagaa catgtgagca aaaggccagc 5280aaaaggccag
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
5340ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
acaggactat 5400aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt ccgaccctgc 5460cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag cgtggcgctt tctcatagct 5520cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5580aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
5640cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
agcagagcga 5700ggtatgtagg cggtgctaca gagttcttga agtggtggcc
taactacggc tacactagaa 5760ggacagtatt tggtatctgc gctctgctga
agccagttac cttcggaaaa agagttggta 5820gctcttgatc cggcaaacaa
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 5880agattacgcg
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
5940acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
tcaaaaagga 6000tcttcaccta gatcctttta aattaaaaat gaagttttaa
atcaatctaa agtatatatg 6060agtaaacttg gtctgacagt taccaatgct
taatcagtga ggcacctatc tcagcgatct 6120gtctatttcg ttcatccata
gttgcctgac tccccgtcgt gtagataact acgatacggg 6180agggcttacc
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
6240cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
ggtcctgcaa 6300ctttatccgc ctccatccag tctattaatt gttgccggga
agctagagta agtagttcgc 6360cagttaatag tttgcgcaac gttgttgcca
ttgctacagg catcgtggtg tcacgctcgt 6420cgtttggtat ggcttcattc
agctccggtt cccaacgatc aaggcgagtt acatgatccc 6480ccatgttgtg
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
6540tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
actgtcatgc 6600catccgtaag atgcttttct gtgactggtg agtactcaac
caagtcattc tgagaatagt 6660gtatgcggcg accgagttgc tcttgcccgg
cgtcaatacg ggataatacc gcgccacata 6720gcagaacttt aaaagtgctc
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 6780tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
6840catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
aatgccgcaa 6900aaaagggaat aagggcgaca cggaaatgtt gaatactcat
actcttcctt tttcaatatt 6960attgaagcat ttatcagggt tattgtctca
tgagcggata catatttgaa tgtatttaga 7020aaaataaaca aataggggtt
ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7080aaaccattat
tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
7140tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
gagacggtca 7200cagcttgtct gtaagcggat gccgggagca gacaagcccg
tcagggcgcg tcagcgggtg 7260ttggcgggtg tcggggctgg cttaactatg
cggcatcaga gcagattgta ctgagagtgc 7320accatatgcg gtgtgaaata
ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 7380attcgccatt
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
7440tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
acgccagggt 7500tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctg
7544
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