U.S. patent application number 16/679445 was filed with the patent office on 2020-06-25 for extracellular vesicles for agent delivery.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to STEPHEN J. GOULD, LING LI, FLORIN M. SELARU.
Application Number | 20200197535 16/679445 |
Document ID | / |
Family ID | 56544430 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200197535 |
Kind Code |
A1 |
SELARU; FLORIN M. ; et
al. |
June 25, 2020 |
EXTRACELLULAR VESICLES FOR AGENT DELIVERY
Abstract
The present invention relates to the field of extracellular
vesicles. More specifically, the present invention provides methods
and compositions for using extracellular vesicles as a vector for
nucleic acid treatment in vivo of various diseases. In a specific
embodiment, the present invention provides an extracellular vesicle
isolated from a cell comprising one or more microRNAs (miRNAs) that
have been loaded ex vivo into the vesicle so that the miRNAs are
present in a higher concentration than when measured in the same
extracellular vesicle isolated directly from the cell. In another
embodiment, the present invention provides a method for treating
cholangiocarcinoma in a subject comprising the step of
administering to the subject a plurality of exosomes comprising
miR-195.
Inventors: |
SELARU; FLORIN M.;
(BALTIMORE, MD) ; LI; LING; (BALTIMORE, MD)
; GOULD; STEPHEN J.; (BALTIMORE, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
BALTIMORE |
MD |
US |
|
|
Assignee: |
THE JOHNS HOPKINS
UNIVERSITY
BALTIMORE
MD
|
Family ID: |
56544430 |
Appl. No.: |
16/679445 |
Filed: |
November 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15545937 |
Jul 24, 2017 |
10493165 |
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PCT/US2016/015791 |
Jan 29, 2016 |
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16679445 |
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62109764 |
Jan 30, 2015 |
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62150318 |
Apr 21, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/574 20130101;
A61K 9/1075 20130101; C12N 2310/141 20130101; A61K 35/33 20130101;
C12P 19/34 20130101; C12P 21/00 20130101; A61K 9/5068 20130101;
C12P 1/00 20130101; C12N 15/113 20130101; C12N 2320/32 20130101;
A61K 33/24 20130101; A61K 48/0075 20130101; A61K 45/06 20130101;
A61K 31/704 20130101; A61K 48/0008 20130101; A61K 33/243 20190101;
A61K 35/13 20130101; A61K 47/6901 20170801; G01N 33/57438 20130101;
A61K 48/0091 20130101; A61P 25/00 20180101; A61K 31/685 20130101;
A61K 31/7105 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12P 1/00 20060101 C12P001/00; A61K 31/7105 20060101
A61K031/7105; A61K 47/69 20060101 A61K047/69; G01N 33/574 20060101
G01N033/574; A61P 25/00 20060101 A61P025/00; A61K 35/33 20060101
A61K035/33; A61K 35/13 20060101 A61K035/13; C12N 15/113 20060101
C12N015/113; A61K 33/243 20060101 A61K033/243; C12P 21/00 20060101
C12P021/00; A61K 9/107 20060101 A61K009/107; A61K 31/685 20060101
A61K031/685; A61K 31/704 20060101 A61K031/704; A61K 33/24 20060101
A61K033/24; A61K 45/06 20060101 A61K045/06; C12P 19/34 20060101
C12P019/34 |
Claims
1-4. (canceled)
5. An extracellular vesicle isolated from a cancer associated
fibroblast (CAF), wherein the vesicle comprises a small molecule,
and wherein the extracellular vesicle selectively targets a cancer
cell.
6. (canceled)
7. The extracellular vesicle of claim 5, wherein the small molecule
is loaded into the cell or extracellular vesicle ex vivo.
8. The extracellular vesicle of claim 5, further comprising a
recombinant polypeptide or polynucleotide that is heterologously
expressed in the CAF or is loaded into the cell or extracellular
vesicle ex vivo.
9. The extracellular vesicle of claim 8, wherein the recombinant
polynucleotide is a microRNA.
10. The extracellular vesicle of claim 9, wherein the microRNA is
miR-195, miR-126, or miR-192.
11. The extracellular vesicle of claim 5, wherein the small
molecule is a lipid or other hydrophobic small molecule.
12. The extracellular vesicle of claim 5, wherein the small
molecule is doxorubicin, cisplatin, or phosphatidyl
ethanolamine.
13. The extracellular vesicle of claim 12, wherein the phosphatidyl
ethanolamine is derivatized with an agent selected from the group
consisting of rhodamine, fluorescein, biotin, streptavidin, a small
molecule, a polynucleotide, and a polypeptide.
14. The extracellular vesicle of claim 5, wherein the small
molecule can be used for imaging purposes.
15-25. (canceled)
26. The extracellular vesicle of claim 5, wherein the vesicle
expresses increased levels of one or more markers selected from the
group consisting of alpha-SMA, Collagen, Vimentin (FSP-1), S100,
Metalloproteinases, NG2, PDGFR-B, SDF1/CXCL12, CD34, Fibroblast
activation protein (FAP), FSP-1, CD31, Thy-1, and Gremlin, and/or
expresses reduced levels of laminin.
27. (canceled)
28. The extracellular vesicle of claim 5, wherein the CAF is
derived from a fibroblast cultured for at least 1-14 days in the
presence of a cancer cell or in the presence of conditioned media
derived from a cancer cell culture.
29-32. (canceled)
33. A method for obtaining the extracellular vesicle of claim 5,
the method comprising culturing a fibroblast or stromal cell in
conditioned media obtained from a cancer cell culture, and
isolating extracellular vesicles from the media.
34-29. (canceled)
40. An extracellular vesicle produced according to the method of
claim 33.
41. A pharmaceutical composition comprising the extracellular
vesicle of claim 5.
42. A method of delivering a small molecule to a cell, the method
comprising contacting the cell with the extracellular vesicle of
claim 5, thereby delivering the small molecule agent to the
cell.
43. (canceled)
44. The method of claim 46, wherein the cancer is breast cancer,
pancreatic cancer, glioblastoma, melanoma, lung cancer, ovarian
cancer, or liver cancer.
45. A method of altering gene expression in a cell, the method
comprising contacting the cell with the extracellular vesicle of
claim 5.
46. A method for treating cancer in a subject comprising
administering to the subject a pharmaceutical composition
comprising an effective amount of the extracellular vesicle of
claim 5.
47. The method of claim 46, wherein the cancer is
cholangiocarcinoma, hepatocellular carcinoma, or hepatoma.
48. An extracellular vesicle isolated from a cancer associated
fibroblast (CAF), wherein the extracellular vesicle comprises a
small molecule and a heterologous polynucleotide comprising
miR-195, miR-126, or miR-192, and wherein the extracellular vesicle
selectively targets a cancer cell.
49. A method for treating cancer in a subject comprising
administering to the subject a pharmaceutical composition
comprising an effective amount of the extracellular vesicle of
claim 48.
50. A pharmaceutical composition comprising an effective amount of
a first and a second extracellular vesicle, wherein the first
extracellular vesicle comprises a small molecule and the second
extracellular vesicle comprises a heterologous polynucleotide
comprising miR-195, miR-126, or miR-192.
51. A method for treating cancer in a subject comprising
administering to the subject the pharmaceutical composition of
claim 50.
52. (canceled)
53. A composition for imaging studies, the composition comprising
the extracellular vesicle of claim 5, wherein the vesicle comprises
a detectable or imaging agent.
54. (canceled)
55. The composition of claim 54, wherein the imaging agent is a
nanoparticle, magnetite, nanoparticle, paramagnetic particle,
microsphere, nanosphere, and is selectively targeted to cancer
cells.
56-57. (canceled)
58. A kit for delivering an agent to a cell the kit, wherein the
agent comprises the extracellular vesicle of claim 5.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/545,937, filed Jul. 24, 2017, which is the U.S.
national phase application, pursuant to 35 U.S.C. .sctn. 371, of
PCT International Application Ser. No.: PCT/US2016/015791, filed
Jan. 29, 2016, designating the United States and published in
English, which claims priority to and the benefit of U.S.
Provisional Application No. 62/109,764, filed Jan. 30, 2015, and
U.S. Provisional Application No. 62/150,318, filed Apr. 21, 2015,
each of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been filed electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 5, 2018, is named 167689_011203_US_SL.txt and is 1,452
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of extracellular
vesicles (e.g., exosomes, microvesicles, macrovesicles). More
specifically, the present invention provides compositions
comprising extracellular vesicles for delivery of agents (e.g.,
polynucleotides, polypeptides, small molecules) and methods of
using such compositions, for example, in therapeutic, imaging, and
research methods.
BACKGROUND OF THE INVENTION
[0004] Cholangiocarcinoma (CCA) is the second most common primary
liver cancer in the United States. The survival of CCA patients is
dismal, usually measured in months. Primary therapy with surgery is
applicable to fewer than 20% of patients. Photodynamic therapy and
chemotherapy provide responses in a minority of patients without
curative intent. Thus there is an urgent need for improved
treatment for CCA, and novel treatment modalities for CCA are
potentially translatable to other types of cancer. In addition,
there exists a need for methods that selectively deliver
therapeutics to cancer cells. Such compositions and methods could
be translated to a wide array of disease treatments.
SUMMARY OF THE INVENTION
[0005] The present invention provides extracellular vesicles (EVs)
derived from a cancer associated cell (e.g., fibroblast-like cell,
stromal cell) comprising an agent (e.g., polypeptide,
polynucleotide, small molecule), and methods of using such EVs to
deliver the agent to a target cell.
[0006] The invention generally provides an extracellular vesicle
isolated from a cancer associated fibroblast (CAF), where the
vesicle contains an exogenous agent.
[0007] In one aspect, the invention provides an extracellular
vesicle isolated from a cancer associated fibroblast (CAF), where
the vesicle contains a heterologous polynucleotide identified as
being down-regulated in the CAF, and where the extracellular
vesicle selectively targets a cancer cell.
[0008] In various embodiments of the above-aspects or any other
aspect of the invention delineated herein, the agent is an
exogenous polynucleotide. In various embodiments of the
above-aspects the polynucleotide is miR-195, miR-126, or miR-192 or
is a polynucleotide encoding miR-195, miR-126, or miR-192. In
various embodiments of the above-aspects the polynucleotide is a
vector encoding miR-195, miR-126, or miR-192. In various
embodiments of the above-aspects, the polypeptide is a recombinant
polypeptide heterologously expressed in the CAF or loaded into the
cell or extracellular vesicle ex vivo. In various embodiments of
the above-aspects, the polynucleotide is a recombinant
polynucleotide that is heterologously expressed in the cell or is
loaded into the cell ex vivo. In various embodiments of the
above-aspects, the recombinant polynucleotide is a microRNA. In
various embodiments of the above-aspects the microRNA is miR-195,
miR-126, or miR-192. In various embodiments of the above-aspects,
the small molecule is a lipid or other hydrophobic small molecule.
In various embodiments of the above-aspects, the small molecule is
doxorubicin, cisplatin, or phosphatidyl ethanolamine. In various
embodiments of the above-aspects, the phosphatidyl ethanolamine is
derivatized with an agent selected from the group consisting of
rhodamine, fluorescein, biotin, streptavidin, a small molecule, a
polynucleotide, and a polypeptide. In various embodiments of the
above-aspects, the polypeptide is an antibody, a polypeptide that
localizes to a specific cell type, a therapeutic protein, or
protein that can be used for imaging purposes. In various
embodiments of the above-aspects, the agent is a nanoparticle,
paramagnetic particle, microsphere, or nanosphere for magnetic
imaging. In various embodiments of the above-aspects, the cancer
associated fibroblast is a stromal cell. In various embodiments of
the above-aspects, the stromal cell is derived from a tumor
microenvironment. In various embodiments of the above-aspects, the
tumor is a cholangiocarcinoma, hepatocellular carcinoma, or
hepatoma. In various embodiments of the above-aspects, the tumor is
a breast cancer tumor, pancreatic tumor, glioblastoma, melanoma,
lung cancer tumor, ovarian cancer tumor, or any other type of
cancer. In various embodiments of the above-aspects, the
extracellular vesicle is isolated from a bodily fluid selected from
the group consisting of blood, plasma, serum, urine, stool, semen,
cerebrospinal fluid, prostate fluid, lymphatic drainage, bile
fluid, and pancreatic secretions. In various embodiments of the
above-aspects, the extracellular vesicle is isolated from cell
culture media. In various embodiments of the above-aspects, the
extracellular vesicle is isolated from cells cultured in
conditioned media obtained from a culture containing cancer cells.
In various embodiments of the above-aspects, the extracellular
vesicle is isolated from a culture containing a CAF derived from a
fibroblast, fibroblast-like cell, stellate cell, or myofibroblast.
In various embodiments of the above-aspects, the CAF expresses one
or more of alpha smooth muscle actin and/or collagen. In various
embodiments of the above-aspects, the fibroblast-like cell has a
fibroblast morphology. In various embodiments of the above-aspects,
the vesicle expresses increased levels of one or more markers
selected from the group consisting of alpha-SMA, Collagen, Vimentin
(FSP-1), S100, Metalloproteinases, NG2, PDGFR-B, SDF1/CXCL12, CD34,
Fibroblast activation protein (FAP), FSP-1, CD31, Thy-1, and
Gremlin. In various embodiments of the above-aspects, the vesicle
expresses reduced levels of laminin. In various embodiments of the
above-aspects, the CAF is derived from a fibroblast cultured for at
least 1-14 days in the presence of a cancer cell or in the presence
of conditioned media derived from a cancer cell culture. In various
embodiments of the above-aspects, the vesicle is isolated from
mammalian cells. In various embodiments of the above-aspects, the
vesicle is an exosome or a microvesicle.
[0009] In another aspect, the invention provides a method for
obtaining an extracellular vesicle, the method involving culturing
a fibroblast or stromal cell in conditioned media obtained from a
cancer cell culture, and isolating extracellular vesicles from the
media.
[0010] In another aspect, the invention provides an extracellular
vesicle produced according to the method of the above aspects.
[0011] In another aspect, the invention provides a pharmaceutical
composition containing a vesicle of any of the above aspects.
[0012] In another aspect, the invention provides a method of
delivering an agent to a cell, the method involving contacting the
cell with a vesicle of any of the above-aspects, thereby delivering
the agent to the cell.
[0013] In another aspect, the invention provides a method of
reducing a tumor in a subject, the method involving contacting the
cell with the vesicle of any of the above aspects.
[0014] In another aspect, the invention provides a method of
altering gene expression in a cell, the method involving contacting
the cell with a vesicle of any previous aspect.
[0015] In another aspect, the invention provides a method for
treating cancer in a subject comprising administering to the
subject a pharmaceutical composition comprising an effective amount
of the vesicle of any previous aspect.
[0016] In another aspect, the invention provides a method for
treating cholangiocarcinoma, hepatocellular carcinoma, or hepatoma
in a subject comprising administering to the subject a
pharmaceutical composition comprising an effective amount of an
extracellular vesicle isolated from a CAF over-expressing a
recombinant polynucleotide encoding miR-195, miR-192, or
miR-126.
[0017] In another aspect, the invention provides a pharmaceutical
composition comprising a first and a second extracellular vesicle,
where each vesicle contains a different agent. In one embodiment,
each vesicle comprises a different miRNA.
[0018] In another aspect, the invention provides a pharmaceutical
composition comprising a plurality of exosomes, where each exosome
contains one of miR-195, miR-192, or miR-126.
[0019] In another aspect, the invention provides a composition for
imaging studies, the composition comprising an extracellular
vesicle isolated from a cancer associated fibroblast (CAF) or
fibroblast-like cell, where the vesicle contains a detectable
agent. In one embodiment, the detectable agent is an imaging agent.
In another embodiment, the imaging agent is a nanoparticle,
magnetite, nanoparticle, paramagnetic particle, microsphere,
nanosphere, and is selectively targeted to cancer cells.
[0020] In another aspect, the invention provides a kit for
delivering an agent to a cell the kit comprising an extracellular
vesicle isolated from a cancer associated fibroblast (CAF) or
fibroblast-like cell, where the vesicle contains an agent.
[0021] In various embodiments of the above-aspects, the method
inhibits tumor cell proliferation. In various embodiments of the
above-aspects, the extracellular vesicle is an exosome. In various
embodiments of the above-aspects, the cancer cells are derived from
a liver cancer or breast cancer. In various embodiments of the
above-aspects, the cell is cultured for between about 3-days and 2
weeks in conditioned media. In various embodiments of the
above-aspects, the method further contains incubating the isolated
extracellular vesicle in a solution comprising an agent. In various
embodiments of the above-aspects, the extracellular vesicle is
incubated for between about 1 and 4 hours. In various embodiments
of the above-aspects, the fibroblast or stromal cell contains a
vector encoding a recombinant protein or microRNA. In various
embodiments of the above-aspects, the extracellular vesicle
contains an increased level of a recombinant protein,
polynucleotide, or small molecule than a corresponding control cell
not cultured in conditioned media. In various embodiments of the
above-aspects, the extracellular vesicle is a microvesicle.
Definitions
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them below, unless specified otherwise.
[0023] By "cancer associated fibroblast (CAF)" is meant a
fibroblast that expresses increased levels of alpha-smooth muscle
actin (SMA), PDGFRbeta, and/or collagen relative to a control
fibroblast. In one embodiment, a CAF expresses at least about
2-fold, 5-fold, 10-fold more alpha-SMA, PDGFRbeta, and collagen
relative to a non-CAF fibroblast (i.e., a fibroblast derived from
healthy non-cancerous tissue, or that has not been cultured in
conditioned media derived from cancer cells). A CAF derived EV
promotes tumor growth and metastasis. In contrast, CAFs of the
invention comprise agents that inhibit tumor growth. In another
embodiment, a CAF expresses reduced levels of miR-195, miR-192 and
miR-126 relative to a reference. In another embodiment, a CAF
overexpresses any one or more of the following markers: Actin
(a-SMA), Collagen, Vimentin (FSP-1), S100, Metalloproteinases, NG2,
PDGFR-B, SDF1 (CXCL12), CD34, Fibroblast activation protein (FAP)
and FSP-1 (as well as CD31), Thy-1, and Gremlin relative to a
reference. In another embodiment, a CAF expresses reduced levels of
laminin relative to a reference. In addition to stromal cells, CAFs
may be derived from cells having proximity to the tumor in vivo.
Thus, CAFs may be derived from cells associated with blood vessels
or local deposits of fat near the term. In some instances, a CAF is
identified at a site distant from the tumor. Such CAFs are
identified as CAFs or their subtypes by marking studies. In
particular embodiments, a cancer associated cell (CAC) may be used
in place of a CAF. CACs include brain derived glia,
oligodendroglia, and microglia. Other CACs include Breast-EMT and
bone marrow stem cells which have become CAFs. Other cells useful
in the invention include reactive cell populations associated with
cancer that express in various proportions FSP-1, S100,
Metalloproteinases, NG2 a-SMA, and PDGFR-B.
[0024] As used herein, the term "microRNA," "miRNA," or "miR"
refers to RNAs that function post-transcriptionally to regulator
expression of genes, usually typically by binding to complementary
sequences in the three prime (3') untranslated regions (3' UTRs) of
target messenger RNA (mRNA) transcripts, usually resulting in gene
silencing. miRNAs are typically small regulatory RNA molecules, for
example, 21 or 22 nucleotides long. The terms "microRNA," "miRNA,"
and "miR" are used interchangeably.
[0025] By "miR-195" is meant a polynucleotide or fragment thereof
having at least about 85% or greater nucleic acid sequence identity
to the polynucleotide sequence provided at NCBI Accession No.
NR_029712 that is capable of modulating gene expression. In one
embodiment, the miRNA affects the stability and/or translation of
mRNAs.
[0026] An exemplary miR-195 nucleic acid sequence is provided
below:
TABLE-US-00001 Homo sapiens miR-195 (SEQ ID NO: 1) 1 agcttccctg
gctctagcag cacagaaata ttggcacagg gaagcgagtc tgccaatatt 61
ggctgtgctg ctccaggcag ggtggtg
The exemplary sequence represents the predicted microRNA stem-loop.
Some sequence at the 5' and 3' ends may not be included in the
intermediate precursor miRNA produced by Drosha cleavage.
[0027] By "miR-195 gene" is meant the polynucleotide sequence
encoding the miR-195 miRNA.
[0028] By "miR-192" is meant a polynucleotide or fragment there of
having at least about 85% or greater identity to the polynucleotide
sequence provided at NCBI Accession No. NR_029578 that is capable
of modulating gene expression. In one embodiment, the miRNA affects
the stability and/or translation of mRNAs. An exemplary miR-192
nucleotide sequence is provided below:
TABLE-US-00002 Homo sapiens miR-192 (SEQ ID NO: 2) 1 gccgagaccg
agtgcacagg gctctgacct atgaattgac agccagtgct ctcgtctccc 61
ctctggctgc caattccata ggtcacaggt atgttcgcct caatgccagc
The exemplary sequence represents the predicted microRNA stem-loop.
Some sequence at the 5' and 3' ends may not be included in the
intermediate precursor miRNA produced by Drosha cleavage.
[0029] By "miR-192 gene" is meant the polynucleotide sequence
encoding the miR-192 miRNA.
[0030] By "miR-126" is meant a polynucleotide or fragment there of
having at least about 85% or greater identity to the polynucleotide
sequence provided at NCBI Accession No. NR_029695 that is capable
of modulating gene expression. In one embodiment, the miRNA affects
the stability and/or translation of mRNAs. An exemplary miR-126
nucleotide sequence is provided below:
TABLE-US-00003 Homo sapiens miR-126 (SEQ ID NO: 3) 1 cgctggcgac
gggacattat tacttttggt acgcgctgtg acacttcaaa ctcgtaccgt 61
gagtaataat gcgccgtcca cggca
The exemplary sequence represents the predicted microRNA stem-loop.
Some sequence at the 5' and 3' ends may not be included in the
intermediate precursor miRNA produced by Drosha cleavage.
[0031] By "miR-126 gene" is meant the polynucleotide sequence
encoding the miR-126 miRNA.
[0032] By "agent" is meant a polypeptide, polynucleotide, or
fragment, or analog thereof, small molecule, or other biologically
active molecule.
[0033] By "alteration" is meant a change (increase or decrease) in
the expression levels of a gene or polypeptide as detected by
standard art known methods such as those described above. As used
herein, an alteration includes a 10% change in expression levels,
preferably a 25% change, more preferably a 40% change, and most
preferably a 50% or greater change in expression levels.
[0034] As used herein, the term "animal" refers to any member of
the animal kingdom. The term "animal" may refer to humans at any
stage of development or any non-human animal at any stage of
development. In some embodiments, the term "animal" may refer to a
transgenic or genetically engineered animal or a clone.
[0035] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Methods of preparing antibodies are well known to those of ordinary
skill in the science of immunology. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. Tetramers may be naturally occurring or
reconstructed from single chain antibodies or antibody fragments.
Antibodies also include dimers that may be naturally occurring or
constructed from single chain antibodies or antibody fragments. The
antibodies in the present invention may exist in a variety of forms
including, for example, polyclonal antibodies, monoclonal
antibodies, Fv, Fab and F(ab') 2, as well as single chain
antibodies (scFv), humanized antibodies, and human antibodies
(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In:
Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,
1988, Science 242:423-426). In some embodiments, the antibody
specifically binds to C4A polypeptide.
[0036] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab') 2, and Fv
fragments, linear antibodies, scFv antibodies, single-domain
antibodies, such as camelid antibodies (Riechmann, 1999, Journal of
Immunological Methods 231:25-38), composed of either a VL or a VH
domain which exhibit sufficient affinity for the target, and
multispecific antibodies formed from antibody fragments. The
antibody fragment also includes a human antibody or a humanized
antibody or a portion of a human antibody or a humanized
antibody.
[0037] As used herein, the term "approximately" or "about," as
applied to one or more values of interest, refers to a value that
is similar to a stated reference value. In some embodiments, the
term "approximately" or "about" refers to a range of values that
fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either
direction of the stated reference value unless otherwise stated or
otherwise evident from the context.
[0038] By "control" is meant a standard or reference condition. The
term "control" refers to a standard against which results are
compared. In some embodiments, a control is used at the same time
as a test variable or subject to provide a comparison. In some
embodiments, a control is a historical control that has been
performed previously, a result or amount that has been previously
known, or an otherwise existing record. A control may be a positive
or negative control.
[0039] By "decreases" is meant a reduction by at least about 5%
relative to a reference level. A decrease may be by 5%, 10%, 15%,
20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more.
[0040] By "an effective amount" is meant the amount of an agent
required to ameliorate the symptoms of a disease relative to an
untreated patient. In one embodiment, the disease is cancer (e.g.,
cholangiocarcinoma, hepatocellular carcinoma, hepatoma). In other
embodiments, the disease is a single gene disorder including, but
not limited to, cystic fibrosis, sickle cell anemia, Tay-Sachs
disease, myotonic dystrophy, Duchenne muscular dystrophy, Fragile X
syndrome, glycogen storage diseases, and spinal muscular atrophy.
As would be appreciated by one of ordinary skill in the art, the
exact amount required to treat a disease will vary from subject to
subject, depending on age, general condition of the subject, the
severity of the condition being treated, the particular compound
and/or composition administered, and the like. The effective amount
of active agent(s) used to practice the present invention for
therapeutic treatment of a disease varies depending upon the manner
of administration, the age, body weight, and general health of the
subject. Ultimately, the attending physician or veterinarian will
decide the appropriate amount and dosage regimen. Such amount is
referred to as an "effective" amount.
[0041] By "exogenous" is meant foreign. An exogenous agent is one
that is not naturally occurring in the cell, such as a protein that
is recombinantly expressed.
[0042] As used herein, the term "exosome" refers to a small
membrane extracellular vesicle of .about.30-300 nm diameter that is
secreted from producing cells into the extracellular environment,
as described initially by Trams et al., 1981, BBA. The surface of
an exosome comprises a lipid bilayer from the membrane of the donor
cell, and the lumen of the exosome is topologically the same as the
cytosol from the cell that produces the exosome. The exosome
contains proteins, RNAs, lipids, and carbohydrates of the producing
cell, though some may be modified or added to the exosome after its
release from the cell, either through natural processes or by
experimental manipulation.
[0043] As used herein, the term "exosome" refers to a small
membrane extracellular vesicle of .about.30-300 nm diameter that is
secreted from producing cells into the extracellular environment,
as described initially by Trams et al., 1981, BBA. The surface of
an exosome comprises a lipid bilayer from the membrane of the donor
cell, and the lumen of the exosome is topologically the same as the
cytosol from the cell that produces the exosome. The exosome
contains proteins, RNAs, lipids, and carbohydrates of the producing
cell, though some may be modified or added to the exosome after its
release from the cell, either through natural processes or by
experimental manipulation.
[0044] By "fragment" is meant a portion (e.g., at least 10, 25, 50,
100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or
nucleic acids) of a protein or nucleic acid molecule that is
substantially identical to a reference protein or nucleic acid and
retains the biological activity of the reference.
[0045] By "heterologous" is meant originating in a different cell
type or species from the recipient.
[0046] A "host cell" is any prokaryotic or eukaryotic cell that
contains either a cloning vector or an expression vector. This term
also includes those prokaryotic or eukaryotic cells that have been
genetically engineered to contain the cloned gene(s) in the
chromosome or genome of the host cell.
[0047] By "inhibits a neoplasia" is meant decreases the propensity
of a cell to develop into a neoplasia or slows, decreases, or
stabilizes the growth or proliferation of a neoplasia.
[0048] As used herein, the term "in vitro" refers to events or
experiments that occur in an artificial environment, e.g., in a
petri dish, test tube, cell culture, etc., rather than within a
multicellular organism.
[0049] As used herein, the term "in vivo" refers to events or
experiments that occur within a multicellular organism.
[0050] As used herein, the term "isolated" refers to a substance,
molecule, or entity that has been either separated from at least
some of the components with which it was associated when initially
produced in nature or through an experiment, and/or produced,
prepared, or manufactured by the hand of man. Isolated substances
and/or entities may be separated from at least about 10%, about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, about 90%, about 95%, about 98%, about 99%, substantially
100%, or 100% of the other components with which they were
initially associated. In some embodiments, isolated agents are more
than about 80%, about 85%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, about
99%, substantially 100%, or 100% pure. As used herein, a substance
is "pure" if it is substantially free of other components.
[0051] By "inhibitory nucleic acid molecule" is meant a single
stranded or double-stranded RNA, siRNA (short interfering RNA),
shRNA (short hairpin RNA), or antisense RNA, or a portion thereof,
or an analog or mimetic thereof, that when administered to a
mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%,
or even 90-100%) in the expression of a target sequence. Such
inhibitory nucleic acid molecules may delivered using compositions
of the invention. Typically, a nucleic acid inhibitor comprises or
corresponds to at least a portion of a target nucleic acid
molecule, or an ortholog thereof, or comprises at least a portion
of the complementary strand of a target nucleic acid molecule.
[0052] By "marker" is meant any protein or polynucleotide having an
alteration in expression level or activity that is associated with
a disease or disorder.
[0053] By "modification" is meant any biochemical or other
synthetic alteration of a nucleotide, amino acid, or other agent
relative to a naturally occurring reference agent.
[0054] By "neoplasia" is meant any disease that is caused by or
results in inappropriately high levels of cell division,
inappropriately low levels of apoptosis, or both. For example,
cancer is a neoplasia. Examples of cancers include, without
limitation, leukemias (e.g., acute leukemia, acute lymphocytic
leukemia, acute myelocytic leukemia, acute myeloblastic leukemia,
acute promyelocytic leukemia, acute myelomonocytic leukemia, acute
monocytic leukemia, acute erythroleukemia, chronic leukemia,
chronic myelocytic leukemia, chronic lymphocytic leukemia),
polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's
disease), Waldenstrom's macroglobulinemia, heavy chain disease, and
solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
cholangiocarcinoma (also termed bile duct carcinoma),
choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, uterine cancer, testicular cancer, lung carcinoma,
small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma,
and retinoblastoma). Lymphoproliferative disorders are also
considered to be proliferative diseases.
[0055] In some embodiments, "cancer" can include histologic and
molecular subtypes of liver cancer, pancreatic cancer, prostate
cancer, breast cancer, hepatocellular carcinoma, colon cancer, lung
cancer, lymphoma, leukemia, melanoma, basal cell cancer, cervical
cancer, colorectal cancer, stomach cancer, bladder cancer, anal
cancer, bone cancer, brain tumor, esophageal cancer, gall bladder
cancer, gastric cancer, testicular cancer, Hodgkin Lymphoma,
intraocular melanoma, kidney cancer, oral cancer, melanoma,
neuroblastoma, Non-Hodgkin Lymphoma, ovarian cancer,
retinoblastoma, skin cancer, throat cancer, and thyroid cancer.
Fibroblasts having proximity to any of the aforementioned cancer
types or grown in a culture comprising such cancer cells are CAFs.
For example, breast cancer associated fibroblasts are those growing
in a culture that also comprises a cancer cell. Cholangiocarcinoma
or hepatocellular cancer associated fibroblasts are those growing
in a culture that also comprises a cancer cell.
[0056] As used herein, the term "microvesicle" refers to a single
membrane vesicle secreted by cells that may have a larger diameter
than those which some refer to as exosomes. Microvesicles may have
a diameter (or largest dimension where the particle is not
spheroid) of between about 10 nm to about 5000 nm (e.g., between
about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between
about 75 nm and 1250 nm, between about 50 nm and 1250 nm, between
about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between
about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.).
Microvesicles suitable for use in the present invention originate
from cells yet different subpopulations of microvesicles may
exhibit different surface/lipid characteristics. Alternative names
for microvesicles include, but are not limited to, exosomes,
ectosomes, membrane particles, exosome-like particles, and
apoptotic vesicles. As used herein, an abbreviated form "MV" is
sometime used to refer to microvesicle.
[0057] As used herein, the term "microvesicle" refers to a
membranous particle comprising fragments of plasma membrane that is
derived from various cell types. Typically, microvesicles have a
diameter (or largest dimension where the particle is not spheroid)
of between about 10 nm to about 5000 nm (e.g., between about 50 nm
and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm
and 1250 nm, between about 50 nm and 1250 nm, between about 30 nm
and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm
and 1000 nm, between about 50 nm and 750 nm, etc.). Typically, at
least part of the membrane of the microvesicle is directly obtained
from a cell (also known as a donor cell). Microvesicles suitable
for use in the present invention may originate from cells by
membrane inversion, exocytosis, shedding, blebbing, and/or budding.
Depending on the manner of generation (e.g., membrane inversion,
exocytosis, shedding, or budding), the microvesicles contemplated
herein may exhibit different surface/lipid characteristics.
[0058] Alternative names for microvesicles include, but are not
limited to, exosomes, ectosomses, membrane particles, exosome-like
particles, and apoptotic vesicles. As used herein, an abbreviated
form "MV" is sometime used to refer to microvesicle.
[0059] As used herein, an individual "suffering from" a disease,
disorder, or condition means that the person has been diagnosed
with or displays one or more symptoms of the disease, disorder, or
condition
[0060] By "nucleic acid molecule" is meant an oligomer or polymer
of ribonucleic acid or deoxyribonucleic acid, or analog thereof.
This term includes oligomers consisting of naturally occurring
bases, sugars, and intersugar (backbone) linkages as well as
oligomers having non-naturally occurring portions which function
similarly. Such modified or substituted oligonucleotides are often
preferred over native forms because of properties such as, for
example, enhanced stability in the presence of nucleases. In
certain embodiments, the term "nucleic acid molecule" refers to
genetic material that can be transferred via EVs including, but not
limited to, miRNA, mRNA, tRNA, rRNA, siRNA, shRNA, DNA (including
fragments, plasmids, and the like). Such genetic materials can be
transferred to EVs via transfection, transformation,
electroporation, and microinjection.
[0061] By "obtaining" as in "obtaining the inhibitory nucleic acid
molecule" is meant synthesizing, purchasing, or otherwise acquiring
the inhibitory nucleic acid molecule.
[0062] By "operably linked" is meant that a first polynucleotide is
positioned adjacent to a second polynucleotide that directs
transcription of the first polynucleotide when appropriate
molecules (e.g., transcriptional activator proteins) are bound to
the second polynucleotide.
[0063] By "positioned for expression" is meant that the
polynucleotide of the invention (e.g., a DNA molecule) is
positioned adjacent to a DNA sequence that directs transcription
and translation of the sequence (i.e., facilitates the production
of, for example, a recombinant microRNA molecule described
herein).
[0064] By "portion" is meant a fragment of a polypeptide or nucleic
acid molecule. This portion contains, preferably, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of
the reference nucleic acid molecule or polypeptide. A fragment may
contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
or 21 nucleotides.
[0065] By "reference" is meant a standard or control condition.
[0066] By "reporter gene" is meant a gene encoding a polypeptide
whose expression may be assayed; such polypeptides include, without
limitation, glucuronidase (GUS), luciferase, chloramphenicol
transacetylase (CAT), and beta-galactosidase.
[0067] By "selectively deliver" is meant that the majority of the
EV is delivered to a targeted cell type relative to non-target
cells present in the culture, tissue, or organ. In embodiments,
greater than about 50%, 60%, 70%, 80%, 90%, 95% or even approaching
100% of the EVs are delivered to a desired cell type. In other
embodiments, only about 10%, 15%, 20% 25%, 30%, 35%, or 40% of the
EVs are delivered to non-target cells.
[0068] The term "siRNA" refers to small interfering RNA; a siRNA is
a double stranded RNA that "corresponds" to or matches a reference
or target gene sequence. This matching need not be perfect so long
as each strand of the siRNA is capable of binding to at least a
portion of the target sequence. SiRNA can be used to inhibit gene
expression, see for example Bass, 2001, Nature, 411, 428 429;
Elbashir et al., 2001, Nature, 411, 494 498; and Zamore et al.,
Cell 101:25-33 (2000).
[0069] "As used herein, the term "stromal cell" refers to
non-vascular, non-inflammatory, non-epithelial connective tissue
cells of any organ that surround a tumor. Stromal cells are also
known as cancer-associated fibroblasts. Stromal cells support the
function of the parenchymal cells of that organ. Fibroblasts and
pericytes are among the most common types of stromal cells. The
stromal cells can be derived from numerous body tissue types,
including, but not limited to, breast tissue, thymic tissue, bone
marrow tissue, bone tissue, dermal tissue, muscle tissue,
respiratory tract tissue, gastrointestinal tract tissue,
genitourinary tissue, central nervous system tissue, peripheral
nervous system tissue, reproductive tract tissue.
[0070] As used herein, the term "subject" refers to a human or any
non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle,
swine, sheep, horse or primate). A human includes pre and post
natal forms. In many embodiments, a subject is a human being. A
subject can be a patient, which refers to a human presenting to a
medical provider for diagnosis or treatment of a disease. The term
"subject" is used herein interchangeably with "individual" or
"patient." A subject can be afflicted with or is susceptible to a
disease or disorder but may or may not display symptoms of the
disease or disorder.
[0071] The term "pharmaceutically-acceptable excipient" as used
herein means one or more compatible solid or liquid filler,
diluents or encapsulating substances that are suitable for
administration into a human.
[0072] By "specifically binds" is meant a molecule (e.g., peptide,
polynucleotide) that recognizes and binds a protein or nucleic acid
molecule of the invention, but which does not substantially
recognize and bind other molecules in a sample, for example, a
biological sample, which naturally includes a protein of the
invention.
[0073] By "substantially identical" is meant a protein or nucleic
acid molecule exhibiting at least 50% identity to a reference amino
acid sequence (for example, any one of the amino acid sequences
described herein) or nucleic acid sequence (for example, any one of
the nucleic acid sequences described herein). Preferably, such a
sequence is at least 60%, more preferably 80% or 85%, and still
more preferably 90%, 95% or even 99% identical at the amino acid
level or nucleic acid to the sequence used for comparison.
[0074] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining
the degree of identity, a BLAST program may be used, with a
probability score between e.sup.-3 and e.sup.-100 indicating a
closely related sequence.
[0075] By "targets" is meant alters the biological activity of a
target polypeptide or nucleic acid molecule.
[0076] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a polynucleotide molecule encoding (as used herein) a
protein of the invention.
[0077] By "vector" is meant a nucleic acid molecule, for example, a
plasmid, cosmid, or bacteriophage, that is capable of replication
in a host cell. In one embodiment, a vector is an expression vector
that is a nucleic acid construct, generated recombinantly or
synthetically, bearing a series of specified nucleic acid elements
that enable transcription of a nucleic acid molecule in a host
cell. Typically, expression is placed under the control of certain
regulatory elements, including constitutive or inducible promoters,
tissue-preferred regulatory elements, and enhancers.
BRIEF DESCRIPTION OF THE FIGURES
[0078] FIG. 1. An image of a co-culture of cancer cells and the
fibroblast-like (e.g. fibroblasts, stellate cells, etc.) cell, LX-2
(. HuCCT1 CCA cells are marked with EGFP and LX2 fibroblasts are
unstained.
[0079] FIG. 2. Table showing downregulation of multiple miRs in the
fibroblast-like LX2 following their co-culture with CCA cells. The
table presents the Ct value and the qRT-PCR value normalized to U6.
The ratio of qRT-PCR expression in LX2 cells cultured alone
(control) or in the presence of cancer cells (F/CAFs) is
highlighted in the right column.
[0080] FIG. 3. Restoration of miR-195 in the LX-2 fibroblast-like
cell is sufficient to inhibit invasiveness of co-cultured cancer
cells. Four different human and rat CCA cells were co-cultured with
LX2-NSM or LX2-miR-195 cells. Invading cells were visualized by
Crystal Violet staining.
[0081] FIG. 4. Up-regulation of miR-195 in fibroblast-like cells
inhibits co-cultured cancer cells that were permitted to exchange
media, but were not in direct contact. From left to right, the
slides demonstrate decreased invasion, migration and growth of
cancer cells induced by mediators released in media by LX2-195
cells vs. LX2-control.
[0082] FIG. 5. LX2-miR-195 fibroblast-like cells release soluble
factors that cause elevated levels of miR-195 in cancer cells.
Levels of miR-195 were measured in three different CCA cancer cells
following their exposure to soluble factors from either (left bars)
LX2 fibroblasts or (right bars) LX2-195 cells that overexpress
miR-195.
[0083] FIG. 6. LX2-miR-195 cells secrete .about.60-fold higher
levels of miR-195 in exosomes/EVs than control LX2 cells.
[0084] FIG. 7. EVs derived from a hepatic fibroblast-like cell are
targeted to CCA cancer cells in vivo., and selectively deliver a
protein cargo to the cancer cells but not to surrounding parts of
the liver or to other organs of the body. EVs (indicated by the
anti-mCherry staining in the figure from the presence of the
expression of the TSG101/mCherry fusion protein which is expressed
in cells and secreted to) are selectively enriched in pockets of
the tumor (DAPI stain), that are surrounded by the endogenous
fibroblasts (indicated by the .alpha.-SMA staining for active
fibroblast). EVs are visualized by staining for an EV cargo protein
that was expressed in the fibroblast like cells, demonstrating
selective delivery of protein to cancer cells in vivo.
[0085] FIG. 8. EV-carried plasmid designed to express Cre
recombinase is selectively delivered to rats via tail vein
injections. The tumor area was stained with antibodies to detect
alpha-SMA (a marker of activated fibroblasts), DAPI (nuclear stain,
which detects all cells), and also visualized to detect GFP, which
is only expressed if the introduced EVs delivered Cre-expressing
DNA into the CCA cells. CCA cells that did not take up functional
Cre remained red in these experiments . We observed cords of
fibroblasts (stained with anti-alpha SMA), as well as pockets of
cancer cells, many of which were expressing GFP, establishing
selective delivery of DNA into the cancer cells in vivo.
[0086] FIG. 9. miR-195-loaded EVs inhibit CCA growth in vivo. EVs
were loaded with (left panels) a non-specific miR mimic or (right
panels) a miR-195 mimic and injected into rats with CCA. 30 days
later, the rats were sacrificed. Tumors were significantly smaller
in animals that had been injected with miR-195-loaded EVs.
[0087] FIG. 10. miR-195-loaded EVs inhibit CCA tumor growth, as
measured by volume (left graph), as well as weight (right graph).
The tumors resected from rats were measured and weighed. The first
3 bars (front the left) in each graph represent 3 tumors from rats
treated with the negative control (EVs-NSM), while the 3 bars on
the right in each graph represent 3 tumors from rats treated with
EVs-miR-195.
[0088] FIG. 11. miR-195 downregulates CDK6 and VEGF when directly
transfected into BDEneu cells (left panel), when conditioned media
from LX2 cells (treated with miR-195 or NSM) is utilized (middle
panel), and when treated with exosomes loaded with miR-195 vs. NSM
(right panel).
[0089] FIG. 12. Tail vein treatment of CCA with EVs-miR-195
increases the survival in rats by 50% vs. control.
[0090] FIG. 13. LX2 cells expressing miR-126 inhibit CCA
invasiveness in vitro. HuCCT1 cells were co-cultured directly with
LX2 cells expressing either (upper image) a control miR, or (lower
image) miR-126. Invasiveness of HuCCT1 cells was decreased 3.2 fold
when co-cultured with LX2-126 cells.
[0091] FIG. 14. LX2 cells expressing miR-126 inhibit CCA migration
4-fold in vitro. HuCCT1 cells were co-cultured directly with LX2
cells expressing either a controls miR or miR-126. Migration was
measured in a scratch assay.
[0092] FIGS. 15A-15C. Mammary fibroblast-derived EVs deliver a
small molecule to breast cancer cells. MDA-MB-231 cells (stably
expressing the fluorescent protein tdTomato) were grown in the
presence of primary human mammary fibroblast cells that had
previously been labeled with a fluorescent lipid (N-F-PE;
N-fluorescein-phosphatidylethanolamine (Avanti polar lipids)) that
is selectively secreted from human cells in EVs (Booth et al., J.
Cell Biol. 2006; Fang et al., PLoS Biol. 2007). Over the course of
2-3 days, the (FIG. 15A) tdTomato-expressing human breast cancer
(seen as white or light signal emitting cells on black and white
drawings) cells took up the (FIG. 15B) EVs that had been released
from the primary mammary fibroblast cell line. Cells were also
stained with (FIG. 15C) DAPI to visualize the nucleus.
[0093] FIGS. 16A-16D. Mammary fibroblast-derived EVs deliver a
protein to breast cancer cells. MDA-MB-231 cells (stably expressing
the fluorescent protein tdTomato) were grown in the presence of
primary human mammary fibroblast cells that had previously been
transfected with a plasmid designed to express Acyl-GFP, a form of
GFP that is secreted from human cells in EVs. Over the course of
2-3 days, the (FIG. 16A) tdTomato-expressing human breast cancer
cells took up the (FIG. 16B) the fluorescent lipid, N-F-PE labeled
EVs that had been released from the primary mammary fibroblast cell
line. Cells were also stained with (FIG. 16C) DAPI to visualize the
nucleus, and (FIG. 16D) the images were merged to show the presence
of CAF-derived EVs in the breast cancer cell (in this case, in the
nucleus).
[0094] FIGS. 17A-17B. Mammary fibroblasts promote the neoplastic
phenotype of MDA-MB-231 breast cancer cells. MDA-MB-231 cells
(stably expressing the fluorescent protein td Tomato) were grown
alone or in the presence of primary human mammary fibroblast cells.
(FIG. 17A) The diameter of MDA-MB-231 cells grown on their own was
approximately 200 relative units, but increased .about.7-fold upon
co-culture with CAFs, an increase in cell size that was apparent as
early as 3 hours after co-culture with mammary fibroblasts and was
complete within 1 day. Experiments were performed in triplicate,
followed by calculation of average and standard deviation.
Significant difference from t=1 hr (p, 0.05) were observed for all
but the 2 hr sample. (FIG. 17B) Growth of MDA-MB-231 cells was
induced .about.2-fold by co-culture with mammary fibroblasts.
MDA-MB-231 cells were plated on culture dishes. The next day, the
dishes were either (1) grown on their own, or (2,3) were populated
with mammary fibroblast (2) HMF line or (3) MMF line, to a density
of .about.20%. The next day the number of red MDA-MB-231 cells in
each dish was counted. Experiments were performed in triplicate,
and the averages and standard deviations showed significant
differences between each experimental sample (p<0.05) from that
of the control cancer cells grown on their own.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The invention provides extracellular vesicles (EVs) derived
from a cancer associated cell (e.g., fibroblast-like cell, stromal
cell) comprising an agent (e.g., polypeptide, polynucleotide, small
molecule), and methods of using such EVs to deliver the agent to a
target cell.
[0096] The invention is based, at least in part, on the discovery
that fibroblast gene expression is altered in fibroblasts that grow
in proximity to cancer cells (e.g., in stroma) or in conditioned
media where cancer cells had previously been cultured. Such cells
are termed cancer associated fibroblasts (CAF). As reported in
detail below, the gene expression of CAFs is altered following
their growth in cancer conditioned media or in stroma. For example,
CAFs have increased expression of CAF markers: alpha-smooth muscle
actin (SMA), PDGFRbeta, and collagen. In one embodiment, a CAF
expresses at least about 2-fold, 5-fold, 10-fold more alpha-SMA,
PDGFRbeta, and collagen relative to a non-CAF fibroblast (i.e., a
fibroblast derived from healthy non-cancerous tissue, or that has
not been cultured in conditioned media derived from cancer cells).
We show here that there is also a significant decrease in multiple
miRs, including miR-195, miR-192 and miR-126. These microRNAs are
involved in the transition from normal fibroblasts to CAFs. The
overexpression of miR-195 in the CAF reverses many of the changes
observed in not only in CAFs expressing mir-195, but in neighboring
cells as well. Surprisingly, this effect was mediated by
extracellular vesicles isolated from the mir-195 overexpressing
cells. Levels of miR-195 were >60-fold higher in these EVs than
in EVs isolated from control cells that were not over-expressing
mir-195. In further experiments, cells over-expressing polypeptides
and polynucleotides were found to shed EVs comprising increased
levels of the over-expressed polypeptide or polynucleotide. When
injected into rats having CCA, these fibroblast-derived vesicles
were highly enriched within the CCA cells relative to non-cancer
cells.
[0097] Accordingly, the invention provides extracellular vesicles
(EVs) derived from CAFs that comprise an agent (e.g., polypeptide,
polynucleotide, small molecule), and methods of using such EVs to
selectively deliver the agent to a target cell (e.g., cancer cell)
in vivo or in vitro.
Cholangiocarcinoma
[0098] Cholangiocarcinoma (CCA) is the second most common primary
liver cancer. CCAs are very desmoplastic cancers (similar to
pancreatic cancer, and some breast cancers). As described herein,
we identified microRNA species that are relatively downregulated in
fibroblast-like cells, along the continuum of inactive-to
activated-to cancer associated-fibroblasts (CAFs). Studies in vitro
showed that `therapeutic` upregulation of these miR species in
fibroblast-like cells resulted in less growth and invasiveness of
neighboring cancer cells. Without intending to be bound by theory,
it is likely that cancer-associated fibroblast-like cells play a
regulatory role in CCA and other tumors. Thus, we have demonstrated
that our therapy interferes with the signaling between
fibroblast-like cells and cancer cells. The result is to restrict
the growth and invasion of cancer. In understanding this signaling,
as described herein, we demonstrated that transport of
extracellular vesicles (EVs) between fibroblast like cells and
cancer cells, in both the CCA model and in a breast cancer model,
constitutes a rich signaling network which involves miRNAs and can
also involve the transfer of proteins and lipids. We then
engineered such EVs to contain as cargo the desired miR species,
the desired protein, or the desired small molecule. The fibroblast
cell-derived EVs are used to interfere with the signaling network
that influences proliferation or invasion by cancer cells. Results
described herein below indicate that EVs derived from
fibroblast-like cells and loaded with microRNAs can affect the
growth and invasion of cancer cells. Moreover, in vivo experiments
demonstrated that EVs loaded with miRs can be systemically
delivered and then selectively concentrate in liver tumors. This
delivery was sufficient to decrease cancer growth and increase the
overall survival (statistically significant) of treated animals.
These fibroblast-like cell-derived EVs do not accumulate in normal
liver cells, nor do these EVs accumulate in other tissues (e.g.
kidney, lung, etc.).
[0099] In conclusion, our studies demonstrate the existence and
functioning of EV exchange between fibroblast-like cells and cancer
cells in two cancer models. We show that miRs loaded into EVs from
fibroblast-like cells can have a functional role in control of the
cancer cells. We show that EVs of fibroblast-like cell origin can
be loaded with functional miRs, DNAs, proteins, and lipids. In
addition, we show that EVs of fibroblast-like cell origin when
loaded with miRs selectively target cancer cells in vivo and
diminish their growth. Finally, EVs of fibroblast-like cell origin
loaded with miRs can be systemically administered to animals
bearing cancers with resulting reduction of tumor growth and
resulting survival benefit.
Polynucleotides for Delivery
[0100] EVs derived EVs containing a microRNA may be used to deliver
the microRNA to a target cell. MicroRNAs (miRNAs) are 20-24
nucleotide RNA molecules that regulate the stability or
translational efficiency of target mRNAs. miRNAs have diverse
functions including the regulation of cellular differentiation,
proliferation, and apoptosis (Ambros, Nature 431, 350-5 (2004)).
Although strict tissue- and developmental-stage-specific expression
is critical for appropriate miRNA function, few mammalian
transcription factors that regulate miRNAs have been
identified.
[0101] In general, EVs of the invention comprise a polynucleotide
that is downregulated in a cell of interest (e.g., cancer cell).
The EV rescues the down regulation by increasing levels of the
polynucleotide. In other embodiments, the EV provides a replacement
polynucleotide that replaces or corrects a defective polynucleotide
present in the cell.
[0102] In one embodiment, an EV derived from a fibroblast-like cell
comprises a miR-195, miR-192, or miR-126 microRNA. In another
embodiment, EV derived from a fibroblast-like cell comprises a
nucleic acid sequence encoding a microRNA, such as miR from
fibroblast-like cells can be used to deliver virtually any
polynucleotide, including RNA, DNA, an antisense oligonucleotide, a
short interfering RNA (siRNA), a short hairpin RNA (shRNA), or
plasmid DNA polynucleotides and modified oligonucleotides.
Exemplary siRNAs include siRNAs targeting Anti-RhoA/C,
geranylgeranyl (or farnesyl) and transferase inhibitors of Ras
activation, cerivastatin, palbococlib, also siRNA to CXCR4 in
breast cancer metastases.
[0103] Polynucleotides provided in EVs include Mir -195, miR-192,
or miR-126, as well as nucleic acid molecules.
[0104] In one embodiment, we have found that CCA cells alter the
gene expression profile of surrounding fibroblasts, including
reduced expression of miR-195; overexpression of miR-195 in CAFs is
sufficient to inhibit CCA growth, migration, and invasion in vitro;
miR-195 is secreted from CAFs within EVs; elevating miR-195 levels
in CAFs is sufficient to up-regulate the levels of miR-195 in
neighboring cancer cells; and intravenous injection of
miR-195-loaded EVs inhibit CCA growth and extends survival in
vivo.
[0105] Expression vectors having a polynucleotide with therapeutic
function can be delivered to cells of a subject having a disease
(e.g., cancer) using the EVs of the invention.
[0106] In a specific embodiment, the DNA encodes a protein with a
specific function, either of diagnostic or therapeutic potential,
such as Cre recombinase. In another embodiment, the nucleic acid
molecule inhibits expression of a tumor suppressor gene as a way to
induce a large animal model of cancer biology. In a more specific
embodiment, the tumor suppressor gene is p53.
[0107] The EV comprising nucleic acid molecules are selectively
delivered to target cells of a subject (e.g., cancer cells) in a
form in which they are taken up and are advantageously expressed so
that therapeutically effective levels can be achieved.
[0108] An isolated nucleic acid molecule can be manipulated using
recombinant DNA techniques well known in the art. Thus, a
nucleotide sequence contained in a vector in which 5' and 3'
restriction sites are known, or for which polymerase chain reaction
(PCR) primer sequences have been disclosed, is considered isolated,
but a nucleic acid sequence existing in its native state in its
natural host is not. An isolated nucleic acid may be substantially
purified, but need not be. For example, a nucleic acid molecule
that is isolated within a cloning or expression vector may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein, because it can be manipulated using standard
techniques known to those of ordinary skill in the art.
[0109] Transducing viral (e.g., retroviral, adenoviral, lentiviral
and adeno-associated viral) vectors can be used for somatic cell
gene therapy, especially because of their high efficiency of
infection and stable integration and expression (see, e.g.,
Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al.,
Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of
Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267,
1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319,
1997). For example, a polynucleotide can be cloned into a
retroviral or other vector and expression can be driven from its
endogenous promoter, from the retroviral long terminal repeat, or
from a promoter specific for a target cell type of interest. Other
viral vectors that can be used include, for example, a vaccinia
virus, a bovine papilloma virus, or a herpes virus, such as
Epstein-Barr Virus (also see, for example, the vectors of Miller,
Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281,
1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et
al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The
Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research
and Molecular Biology 36:311-322, 1987; Anderson, Science
226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et
al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science
259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).
Retroviral vectors are particularly well developed and have been
used in clinical settings (Rosenberg et al., N. Engl. J. Med
323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
[0110] Polynucleotide expression can be directed from any suitable
promoter (e.g., the human cytomegalovirus (CMV), simian virus 40
(SV40), or metallothionein promoters), and regulated by any
appropriate mammalian regulatory element. For example, if desired,
enhancers known to preferentially direct gene expression in
specific cell types can be used to direct the expression of a
nucleic acid. The enhancers used can include, without limitation,
those that are characterized as tissue- or cell-specific
enhancers.
[0111] EVs derived from fibroblast-like cells can also be used to
deliver nucleic acid molecules comprising a modified nucleic acid.
Nucleic acid molecules include nucleobase oligomers containing
modified backbones or non-natural internucleoside linkages.
Oligomers having modified backbones include those that retain a
phosphorus atom in the backbone and those that do not have a
phosphorus atom in the backbone. For the purposes of this
specification, modified oligonucleotides that do not have a
phosphorus atom in their internucleoside backbone are also
considered to be nucleobase oligomers. Nucleobase oligomers that
have modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates,
phosphotriesters, aminoalkyl-phosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates. Various salts,
mixed salts and free acid forms are also included. Representative
United States patents that teach the preparation of the above
phosphorus-containing linkages include, but are not limited to,
U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;
5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;
5,571,799; 5,587,361; and 5,625,050, each of which is herein
incorporated by reference.
[0112] Nucleobase oligomers having modified oligonucleotide
backbones that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, and CH.sub.2
component parts. Representative United States patents that teach
the preparation of the above oligonucleotides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0113] Nucleobase oligomers may also contain one or more
substituted sugar moieties. Such modifications include 2'-O-methyl
and 2'-methoxyethoxy modifications. Another desirable modification
is 2'-dimethylaminooxyethoxy, 2'-aminopropoxy and 2'-fluoro.
Similar modifications may also be made at other positions on an
oligonucleotide or other nucleobase oligomer, particularly the 3'
position of the sugar on the 3' terminal nucleotide. Nucleobase
oligomers may also have sugar mimetics such as cyclobutyl moieties
in place of the pentofuranosyl sugar. Representative United States
patents that teach the preparation of such modified sugar
structures include, but are not limited to, U.S. Pat. Nos.
4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;
5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;
5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;
5,670,633; and 5,700,920, each of which is herein incorporated by
reference in its entirety.
[0114] In other nucleobase oligomers, both the sugar and the
internucleoside linkage, i.e., the backbone, are replaced with
novel groups. Methods for making and using these nucleobase
oligomers are described, for example, in "Peptide Nucleic Acids
(PNA): Protocols and Applications" Ed. P. E. Nielsen, Horizon
Press, Norfolk, United Kingdom, 1999. Representative United States
patents that teach the preparation of PNAs include, but are not
limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,
each of which is herein incorporated by reference. Further teaching
of PNA compounds can be found in Nielsen et al., Science, 1991,
254, 1497-1500.
Polypeptide Delivery
[0115] The invention provides EVs comprising proteins. In a
specific embodiment, the EV-delivered protein corrects a deficiency
of the cell or subject, or induces the death of infected or
deficient cells. Recombinant polypeptides of the invention are
produced using virtually any method known to the skilled artisan.
Typically, recombinant polypeptides are produced by transformation
of a suitable host cell with all or part of a polypeptide-encoding
nucleic acid molecule or fragment thereof in a suitable expression
vehicle.
[0116] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems may be
used to provide the recombinant protein. The precise host cell used
is not critical to the invention. A polypeptide of the invention
may be produced in a prokaryotic host (e.g., E. coli) or in a
eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells,
e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or
preferably COS cells). Such cells are available from a wide range
of sources (e.g., the American Type Culture Collection, Rockland,
Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular
Biology, New York: John Wiley and Sons, 1997). The method of
transformation or transfection and the choice of expression vehicle
will depend on the host system selected. Transformation and
transfection methods are described, e.g., in Ausubel et al.
(supra); expression vehicles may be chosen from those provided,
e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et
al., 1985, Supp. 1987).
[0117] A variety of expression systems exist for the production of
the polypeptides of the invention. EVs derived from fibroblast-like
cells can be loaded with any one or more of the following
expression vectors or with the polypeptides generated using such
vectors. Expression vectors useful for producing polypeptides
include, without limitation, chromosomal, episomal, and
virus-derived vectors, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof.
[0118] One particular bacterial expression system for polypeptide
production is the E. coli pET expression system (e.g., pET-28)
(Novagen, Inc., Madison, Wis). According to this expression system,
DNA encoding a polypeptide is inserted into a pET vector in an
orientation designed to allow expression. Since the gene encoding
such a polypeptide is under the control of the T7 regulatory
signals, expression of the polypeptide is achieved by inducing the
expression of T7 RNA polymerase in the host cell. This is typically
achieved using host strains that express T7 RNA polymerase in
response to IPTG induction. Once produced, recombinant polypeptide
is then isolated according to standard methods known in the art,
for example, those described herein.
[0119] Another bacterial expression system for polypeptide
production is the pGEX expression system (Pharmacia). This system
employs a GST gene fusion system that is designed for high-level
expression of genes or gene fragments as fusion proteins with rapid
purification and recovery of functional gene products. The protein
of interest is fused to the carboxyl terminus of the glutathione
S-transferase protein from Schistosoma japonicum and is readily
purified from bacterial lysates by affinity chromatography using
Glutathione Sepharose 4B. Proteins can be recovered under mild
conditions by elution with glutathione. Cleavage of the glutathione
S-transferase domain from the fusion protein is facilitated by the
presence of recognition sites for site-specific proteases upstream
of this domain. For example, proteins expressed in pGEX-2T plasmids
may be cleaved with thrombin; those expressed in pGEX-3X may be
cleaved with factor Xa.
[0120] Alternatively, recombinant polypeptides of the invention are
expressed in Pichia pastoris, a methylotrophic yeast. Pichia is
capable of metabolizing methanol as the sole carbon source. The
first step in the metabolism of methanol is the oxidation of
methanol to formaldehyde by the enzyme, alcohol oxidase. Expression
of this enzyme, which is coded for by the AOX1 gene is induced by
methanol. The AOX1 promoter can be used for inducible polypeptide
expression or the GAP promoter for constitutive expression of a
gene of interest.
[0121] Once the recombinant polypeptide of the invention is
expressed, it is isolated, for example, using affinity
chromatography. In one example, an antibody (e.g., produced as
described herein) raised against a polypeptide may be attached to a
column and used to isolate the recombinant polypeptide. Lysis and
fractionation of polypeptide-harboring cells prior to affinity
chromatography may be performed by standard methods (see, e.g.,
Ausubel et al., supra). Alternatively, the polypeptide is isolated
using a sequence tag, such as a hexahistidine tag (SEQ ID NO: 4),
that binds to nickel column.
[0122] Once isolated, the recombinant protein can, if desired, be
further purified, e.g., by high performance liquid chromatography
(see, e.g., Fisher, Laboratory Techniques In Biochemistry and
Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
Polypeptides of the invention, particularly short peptide
fragments, can also be produced by chemical synthesis (e.g., by the
methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984
The Pierce Chemical Co., Rockford, Ill.). These general techniques
of polypeptide expression and purification can also be used to
produce and isolate useful peptide fragments or analogs (described
herein).
[0123] The isolated polypeptides or fragments are loaded into EVs
as described herein.
Antibody Delivery
[0124] Like other polypepties, antibodies can be delivered using
EVs derived from fibroblast-like cells or CAFs. Antibodies can be
made by any of the methods known in the art utilizing a polypeptide
interest, or immunogenic fragments thereof, as an immunogen. One
method of obtaining antibodies is to immunize suitable host animals
with an immunogen and to follow standard procedures for polyclonal
or monoclonal antibody production. The immunogen will facilitate
presentation of the immunogen on the cell surface. Immunization of
a suitable host can be carried out in a number of ways. Nucleic
acid sequences encoding a polypeptide of the invention or
immunogenic fragments thereof, can be provided to the host in a
delivery vehicle that is taken up by immune cells of the host. The
cells will in turn express the receptor on the cell surface
generating an immunogenic response in the host. Alternatively,
nucleic acid sequences encoding the polypeptide, or immunogenic
fragments thereof, can be expressed in cells in vitro, followed by
isolation of the polypeptide and administration of the polypeptide
to a suitable host in which antibodies are raised.
[0125] Alternatively, antibodies against the polypeptide may, if
desired, be derived from an antibody phage display library. A
bacteriophage is capable of infecting and reproducing within
bacteria, which can be engineered, when combined with human
antibody genes, to display human antibody proteins. Phage display
is the process by which the phage is made to `display` the human
antibody proteins on its surface. Genes from the human antibody
gene libraries are inserted into a population of phage. Each phage
carries the genes for a different antibody and thus displays a
different antibody on its surface.
[0126] Antibodies made by any method known in the art can then be
purified from the host. Antibody purification methods may include
salt precipitation (for example, with ammonium sulfate), ion
exchange chromatography (for example, on a cationic or anionic
exchange column run at neutral pH and eluted with step gradients of
increasing ionic strength), gel filtration chromatography
(including gel filtration HPLC), and chromatography on affinity
resins such as protein A, protein G, hydroxyapatite, and
anti-immunoglobulin.
[0127] Antibodies can be conveniently produced from hybridoma cells
engineered to express the antibody. Methods of making hybridomas
are well known in the art. The hybridoma cells can be cultured in a
suitable medium, and spent medium can be used as an antibody
source. Polynucleotides encoding the antibody of interest can in
turn be obtained from the hybridoma that produces the antibody, and
then the antibody may be produced synthetically or recombinantly
from these DNA sequences. For the production of large amounts of
antibody, it is generally more convenient to obtain an ascites
fluid. The method of raising ascites generally comprises injecting
hybridoma cells into an immunologically naive histocompatible or
immunotolerant mammal, especially a mouse. The mammal may be primed
for ascites production by prior administration of a suitable
composition (e.g., Pristane).
[0128] In particular embodiments, the EV comprises an antibody
against a tumor antigen (e.g., an antigen associated with breast
cancer tumor, pancreatic tumor, glioblastoma, melanoma, lung cancer
tumor, ovarian cancer tumor). In another embodiment, the antibody
comprises an antibody that targets a protein expressed in the blood
vessels supplying the tumor. In yet another embodiment, the
antibody targets a protein that functions in miRNA maturation,
checkpoint blocking, or that is histone specific.
Small Molecule Delivery
[0129] EVs derived from fibroblast-like cells are used to deliver
therapeutic or imaging agents. In one embodiment, the invention
provides an EV comprising, for example, N-fluorescein
phosphatidylethanolamine (N-F-PE), doxorubicin, or cisplatin. In
other embodiments, an EV described herein a conventional
chemotherapeutic agent including, but not limited to, alemtuzumab,
altretamine, aminoglutethimide, amsacrine, anastrozole,
azacitidine, bleomycin, bicalutamide, busulfan, capecitabine,
carboplatin, carmustine, celecoxib, chlorambucil,
2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,
cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin,
docetaxel, doxorubicin, epirubicin, estramustine phosphate,
etodolac, etoposide, exemestane, floxuridine, fludarabine,
5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab,
goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide,
imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine,
mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed,
rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide,
teniposide, 6-thioguanine, topotecan, toremofine, trastuzumab,
vinblastine, vincristine, vindesine, and vinorelbine.
[0130] In particular embodiments, the EV comprises sirolimus,
evirolimus, lapatinib, or olaparib.
Delivery of Imaging Agents
[0131] EVs comprising a detectable agent are useful for imaging
studies. The invention provides an EV comprising any one of the
following exemplary small molecules useful in imaging:
carbocyanine, indocarbocyanine, oxacarbocyanine, thuicarbocyanine
and merocyanine, polymethine, coumarine, rhodamine, xanthene,
fluorescein, borondipyrromethane (BODIPY), Cy5, Cy5.5, Cy7,
VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660,
AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677,
Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor
647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS,
IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
[0132] In other embodiments, the EV comprises a nanoparticle useful
in imaging studies. In one embodiment, nanoparticles are
synthesized using a biodegradable shell known in the art. In one
embodiment, a polymer, such as poly (lactic-acid) (PLA) or poly
(lactic-co-glycolic acid) (PLGA) is used. Such polymers are
biocompatible and biodegradable, and are subject to modifications
that desirably increase the circulation lifetime of the
nanoparticle. In one embodiment, nanoparticles are modified with
polyethylene glycol (PEG), which increases the half-life and
stability of the particles in circulation (Gref et al., Science
263(5153): 1600-1603, 1994).
[0133] Biocompatible polymers useful in the composition and methods
of the invention include, but are not limited to, polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and copolymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetage
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate),
poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate, poly vinyl chloride polystyrene, polyvinylpryrrolidone,
polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,
polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),
poly(ethyl methacrylates), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecl acrylate) and combinations
of any of these. In one embodiment, the nanoparticles of the
invention include PEG-PLGA polymers.
[0134] In response to the growing need for encapsulation materials,
several different routes to producing hollow polymeric capsules are
available. In one example, the shell is composed of dendrimers
(Zhao, M., et al. J. Am. Chem. Soc. (1998) 120:4877). A dendrimer
is an artificially manufactured or synthesized large molecule
comprised of many smaller ones linked together--built up from
branched units called monomers. Technically, dendrimers are a
unique class of a polymer, about the size of an average protein,
with a compact, tree-like molecular structure, which provides a
high degree of surface functionality and versatility. Their shape
gives them vast amounts of surface area, making them useful
building blocks and carrier molecules at the nanoscale and they
come in a variety of forms, with different physical (including
optical, electrical and chemical) properties. In other embodiments,
the shell comprises block copolymers (Thurmond, K. B., II, et al.
J. Am. Chem. Soc. (1997) 119:6656; Macknight, W. J., et al., Acc.
Chem. Res. (1998) 31:781; Harada, A. and Kataoka, K. Science
(1999), 283:65), vesicles (Hotz, J. and Meier, W. Langmuir (1998)
14:1031; Discher, B. M., et al., Science (1999) 284:1143),
hydrogels (Kataoka, K. et al. J. Am. Chem. Soc. (1998) 120:12694)
and template-synthesized microtubules (Martin, C. R. and
Parthasarathy, R. V. Adv. Mater. (1995) 7:487) that are capable of
encapsuling a photosensitizer.
[0135] In another embodiment, EV of the invention comprises an
isotopic label for positron or scintillation or SPECT imaging.
[0136] In another embodiment, an EV of the invention comprises a
magnetic nanoparticle that has a high magnetic moment to enhance
the selectivity of the nanoparticle for detection. In another
embodiment, a magnetic nanoparticle includes a magnetic core and a
biocompatible outer shell, in which the outer shell both protects
the core from oxidation and enhances magnetic properties of the
nanoparticle. The enhanced magnetic properties can include
increased magnetization and reduced coercivity of the magnetic
core, allowing for highly sensitive detection as well as diminished
non-specific aggregation of nanoparticles. By forming biocompatible
nanoparticles having enhanced magnetic properties, detection of
specific target proteins and cells is provided. In one embodiment,
a nanoparticle core is formed from ferromagnetic materials that are
crystalline, poly-crystalline, or amorphous in structure. For
example, the nanoparticle core can include materials such as, but
not limited to, Fe, Co, Ni, FeOFe.sub.2O.sub.3, NiOFe.sub.2O.sub.3,
CuOFe.sub.2O.sub.3, Fe.sub.2O.sub.3, MgOFe.sub.2O.sub.3, MnBi,
MnSb, MnOFe.sub.2O.sub.3, Y3Fe.sub.5Oi.sub.2, CrO.sub.2, MnAs,
SmCo, FePt, or combinations thereof.
[0137] In another embodiment, the outer shell of the magnetic
nanoparticle partially or entirely surrounds the nanoparticle core.
In some implementations, the shell is formed from a
superparamagnetic material that is crystalline, poly-crystalline,
or amorphous in structure. In some cases, the material used to form
the shell is biocompatible, i.e., the shell material elicits little
or no adverse biological/immune response in a given organism and/or
is nontoxic to cells and organs. Exemplary materials that can be
used for the shell include, but are not limited to, metal oxides,
e.g., ferrite (Fe.sub.3C''4), FeO, Fe203, CoFe.sub.204,
NiFe.sub.204, ZnMnFe.sub.204, or combinations thereof.
[0138] Methods of making and delivering nanoparticles are known in
the art and described, for example, in the following US Patent
Publications: 20150258222, 20140303022, 20130309170, and
20130195767.
Extracellular Vesicle Isolation, Loading, and Targeting
[0139] EVs defined herein are generated as described herein below.
In general, the EVs are released by cells (e.g., CAFs,
fibroblast-like cells) into the extracellular environment. In vivo,
EVs are isolated from a variety of biological fluids, including but
not limited to, blood, plasma, serum, urine, stool, semen,
cerebrospinal fluid, prostate fluid, lymphatic drainage, bile
fluid, and pancreatic secretions. The EVs are then separated using
routine methods known in the art. In one embodiment, EVs are
isolated from the supernatants of cultured cells using differential
ultracentrifugation. In another embodiment, EVs are separated from
nonmembranous particles, using their relatively low buoyant density
(Raposo et al., 1996; Escola et al., 1998; van Niel et al., 2003;
Wubbolts et al., 2003). Kits for such isolation are commercially
available, for example, from Qiagen, InVitrogen and SBI.
[0140] Methods for loading EVs with agent are known in the art and
include lipofection, electroporation, as well as any standard
transfection method.
[0141] In one embodiment, the EVs comprising a polynucleotide or
polypeptide or small molecule of interest are obtained by
over-expressing the polynucleotide or polypeptide or loading the
cells with the small molecule in culture and subsequently isolating
indirectly modified EVs from the cultured cells. In another
embodiment, EVs comprising a polynucleotide or polypeptide or small
molecule of interest are generated by loading previously purified
EVs with the molecule(s) of interest into/onto the EVs by
electroporation (polynucleotide or polypeptide), covalent or
non-covalent coupling to the EV surface (polynucleotide or
polypeptide or small molecule) or simple co-incubation
(polynucleotide or polypeptide or small molecule).
[0142] In general, the physical properties of EVs of the invention
are sufficient to target the EV to a cancer cell of interest (e.g.,
breast cancer tumor, pancreatic tumor, glioblastoma, melanoma, lung
cancer tumor, ovarian cancer tumor). Nevertheless, in particular
embodiments, it may be useful to derivatize the EV with an antibody
that selectively binds to a tumor antigen. Targeted EVs may be
loaded with an agent that is particularly effective against the
targeted cancer cell. Exemplary target factors and agents are
provided in Table 1 (below).
TABLE-US-00004 Target Clinical factor Expression cell Function Drug
Mechanism Trial VEGF tumor cells CAFs, TAMs. Angiogenesis
Bevacizumab Neutralization VEGF Phase II Adsflt Interception of
VEGF Preclinical IMC-1C11 anti-VEGFR-2 antibody Phase I RPI.4610
anti-VEGFR-1 ribozyme Phase II Tenascin-C CAFs, cancer cells cell
adhesion 81C6 radioimmunotherapy Phase II ATN-RNA siRNA Phase I FAP
CAFs, TECs, cancer cells Serine protease PT-100 activity inhibitor
Phase I Sibrotuzumab anti-FAP antibody Phase I Sc40-FasL induce
apoptosis of FAP.sup.+ preclinical cells Rebimastat activity
inhibitor Phase III CTGF CAFs, TECs, cancer cell, Growth factor
FG-3019 anti-CTGF antibody preclinical neural DN-9693 degrade mRNA
preclinical MMPs CAFs, TECs, TAMs, metalloproteinases Marimastat
activity inhibitor Phase III cancer cells Tanomastat activity
inhibitor Phase III Rebimastat activity inhibitor Phase III uPA
CAFs, TAMs, cancer cells Serine protease PAI-2 activity inhibitor
preclinical uPA-UT1 activity inhibitor preclinical CA IX CAFs,
cancer cells Carbonic anhydrase Rencarex WX-G250 induce ADCC Phase
III
Pharmaceutical Compositions
[0143] The invention provides EVs for the delivery of therapeutic
compositions that specifically deliver an agent (e.g.,
polynucleotide, polypeptide, or small molecule for the treatment of
disease. In one embodiment, the present invention provides a
pharmaceutical composition comprising an EV derived from a CAF or
stromal cell. EVs of the invention may be administered as part of a
pharmaceutical composition. In general, EVs are provided in a
physiologically balanced saline solution. The solution comprising
the EVs is stored at room temperature for up to about 24 hours, for
longer than twenty four hours such solutions can be stored at about
four degrees Celsius for days, weeks, or months. EVs are frozen for
long term storage up to 10 years. The compositions should be
sterile and contain a therapeutically effective amount of the EV in
a unit of weight or volume suitable for administration to a
subject.
[0144] EVs of the invention may be administered within a
pharmaceutically-acceptable diluent, carrier, or excipient, in unit
dosage form. Conventional pharmaceutical practice may be employed
to provide suitable formulations or compositions to administer the
compounds to patients suffering from a disease (e.g., cancer).
Administration may begin before the patient is symptomatic. Any
appropriate route of administration may be employed, for example,
administration may be parenteral, intravenous, intraarterial,
subcutaneous, intratumoral, intramuscular, intracranial,
intraorbital, ophthalmic, intraventricular, intrahepatic,
intracapsular, intrathecal, intracisternal, intraperitoneal,
intranasal, aerosol, suppository, or oral administration. For
example, therapeutic formulations may be in the form of liquid
solutions or suspensions; for oral administration, formulations may
be in the form of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, or aerosols.
[0145] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" Ed. A. R. Gennaro, Lippincourt Williams & Wilkins,
Philadelphia, Pa., 2000. Formulations for parenteral administration
may, for example, contain excipients, sterile water, or saline,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or hydrogenated napthalenes. Biocompatible, biodegradable
lactide polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for microRNA molecules include ethylene-vinyl
acetate copolymer particles, osmotic pumps, implantable infusion
systems, and liposomes. Formulations for inhalation may contain
excipients, for example, lactose, or may be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
[0146] The formulations can be administered to human patients in
therapeutically effective amounts (e.g., amounts which prevent,
eliminate, or reduce a pathological condition) to provide therapy
for a disease or condition. The preferred dosage of an EV of the
invention is likely to depend on such variables as the type and
extent of the disorder, the overall health status of the particular
patient, the formulation of the compound excipients, and its route
of administration.
[0147] With respect to a subject having a neoplastic disease or
disorder, an effective amount is sufficient to stabilize, slow, or
reduce the proliferation of the neoplasm. Generally, doses of
active polynucleotide compositions of the present invention would
be from about 0.01 mg/kg per day to about 1000 mg/kg per day. It is
expected that doses ranging from about 50 to about 2000 mg/kg will
be suitable. Lower doses will result from certain forms of
administration, such as intravenous administration. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of EVs
[0148] A variety of administration routes are available. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Other modes of administration include oral, rectal, topical,
intraocular, buccal, intravaginal, intracisternal,
intracerebroventricular, intratracheal, nasal, transdermal,
within/on implants, e.g., fibers such as collagen, osmotic pumps,
or grafts comprising appropriately transformed cells, etc., or
parenteral routes.
Therapy
[0149] Results provided herein below show that conditioned media
from cancer cells can be used to alter a fibroblast-like cell's
gene expression and physiology to promote cancer growth. These
cancer-promoting changes in fibroblast-like cells include changes
in the exosomes/EVs (and other signals) that they deliver to
neighboring cancer cells. The invention provides methods for using
a fibroblast-like cell-derived EVs to reverse these changes and
inhibit cancer cell growth in vitro and in vivo. In one embodiment
of the present invention, fibroblast-like cell-derived exosomes/EVs
can be engineered to deliver anti-neoplastic, therapeutic miRs in
vivo. In this embodiment, the fibroblast-like cells represent both
localized cells of endothelial origin, localized tissue
pleuripotential stem cells which develop fibroblast phenotypes or
endogenous stem cells of bone marrow origin which have migrated to
the site of tumor.
[0150] Yet another embodiment of the present invention is a cancer
therapy that interrupts the support that stroma provides to cancer
cells, in the context of CCA, in the context of breast cancer, and
more broadly with potential to all cancers. Although there are
therapeutic strategies to kill cancer cells (from conventional
chemotherapy to targeted molecular therapies), there are currently
no FDA-approved therapies to interrupt the support that stroma
provides to cancer cells. Another embodiment of the present
invention utilizes EV-mediated miR transfer from stromal cells to
cancer cells to create a therapeutic with anti-neoplastic and
survival-extending properties in vivo.
[0151] Other embodiments of the present invention target other
cancers, including breast cancer, as well as cancers with
pronounced fibrosis. Some of the most aggressive cancers, such as
pancreatic, breast, and hepatocellular carcinoma, develop a close
symbiotic relationship with fibroblast-like cells, and we have
shown that this relationship has strong supporting effects on both
CCA and breast cancer cells
[0152] Therapy may be provided wherever cancer or other disease
therapy is performed: at home, the doctor's office, a clinic, a
hospital's outpatient department, or a hospital. Treatment
generally begins at a hospital so that the doctor can observe the
therapy's effects closely and make any adjustments that are needed.
The duration of the therapy depends on the kind of cancer being
treated, the age and condition of the patient, the stage and type
of the patient's disease, and how the patient's body responds to
the treatment. Drug administration may be performed at different
intervals (e.g., daily, weekly, or monthly). Therapy may be given
in on-and-off cycles that include rest periods so that the
patient's body has a chance to build healthy new cells and regain
its strength.
[0153] Depending on the type of disease and its stage of
development, the therapy can be used to slow the spreading of the
cancer, to slow the cancer's growth, to kill or arrest cancer cells
that may have spread to other parts of the body from the original
tumor, to relieve symptoms caused by the cancer, or to prevent
cancer in the first place. As described above, if desired,
treatment with an agent of the invention may be combined with
conventional therapies, including therapies for the treatment of
proliferative disease (e.g., radiotherapy, surgery, or
chemotherapy). For any of the methods of application described
above, an EV of the invention is desirably administered
intravenously or is applied to the site of neoplasia (e.g., by
injection).
[0154] In particular embodiments, EVs can be used to deliver
therapeutic miRs in vivo, without obvious involvement of normal
liver cells nor development of a cellular inflammatory reaction.
Furthermore, the specific finding is that CAF derived EV-based
therapy utilizing miRs delivered by EVs in particular embodiments
herein targets the cancer-stroma niche interactions, an important
property of cancers that is not currently addressed by prior art
nor any FDA-approved agents. EVs contribute to CAF-mediated support
of CCA, and that miR-loaded, fibroblast-derived EVs can slow the
growth of CCA and prolong survival in vivo. One embodiment of the
present invention is a therapeutic with anti-proliferation,
anti-spread and with survival-extending properties in vivo.
Kits
[0155] Kits of the invention include EVs comprising an agent
formulated for delivery to a cell in vitro or in vivo. Optionally,
the kit includes directions for delivering the EV to a subject. In
other embodiments, the kit comprises a sterile container which
contains the EV; such containers can be boxes, ampules, bottles,
vials, tubes, bags, pouches, blister-packs, or other suitable
container form known in the art. Such containers can be made of
plastic, glass, laminated paper, metal foil, or other materials
suitable for holding nucleic acids. The instructions will generally
include information about the use of the EV. In other embodiments,
the instructions include at least one of the following: description
of the EV; methods for using the enclosed materials for the
treatment of a disease, including a cancer; precautions; warnings;
indications; clinical or research studies; and/or references. The
instructions may be printed directly on the container (when
present), or as a label applied to the container, or as a separate
sheet, pamphlet, card, or folder supplied in or with the
container.
Trangenic Animals
[0156] In another aspect, the EVs of the present invention can be
used to create animal models (including large animals such as
swine, canine, primate and the like) of particular diseases
including, but not limited to, cancer. For example, the EVs can be
manipulated to contain genetic material comprising a transposon
system (e.g., sleeping beauty) encoding an oncogene. In another
embodiment, genetic material comprises a plasmid encoding an
oncogene. In a further embodiment, the genetic material comprises a
viral vector encoding an oncogene. The oncogene can include, but is
not limited to, one or more of c-Myc, K-Ras, N-Ras, c-Met, AKT,
P53, P16, CTNNB1, AXIN1, AXIN2, TP53, PIK3CA, PTEN, MET.
[0157] Another embodiment of the present invention utilizes one or
more of the therapies described in the present patent application
in conjunction with one or more cancer therapies, such as surgery,
chemotherapy, radiation, and targeted molecular therapies.
[0158] Without further elaboration, it is believed that one skilled
in the art, using the preceding description, can utilize the
present invention to the fullest extent. The following examples are
illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
EXAMPLES
[0159] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods described and claimed herein are made and evaluated,
and are intended to be purely illustrative and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for herein. Unless indicated otherwise, parts
are parts by weight, temperature is in degrees Celsius or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, desired solvents, solvent mixtures,
temperatures, pressures and other reaction ranges and conditions
that can be used to optimize the product purity and yield obtained
from the described process. Only reasonable and routine
experimentation will be required to optimize such process
conditions.
[0160] Cholangiocarcinoma (CCA), pancreatic cancer, and other
cancers induce a strong desmoplastic reaction that includes
intimate contact between cancer cells and tissue fibroblasts.
[0161] The vast majority of hepatocellular cancers (HCC) arise in a
fibrotic liver. This fibrotic response was long assumed to have
antineoplastic effects, but recent studies support a new paradigm
in which cancer-associated fibroblasts (CAFs) play a supporting
role in cancer growth and metastasis (Mueller M M, Fusenig N E.
Friends or foes--bipolar effects of the tumor stroma in cancer. Nat
Rev Cancer 2004; 4:839-49). For example, removing CAFs inhibits CCA
growth, while CAF-derived PDGF, Periostin, Tenascin-C,
Thrombospondin-I, and Galectin-1 are known to promote tumor growth
(Sirica A E, Dumur C I, Campbell D J, et al. Intrahepatic
cholangiocarcinoma progression: prognostic factors and basic
mechanisms. Clin Gastroenterol Hepatol 2009; 7:S68-78; Kawahara N,
Ono M, Taguchi K, et al Enhanced expression of thrombospondin-1 and
hypovascularity in human cholangiocarcinoma. Hepatology 1998;
28:1512-7; Shimonishi T, Miyazaki K, Kono N, et al. Expression of
endogenous galectin-1 and galectin-3 in intrahepatic
cholangiocarcinoma. Hum Pathol 2001; 32:302-10). These observations
fit within a broader paradigm in which cancer cells prime stroma to
support cancer growth and metastasis (Wan L, Pantel K, Kang Y.
Tumor metastasis: moving new biological insights into the clinic.
Nat Med 2013; 19:1450-64; Hood J L, San R S, Wickline S A. Exosomes
released by melanoma cells prepare sentinel lymph nodes for tumor
metastasis. Cancer Res 2011; 71:3792-801) in a bidirectional
interplay of signaling reactions (Roccaro A M, Sacco A, Maiso P, et
al. BM mesenchymal stromal cell-derived exosomes facilitate
multiple myeloma progression. J Clin Invest 2013; 123:1542-55).
Example 1: Co-Culturing Fibroblasts and Cancer Cells Leads to
Down-Regulation of Specific miRs within Fibroblasts
[0162] The hypothesis that cancer cells derive support from their
stroma is based on a number of observations, one of which is the
fact that cancer cells alter the physiology and gene expression
patterns of their surrounding cells. In the case of CCA and other
gastrointestinal tumors, tissue fibroblasts and their extracellular
matrix (ECM) are major components of the tumor stroma. To determine
if CCA cancer cells affect fibroblast gene expression patterns, we
generated fluorescent CCA cell lines by infecting them with
MSCV-IRES-EGFP (MIEG3), a retrovirus that expresses enhanced-GFP
(EGFP) (Olaru A V, Ghiaur G, Yamanaka S, et al. A microRNA
downregulated in human cholangiocarcinoma controls cell cycle
through multiple targets involved in the G1/S checkpoint.
Hepatology 2011). These cells were then co-cultured with LX2 cells
(FIG. 1) for 14 days to mimic in vitro the close interactions
between CAFs and cancer cells that occur in vivo. The LX2 cells
were then separated from the fluorescent cancer cells by FACS,
lysed, followed by RNA extraction and qRT-PCR analysis to identify
changes in fibroblast miR abundance induced by CCA cancer
cells.
[0163] We identified significant decreases in multiple fibroblast
miRs, including miR-195, miR-192 and miR-126 (FIG. 2). These miRs
represent candidate genes involved in the transition from normal
fibroblasts to CAFs. We initially focused on the possible role of
miR-195 in this process, since earlier studies of fibroblast
activation had demonstrated that miR-195 is repressed during the
differentiation of quiescent fibroblasts to activated,
collagen-producing fibroblasts (Maubach G, Lim M C, Chen J, et al.
miR studies in in vitro and in vivo activated hepatic stellate
cells. World J Gastroenterol; 17:2748-73; Lakner A M, Steuerwald N
M, Walling T L, et al. Inhibitory effects of microRNA 19b in
hepatic stellate cell-mediated fibrogenesis. Hepatology; 56:300-10;
Chen C, Wu C Q, Zhang Z Q, et al. Loss of expression of miR-335 is
implicated in hepatic stellate cell migration and activation. Exp.
Cell Res.; 317:1714-25).
Example 2: Up-Regulation of miR-195 within Fibroblasts is
Sufficient to Inhibit Cancer Cell Invasiveness
[0164] In vitro, CCA cells display significantly higher invasion in
a matrigel assay when they are co-cultured with LX2 cells. To
determine whether restoring miR-195 expression in LX2 cells had any
effect on the invasiveness of co-cultured CCA cells, we generated
an LX2 fibroblast cell line (LX2-195) that had restored expression
of miR-195, and asked if this had any effect on the behavior of CCA
cells in co-culture. Specifically, we co-cultured LX2-195 cells and
control LX2 cells (expressing a non-specific inhibitor miR mimic
(NSM)) with four different cancer cell lines: HuCCT1, SG231,
BDENeu, and BDESp, all of which are intrahepatic CCA cell lines,
and then assayed them for cancer cell invasiveness by staining the
matrigel with Crystal violet and counting the number of invading
cells (neither LX2 control nor LX2-195 cells invade the matrigel).
Co-culturing cancer cells with LX2-195 cells resulted in a
significant reduction in the invasiveness of all four cancer cell
lines examined (FIG. 3). The most parsimonious interpretation of
these results is that reversing the reduction of a single miR
species in fibroblasts, miR-195, was sufficient to inhibit the
invasion of co-cultured cancer cells.
Example 3: LX2-195 Cells Release a Diffusible Factor that Inhibits
Cancer Cell Invasion, Migration, and Growth
[0165] The inhibition of cancer cell invasiveness by co-culture
with LX2-195 cells (demonstrated above) could be mediated by either
direct cell-cell contact and/or by diffusible factors released from
LX2-195 cells. Given that diffusible factors have the potential for
future therapeutic development, we tested whether LX2-195 cells
impacted cancer cell phenotypes in the absence of direct cell-cell
contact. In brief, cancer cells and fibroblasts were grown on
opposite sides of a transwell apparatus with .about.400 nm dia.
pores for a period of 5 days (FIG. 4). The cancer cells were then
removed and assayed for invasiveness, migration, and growth. Cancer
cells exposed to diffusible factors released from LX2-195 cells
displayed significant reductions in invasion, migration, and
growth, as compared to cancer cells that had been co-cultured with
control LX2 cells, which have much lower levels of miR-195.
Example 4: LX2-195 Cells Release a Diffusible Factor that Causes
Up-Regulation of miR-195 in Neighboring Cancer Cells
[0166] We next tested whether the soluble factors released by
LX2-195 cells affected the levels of miR-195 in neighboring cancer
cells. Using the transwell assay, cancer cells were exposed to
conditioned media from control LX2 or LX2-195 cells. Cancer cells
were then purified away from the LX2 cells by FACS, and RNA was
extracted from the cancer cells and processed for qRT-PCR to
determine the levels of miR-195 and controls. We found that the
level of miR-195 in cancer cells was significantly up-regulated
following exposure to diffusible factors released by LX2-195 cells
(FIG. 5).
Example 5: Fibroblast-Like Cells Secrete miR-195 in Extracellular
Vesicles (EVs)
[0167] To explore the possibility that EVs might contribute to the
CAF-cancer interactions outlined in the previous experiments, we
asked whether fibroblast-like cells release miR-195 in EVs, and
whether the levels of vesicle-associated miR-195 were higher in
vesicles released by LX2-195 cells. EVs were collected from the
supernatant of control LX2 cells and of LX2-195 cell cultures,
followed by RNA extraction and qRT-PCR to determine the relative
abundance of miR-195 in the two EV preparations. We observed that
control LX2 cells (expressing a non-specific mimic (NSM)) secrete
EVs that contain detectable levels of miR-195. However, the levels
of miR-195 were >60-fold higher in the in EVs produced by
LX2-195 cells (FIG. 6).
Example 6: Fibroblast-Like Cell-Derived Extracellular Vesicles
(EVs) are Selectively Targeted to Tumor Cells In Vivo
[0168] The possibility that EVs might contribute to the CAF-cancer
signaling observed above, led us to ask whether fibroblast-like
cell-derived EVs might be targeted to tumor cells in vivo. To this
end, we generated LX2 cells that constitutively express
TSG101/mCherry fusion protein (TSG101 is secreted from the cell in
EVs (Raposo G, Stoorvogel W. Extracellular vesicles: exosomes,
microvesicles, and friends. J Cell Biol 2013; 200:373-83), which
allowed us to selectively detect these fibroblasts-derived EVs).
EVs from the resulting cell line, LX2-TSG101/mCherry, were
collected from the supernatant by standard procedures. The
purified, TSG101/mCherry-labeled, fibroblast-derived EVs were then
injected into the tail vein of rats, which had been injected with
BDEneu tumor cells 24 days earlier and thus had already developed
CCA in their liver (we have extensive experience with this model of
CCA; see Sirica A E, Zhang Z, Lai G H, et al. A novel
"patient-like" model of cholangiocarcinoma progression based on
bile duct inoculation of tumorigenic rat cholangiocyte cell lines.
Hepatology 2008; 47:1178-90). 24 hours after injection, we
sacrificed the rats, removed their livers, lungs, and kidneys,
generated slides of these tissues, and processed them for
immunofluorescence microscopy using antibodies specific for
alpha-Smooth Muscle Actin (stains activated, collagen-producing
fibroblasts) and mCherry to detect the fibroblast-derived,
TSG101-mCherry-containing exosomes/EVs. These experiments revealed
that the fibroblast-derived vesicles were highly enriched in
"pockets" of cancer cells within the fibrotic CCA mass in the liver
(FIG. 7). We were unable to detect significant staining for
TSG101/mCherry in non-cancerous areas of the liver, the lung, or
the kidney. These experiments can be carried out using
EGFP-containing EVs and tdTomato-expressing cancer cells, and the
tissue sections are processed by immunogold label electron
microscopy.
Example 7: Fibroblast-Like Cell-Derived Extracellular Vesicles
(EVs) can Selectively Deliver Heterologous Proteins to Tumor Cells
In Vivo
[0169] EVs from the cell line, LX2-TSG101/mCherry, were collected
from the supernatant by standard procedures. The purified,
TSG101/mCherry-containing, fibroblast-derived EVs were then
injected into the tail vein of rats, which had been injected with
BDEneu tumor cells 24 days earlier and thus had already developed
CCA in their liver (we have extensive experience with this model of
CCA; see Sirica A E, Zhang Z, Lai G H, et al. A novel
"patient-like" model of cholangiocarcinoma progression based on
bile duct inoculation of tumorigenic rat cholangiocyte cell lines.
Hepatology 2008; 47:1178-90). 24 hours after injection, we
sacrificed the rats, removed their livers, lungs, and kidneys,
generated slides of these tissues, and processed them for
immunofluorescence microscopy using antibodies specific for
alpha-Smooth Muscle Actin (stains activated, collagen-producing
fibroblasts) and mCherry to detect the fibroblast-derived,
TSG101-mCherry-containing exosomes/EVs. These experiments revealed
that the fibroblast-derived vesicles were highly enriched in
"pockets" of cancer cells within the fibrotic CCA mass in the liver
(FIG. 7). We were unable to detect significant staining for
TSG101/mCherry in non-cancerous areas of the liver, the lung, or
the kidney (data not shown). This point is also established by our
demonstration that human mammary fibroblast-derived EVs containing
an exosomal form of GFP were taken up by human breast cancer cells
(FIGS. 16A-16D).
Example 8: Fibroblast-Like Cell-Derived Extracellular Vesicles
(EVs) can Selectively Deliver DNA to Tumor Cells In Vivo
[0170] To further investigate the ability of EVs to selectively
deliver cargo molecules to cancer cells, we generated BDEneu cells
carrying a Cre-reporter gene, CAG-loxP-tdTomato-loxp-EGFP. These
cells display bright red fluorescence due to the expression of
tdTomato, (seen as white or light signal emitting cells on black
and white drawings using respective optical filters) as but switch
from red to green fluorescence following the expression of Cre,
which removes the tdTomato gene and places the promoter proximal to
the EGFP gene (seen as white or light signal emitting cells on
black and white drawings using respective optical filters). These
cells were injected into rats, and after tumors were established
the rats were treated with a single set of tail vein injections
with EVs that had been loaded with a plasmid DNA designed to
express Cre recombinase in mammalian cells. As shown in FIG. 8, a
significant number of the Cre-reporter CCA cells switched from red
to green fluorescence, demonstrating that fibroblast-like
cell-derived EVs can deliver DNA to CCA cells in vivo. The fact
that some cancer cells retained their original red fluorescence is
an outstanding control that points to the efficacy of this assay
system for optimizing the variables in the experiment, as well as
an internal control to ensure that the correct cells were used.
Animals that were not injected with plasmid-loaded EVs failed to
produce any green CCA cells. optimize our therapeutic
technology.
Example 9: Fibroblast-Derived, miR-195 Mimic-Loaded EVs Inhibit CCA
Growth In Vivo in a Rat Model
[0171] Taken together, these observations indicate that
fibroblast-derived EVs could be used to deliver therapeutic miRs to
CCA in vivo. To explore this hypothesis, we collected EVs from the
supernatant of LX2 cells, loaded them with a miR-195 mimic by
transfection using Lipofectamine RNAiMAX (Invitrogen), and
re-purified the loaded EVs by size exclusion chromatography. We
also generated control EVs, by transfecting EVs from same LX2 cells
with a non-specific miR mimic (NSM). qRT-PCR analysis indicated
that miR-195 transfection of vesicles resulted in levels of miR-195
that were 500,000 times higher than in the control EVs. To
determine whether these miR-195-loaded EVs might inhibit CCA growth
in vivo, we injected six rats with BDEneu cells, as previously
described (Sirica A E, Zhang Z, Lai G H, et al. A novel
"patient-like" model of cholangiocarcinoma progression based on
bile duct inoculation of tumorigenic rat cholangiocyte cell lines.
Hepatology 2008; 47:1178-90). 5 days later, the rats were injected
(via tail vein) with either miR-195 mimic-loaded or control
miR-loaded EVs (at equivalent numbers of EVs and equivalent dose of
miR mimic). Injections were repeated every other day until day 35,
at which time the animals were sacrificed and livers were excised.
Morphological examination revealed that the tumor size was reduced
in all three animals that had been injected with the miR-195
mimic-loaded EVs, relative to the three animals that had been
injected with the NSM-loaded EVs (FIGS. 9 and 10). Based on our
experience with this animal model, tumor growth in the
control-treated animals was similar to what occurs in untreated
animals (see Sirica A E, Zhang Z, Lai G H, et al. A novel
"patient-like" model of cholangiocarcinoma progression based on
bile duct inoculation of tumorigenic rat cholangiocyte cell lines.
Hepatology 2008; 47:1178-90).
Example 10: Treatment with miR-195 Loaded EVs Downregulates CDK6
and VEGF (Known Targets of miR-195) in Cancer Cells
[0172] To test whether miR-195 causes expected changes in gene
expression, we measured CDK6 and VEGF mRNA, two reported miR-195
targets (FIG. 11). Both mRNAs were downregulated in all
experimental paradigms, including (left panel) direct transfection
of BDEneu cells, (middle panel) exposure of cells to conditioned
medium of LX2-miR-195, and (right panel) cells incubated with
miR-195-loaded EVs.
Example 11: Treatment of Rats with CCA via Tail Vein with
EVs-miR-195 Increases their Survival Significantly
[0173] We treated 20 cancer-bearing rats with EVs-195 and with
EVs-NSM (control), respectively. The treatment was commenced post
cancer cell transplantation Day 15 (as to not interfere with the
implantation of tumor cells, nor with the early steps of cancer
development and growth). Treatment was continued till rats died due
to cancer. All experiments had been approved by the Hopkins IACUC.
As shown in FIG. 12, rats treated with EVs-195 displayed a
statistically significant, 50% increase in survival, providing
solid evidence that miR-loaded EVs can have a positive therapeutic
effect in vivo.
Example 12: To Identify the Optimal Parameters of miR-195 Loaded EV
Therapy in a Rat CCA Model
[0174] The purpose of the in vivo experiments was to assess if
intravenous treatment with EVs loaded with a miR species works in
treating CCA. As described herein, we will test several conditions
with the purpose of elucidating the rate-limiting factors playing a
role in the efficiency of this treatment.
[0175] Frequency of treatment: In the preliminary experiments, we
have treated rats via tail vein every other day. We now propose to
assess if less frequent treatment works equally well. We will do
this study on 18 rats, as follows: 6 rats will be the control group
(treated as in our preliminary experiments every other day), 6 rats
will be treated every 4 days and 6 rats will be treated every 7
days. After 30 days of treatment, all rats will be euthanized and
tumors measured.
[0176] Timing of treatment: In our preliminary experiments we
started treating rats 5 days after BDEneu cancer cell implantation
in rat livers. Although not very likely, it is possible that
miR-195 delivered by EVs affected tumor implantation in addition to
tumor growth. To elucidate this aspect, we will now allow the
tumors to develop for 2 weeks, then the treatment with miR-195-EVs
will commence. We chose 2 weeks because, from our previous
experiments, we know that these rats have tumors developed already
by week 2, and some of them die of cancer at week 4. We will
compare the treatment efficiency with the control arm from the
experiment above. For this experiment we will require 6 rats.
[0177] miR dose and EV dose: For preliminary experiments, for each
rat, we utilized 200 .mu.g miR mimic to transfect 200 .mu.g of EVs
(based on protein weight, ratio 1:1) before delivering in vivo.
However, we would like to test if a different miR dose or different
EV dose is more efficient in treating CCA or, if the efficiency is
maintained while decreasing the dose (with the purpose of cost
saving). First, we will perform an in vitro experiment to determine
the best miR to EV ratio. We propose to utilize miR to transfect
EVs in a weight ratio of 0.5:1, 1:1, 2:2 and 4:1. Next, we will
utilize these transfected EVs to treat HuCCT1 cells in vitro and
then determine by qRT-PCR the level of miR upregulation as a
function of miR quantity used to transfect EVs. We utilize a
concentration of miR mimic of 15 .mu.g per .mu.L. We measure the
amount of exosomes based on the weight of the protein content. We
usually extract approximately 30 .mu.g of exosomes from one 150 cm
cell culture dish. Next, we will utilize the weight ratio miR:EVs
that was determined in vitro for all following experiments. We will
then vary the amount of miR delivered per rat (with the associated
quantity of EVs). We will have 4 experimental arms: 50 .mu.g of miR
mimic per injection/rat, 100 .mu.g, 200 .mu.g and 400 .mu.g. Each
experimental arm will include 6 rats for a total of 24 rats. We
will keep all other parameters of the experiments constant as in
our preliminary experiments presented above (injection timing-5
days post-cancer implantation and injections every other day).
[0178] Kaplan-Meyer curves/survival: Once the optimal frequency,
timing and dose of the treatment is established, we will perform an
experiment to determine the survival of rats treated with
EV-miR-195. The control arm will include 12 rats treated with
EV-NSM (negative control, EVs transfected with control miR) and the
treatment arm will include 12 rats treated with EV-miR195. We will
record the date of death and therefore be able to perform
Kaplan-Meyer curves. These experiments will offer valuable
information from a clinical perspective, as the size of the tumor
is important, however, survival is also of utmost importance. While
we have already demonstrated that EV injections can prolong
survival (FIG. 11), survival studies will remain a key end-point as
we strive to develop an effective anti-cancer therapy based on
miR-loaded EVs.
Example 13: To Characterize the CCA Phenotype Induced by EVs Loaded
with miR-126, and -192, Respectively
[0179] miR-126 and -192 were among the top 3 candidate miRs in our
screen. In fact, miR-126 and -192 were more strongly depleted in
response to CCA cells than miR-195. We will pursue the same sets of
experiments on miR-126 and miR-192 that we have performed and
proposed for miR-195. For example, we will generate LX2 cells that
express miR-126, and cells that express miR-192, and compare the
effect that these cells have on the neoplastic properties of
co-cultured CCA cells, relative to control LX2 cells expressing a
non-specific mimic (NSM)), both in direct co-culture assays and in
transwell co-culture assays. In fact, we have already generated a
LX2-126 line, and our initial experiments indicates that LX2-126
cells inhibit CCA invasiveness (FIG. 13). FIG. 13 shows LX2 cells
expressing miR-126 inhibit CCA invasiveness in vitro. HuCCT1 cells
were co-cultured directly with LX2 cells expressing either (upper
image) a control miR, or (lower image) miR-126. Invasiveness of
HuCCT1 cells was decreased 3.2 fold when co-cultured with LX2-126
cells.
[0180] In addition, the LX2-126 cells appeared to inhibit CCA
migration in a scratch assay test (FIG. 14). FIG. 14 shows LX2
cells expressing miR-126 inhibit CCA migration 4-fold in vitro.
HuCCT1 cells were co-cultured directly with LX2 cells expressing
either a controls miR or miR-126. Migration was measured in a
scratch assay.
[0181] As we move forward with these studies, from cells to
miR-loaded EVs, and from in vitro experiments to in vivo
experiments, we will also incorporate similar controls as those
outlined previously, including characterization of miR-loaded EVs
(by immunoEm, differential centrifugation & immunoblot,
etc.).
[0182] As for the in vivo experiments, they will mirror those shown
and proposed for miR-195-loaded EVs. Specifically, we will inject
miR-loaded EVs into the tail vein of rats that were previously
induced to develop iCCA. We will next determine differences in size
of tumors in the treatment vs. control arm. We will also perform
experiments to determine the optimal dose of EVs, miR loaded into
EVs, the duration of treatment, frequency of injections and finally
derive Kaplan-Meyer curves to indicate if there is a change in
survival of rats treated with EV-miRs by tail vein. To determine
the molecular & cellular impact of treatment with EV-loaded
miR-126 and -192, we will perform experiments as outlined under Aim
1a. In brief, we will collect CCA cells (in vivo experiments and in
vitro culture), extract RNA, measure the levels of known miR-126
and miR-192 targets, perform RNA-seq, followed by pathway analyses,
and follow-on experiments to identify the mechanisms by which
injected EVs inhibit cancer growth (if they do).
Example 14: Delivery of a Small Molecule to Cancer Cells
[0183] MDA-MB-231 breast cancer cells (red) were incubated with EVs
obtained from human mammary fibroblast cells that had been
incubated previously with the exosomal lipid N-F-PE. These EVs were
taken up by the breast cancer cells, demonstrating that
fibroblast-like cell-derived EVs can be loaded with small molecules
and selectively deliver the small molecules to cancer cells.
[0184] The results reported herein above were carried out using the
following methods and materials.
Cell Culture
[0185] LX2 is a human liver stellate cell line derived to study
fibrogenesis. HuCCT1 was derived from a patient with a moderately
differentiated adenocarcinoma of the intrahepatic biliary tree
HuCCT1 was established from a patient with moderately
differentiated adenocarcinoma of the intrahepatic biliary tree.
SG231 is a cholangiocarcinoma cell line derived as described 41.
TFK1 is an extrahepatic CCA cell line. BDENeu and BDEsp are rat
intrahepatic CCA cell lines derived as described. RGF is a rat
portal fibroblast cell line established by Fausther et al 21, 22.
H69, a gift from Dr. Jefferson (Tufts University, Boston, Mass.),
are normal human intrahepatic cholangiocytes derived from a normal
liver prior to liver transplantation. All cell lines were
maintained and grown as described previously.
EVs Preparation and Characterization
[0186] EVs were separated via ultracentrifugation as described
before from LX2 cell culture medium that had been cultured for 72
hours with EV free FBS. Multi-parameter nanoparticle optical
analysis (Nanosight) and Transmission Electron Microscope (TEM)
were utilized to determine the shape, size and tracking the
brownian movement of EVs. Western blot for EV-specific proteins was
performed with anti-CD63 antibody (Santa Cruz Dallas, Tex.) and
anti-TSG-101 antibody (Abcam Cambridge, Mass.) as described
before.
miRNA Mimic Loading of EVs
[0187] Lipofectamine RNAiMAX (Life Technologies, NY) was used to
transfect miR mimic into EVs with an adjusted protocol according to
manufacturer's instruction. MiR-195 mimic and NSM were purchased
from Dharmacon GE Healthcare. Then free miR-195 mimics were
isolated with a micropartition system (Vivaspin 2, 50 kDa MWCO PES,
GE Healthcare, Laurel, Md.). The mixed suspension was added into
the filter and centrifuged at 1500 g for 5 min, the supernatant
collected and either placed on the top of cells for in vitro
experiments or used to inject into the CCA rat model in the in vivo
experiments.
RNA Extraction
[0188] RNA from EVs was extracted by a modified Trizol method while
spiking in cel-miR-39 during the lysis step. Cells were lysed with
Trizol reagent (Invitrogen, Carlsbad, Calif.) according to the
manufacture's protocol.
MicroRNA Real Time PCR Array
[0189] LX2 and HuCCT1 MIEG3-EGFP were seeded at same amounts in
flasks, then directly co-cultured for 14 days, EGFP-negative cells
were sorted and collected by FACS, followed by RNA extraction. 100
m RNA were used for RT-PCR array for co-cultured LX2 cells with LX2
cultured alone as control. Following analysis, select miRs that
were down-regulated in co-cultured LX2 cells were selected for
follow-up experiments.
Quantitative RT-PCR (qRT-PCR) for miRs Expression
[0190] qRT-PCR was performed to detect the miR-195 expression in
EVs, CCA cell lines, and CCA tumor mass cells. For miR-195
expression in EVs, we used cel-miR-39 as control, while for miR-195
expression in cells, RNU6B was used to normalize the data as
described before.
qRT-PCR for mRNA Expression
[0191] RNA was reversed transcribed according to the manufacture
protocol (Thermo Scientific), IQ SYBR Green Supermix (Bio-Rad,
Hercules, Calif.) was for used real time PCR amplification, GAPDH
was used to normalize mRNA expression level, and melting curve
analysis was used to confirm the PCR results.
Plasmids, Transfection and Lentivirus/Retrovirus Infection
[0192] Vectors were purchased from Addgene (Cambridge, Mass.).
Viral supernatants were produced by transfection of HEK-293T cells
with a packaging plasmid (pVSV-G). BDEneu cells were infected with
viral supernatant with Polybrene at a final concentration of 8
.mu.g/.mu.l. pCDH1-EF1-mCherry-TSG101-IRES-GFP was used to infecft
LX2 cells. GFP positive cells were sorted and used to isolate EVs
with mCherry on the surface. pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE
was used as above to produce viral particles, that were then used
to infect BDEneu cells. Puromycin was used to select for stably
infected cells. When a Cre-recombinase encoding plasmid is
detected, cells switch color from RFP to GFP after excision of the
loxp-dsRed-loxP element.
Conditioned Media Preparation
[0193] Confluent LX2-miR-195 mimic/NSM cells were incubated with
DMEM supplemented with penicillin/streptomycin, 0% FBS. Three days
later, the conditioned media was collected, filtered, and used
immediately.
Cell Invasion/Migration Assay
[0194] Cell invasion assays were performed using invasion chambers
(Cat #354480, Corning, Tewksbury, Mass.) while for cell migration
assays 8 .mu.m Transwell plates (Cat #3422, Corning, Tewksbury,
Mass.) were used. For both assays, DMEM with 10% FBS was placed in
the lower chamber as chemoattractant. For directed transfection of
CCA lines, 2 days after transfection, 3.times.10.sup.4 were seed
into invasion chamber. For co-culture invasion assay, either
3.times.10.sup.4 HuCCT1, BDEneu, SG231 or BDEsp were co-cultured
with 3.times.10.sup.4 LX2-NSM or LX2-miR-195 mimc cells or
RGF-NSM/miR-195 mimic at 37.degree. C. for 2 days, then
6.times.10.sup.4 cells were diluted in serum-free medium and placed
into the upper chambers. After 48 hours the
non-invading/non-migrating cells were removed from the membrane
upper surface with cotton swabs, and invaded/migrated cells on the
lower side of chamber were stained with crystal violet. Cells were
counted in 3 random fields at a magnification of 200.times..
Proliferation of CCA Cell Lines Co-Cultured with LX2/RGF miR-195
Mimic/NSM, and CCA Cell Lines Directly Transfected with miR-195
Mimic/NSM
[0195] LX2 or RGF cells transfected with miR-195 mimic or NSM were
cultured for 2 days, then washed and trypsinised, centrifuged and
washed 3 times with PBS. After cell counting, 5.times.10.sup.4 LX2
or RGF cells were seeded into the upper well of 12 well 0.4 .mu.m
transwell co-culture system (cat #3460, Corning, Tewksbury, Mass.).
For the bottom chamber, 1.times.10.sup.5 CCA cells (BDEneu, HuCCT1)
were seeded at the same time after trypinization and counting as
above. After 5 days, the CCA cell number was determined by
counting. For directly transfected BDEneu and HuCCT1 with miR-195
mimic or NSM, 1000 cells were seeded into 96 well plates, MTs Assay
(CellTiter 96 Aqueous One solution Cell Proliferation Assay Cat #
G3580, Promega Corporation, Sunnyvale, Calif.) was used to
determine the proliferation.
Animal Studies
[0196] Fischer 344 male (150-170 g) were purchased from Harlan
(Frederick, Md.) and housed in the animal facility at Johns Hopkins
University. All animal work was approved by and conducted in
accordance with the guidelines of the Institutional Animal Care and
Use Committee at the Johns Hopkins University. For the CCA rat
model Fischer 344 male rats were anesthetized and then inoculated
with 1.times.10.sup.6 BDEneu cells in 100 .mu.l HBSS injected into
the left liver lobe, followed by ligation of the common bile duct.
Rats were monitored daily until day 5 or day 15, when the treatment
with miR-loaded EVs started.
Treatment of CCA Rats with miR-195-Loaded EVs
[0197] Initially 6 CCA rats were randomized into 2 groups. Starting
at day 5, the rats received EV loaded with miR-195 mimic/NSM via
tail veil injections every 2 days. After 25 days, rats were
sacrificed, tumor weight and size were measured, and tissue
specimen frozen in O.C.T. compound. Tissue sections were stained
with primary antibodies and detected using Alexa Fluor
dye-conjugated secondary antibodies. Microphotographs were obtained
using a Zeiss laser scanning microscope (LSM 510). In addition,
single cell suspension from tumor masses were used to sort the
BDEneu cells with RFP fluorescence. MiR-195 levels in miR-195
treated rats and NSM treated controls were measured via RT-PCR.
Kaplan-Meyer Survival Curves
[0198] In a follow up animal in vivo experiment, 20 CCA rats were
randomized in a control group of 9 CCA rats treated with NSM and a
treatment group of 11 rats, treated with miR-195 mimic, both
starting from Day 15. Animals were monitored daily and the date of
death of each rat recorded and the data incorporated into a
Kaplan-Meyer curve. Data were analyzed with the log-rank
(Mantel-Cox) test.
Detection of the Location of EVs in CCA Mass of Liver
[0199] Isolated EVs from LX2-pCDH1-EF1-mCherry-TSG101-IRES-GFP
cells were injected into tail veins of CCA rats. After 24 hours the
rats were sacrificed and tissue specimen of the tumor mass were
frozen in O.C.T. compound. Tissue sections were stained with
primary antibodies against mCherry (cat #632496 Clontech, Mountain
View, Calif.) and alpha-SMA (cat # A2547, Sigma-Aldrich, St. Louis,
Mo.) to detect the injected EVs and to measure the degree of the
fibrotic change in the tumor mass. Furthermore, BDEneu cells
infected with pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE lentiviral
construct were injected into rat livers as described above to
generate the CCA model, and after 20 days, EVs transfected with Cre
plasmid were administered to the rats via tail veil injections. 4-6
days later, rats were sacrificed and tumor sections obtained as
described above. Cells that switched color from dsRed to EGFP
indicate BDEneu tumor cells that have taken up EVs loaded with
Cre-recombinase plasmid.
Proliferation and Apoptosis Measurement In Vivo
[0200] Tumor mass specimens were embedded in paraffin, sections
were stained with primary antibody Ki67 (cat #550609,BD San Jose,
Calif.), caspase 3 (cat #9661S, Cell Signaling Technology, Dancers,
Mass.), alpha-SMA and TUNEL in situ cell death fluorescein
(Sigma-Aldrich, St. Louis, Mo.) to determine the levels of
proliferation, apoptosis, as well as fibrotic infiltrate. Image J
was used to identify the florescence intensity.
OTHER EMBODIMENTS
[0201] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0202] The recitation of a listing of elements in any definition of
a variable herein includes definitions of that variable as any
single element or combination (or subcombination) of listed
elements. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0203] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1
1
5187DNAHomo sapiens 1agcttccctg gctctagcag cacagaaata ttggcacagg
gaagcgagtc tgccaatatt 60ggctgtgctg ctccaggcag ggtggtg 872110DNAHomo
sapiens 2gccgagaccg agtgcacagg gctctgacct atgaattgac agccagtgct
ctcgtctccc 60ctctggctgc caattccata ggtcacaggt atgttcgcct caatgccagc
110385DNAHomo sapiens 3cgctggcgac gggacattat tacttttggt acgcgctgtg
acacttcaaa ctcgtaccgt 60gagtaataat gcgccgtcca cggca
8546PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 4His His His His His His1 55111DNAHomo sapiens
5agacgccacg cctccgctgg cgacgggaca ttattacttt tggtacgcgc tgtgacactt
60caaactcgta ccgtgagtaa taatgcgccg tccacggcac cgcatcgaaa a 111
* * * * *