U.S. patent application number 15/515570 was filed with the patent office on 2017-10-19 for compositions and methods for treating hepatic fibrosis.
This patent application is currently assigned to Research Institute at Nationwide Children's Hospit al. The applicant listed for this patent is Research Institute at Nationwide Children's Hospital. Invention is credited to David Brigstock, Li Chen.
Application Number | 20170298352 15/515570 |
Document ID | / |
Family ID | 55631385 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170298352 |
Kind Code |
A1 |
Brigstock; David ; et
al. |
October 19, 2017 |
COMPOSITIONS AND METHODS FOR TREATING HEPATIC FIBROSIS
Abstract
This disclosure provides pharmaceutical compositions and
purified or isolated naturally occurring exosome products that have
therapeutic use for treating an unmet medical need. The exosome
compositions contain an effective amount of exosomes isolated from
a body fluid of a non-diseased subject. The compositions are useful
in the treatment of a variety of fibrotic diseases.
Inventors: |
Brigstock; David; (Dublin,
OH) ; Chen; Li; (Dublin, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Research Institute at Nationwide Children's Hospital |
Columbus |
OH |
US |
|
|
Assignee: |
Research Institute at Nationwide
Children's Hospit al
Columbus
OH
|
Family ID: |
55631385 |
Appl. No.: |
15/515570 |
Filed: |
September 29, 2015 |
PCT Filed: |
September 29, 2015 |
PCT NO: |
PCT/US15/53019 |
371 Date: |
March 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62057971 |
Sep 30, 2014 |
|
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/141 20130101; C12Q 2600/178 20130101; A61K 9/19 20130101;
C12N 2320/32 20130101; C12N 2330/10 20130101; A61K 31/00 20130101;
C12Q 1/68 20130101; C12Q 1/6883 20130101; C12N 2310/20 20170501;
A61K 45/06 20130101; A61K 31/7088 20130101; G01N 2800/52 20130101;
C12N 15/11 20130101; C12Q 2600/106 20130101; G01N 2800/7052
20130101 |
International
Class: |
C12N 15/113 20100101
C12N015/113; A61K 45/06 20060101 A61K045/06; A61K 9/19 20060101
A61K009/19; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
STATEMENT OF FEDERAL FUNDING
[0002] This invention was made with government support under Grant
No. 1R01AA021276-01 awarded by National Institute on Alcohol Abuse
and Alcoholism (NIAAA) of National Institutes of Health (NIH). The
government has certain rights in the invention.
Claims
1. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an effective amount of exosomes isolated
from a body fluid of a non-diseased subject, wherein optionally:
(a) the microRNA (miR) profile of the exosomes comprises one or
both of: lack of up-regulation of one or both of the markers
miR-26b and/or -122, as compared to the miR profile of a subject
that is suffering from a fibrotic or hepatic disease or an
associated disorder and/or lack of down-regulation of one or both
of miR-9 and/or -196b as compared to the miR profile of a subject
that is suffering from a fibrotic or hepatic disease or an
associated disorder; or (b) the microRNA (miR) profile of the
exosomes comprises lack of up-regulation of miR-26b and -122, and
lack of down-regulation of miR-9, and -196b, as compared to the miR
profile of a subject that is suffering from a fibrotic or hepatic
disease or an associated disorder; or (c) the microRNA (miR)
profile of the exosomes comprises lack of up-regulation or high
expression of one or more, two or more, three or more, five or
more, six or more, seven or more, eight or more, nine or more, ten
or more, eleven or more, twelve or more, thirteen or more, fourteen
or more, fifteen or more, or sixteen or more, or seventeen or more,
or eighteen or more, or all of -7a, -26b, -122, -1906, -21, -22,
-24, -34a, -155, -195, -677, -541, -125b, -500, -466j, -544, -1a-2,
-1194, and/or -700 and/or lack of down-regulation or low expression
of one or more, two or more, three or more, five or more, six or
more, seven or more, eight or more, nine or more, ten or more,
eleven or more, twelve or more, thirteen or more, fourteen or more,
or fifteen or more, or all of miR-27a, -192, -214, -377, -196b, -9,
-455, -23b, -106a, -200b, -532, -34c, -151, -653, -687, and/or
-483, as compared to the miR profile of a subject that is suffering
from a fibrotic or hepatic disease or an associated disorder.
2.-4. (canceled)
5. The pharmaceutical composition of claim 1, wherein the body
fluid comprises one or more of blood, urine, saliva, breast milk,
lymphatic fluid, serum and/or plasma.
6. The pharmaceutical composition of claim 1, wherein the
pharmaceutically acceptable carrier comprises one or more of
saline, glycerol, PBS and optionally, a protease inhibitor.
7. The pharmaceutical composition of claim 1, formulated for low
temperature storage below about '70.degree. C., freeze-drying or
lyophilisation.
8. A pharmaceutical composition comprising one or more, two or
more, three or more, four or more, five or more, six or more, seven
or more, eight or more, nine or more, ten or more, eleven or more,
twelve or more, thirteen or more, fourteen or more, fifteen or
more, or alternatively all of miR-9, -196b, -27a, -192, -214, -377,
-455, -23b, -106a, -200b, -532, -34c, -151, -653, -687, and/or -483
and a pharmaceutically acceptable carrier.
9. The composition of claim 1 further comprising one or more, two
or more, three or more, four or more, five or more, six or more,
seven or more, eight or more, nine or more, ten or more, eleven or
more, twelve or more, thirteen or more, fourteen or more, fifteen
or more, or alternatively all of miR-9, -196b, -27a, -192, -214,
-377, -455, -23b, -106a, -200b, -532, -34c, -151, -653, -687,
and/or -483.
10. A method for one or more of: a) inhibiting the progression of,
b) preventing, and/or c) treating, a fibrotic disease or an
associated disorder in a subject in need thereof, comprising
administering to the subject an effective amount of the
pharmaceutical composition of claim 1, wherein the exosomes are
isolated from a subject that is not diagnosed as having the
fibrotic disease or associated disorder that is to be inhibited,
prevented or treated.
11. The method of claim 10, wherein the fibrotic disease or
associated disorder is a liver disease, a fibrotic liver disease or
an associated disorder and the associated disorder is selected from
the group of: inflammation of one or more of the liver, the heart,
the kidney, the lung, the pancreas, a joint, the eye; high
deposition of lipids, high deposition of insoluble collagen; high
deposition of extracellular matrix components; cirrhosis,
hepatocarcinoma; scleroderma, keloids, rheumatoid arthritis, or
end-stage liver disease.
12. The method of claim 10, further comprising determining the
level of one or more miR-26b, -122, -9, or -196b in a sample
isolated from the patient to monitor the therapy.
13. The method of claim 10, further comprising determining the
expression level of one or more of two or more, three or more, or
four or more, or five or more, or six or more, or seven or more, or
eight or more, or nine or more, or ten or more, or eleven or more,
or twelve or more, or thirteen or more, or fourteen or more, or
fifteen or more, or sixteen or more, or seventeen or more, or
eighteen or more, or nineteen or more, or twenty or more, or
twenty-one or more, or twenty-two or more, or twenty-three or more,
or twenty-four or more, or twenty-five or more, or twenty-six, or
twenty-seven or more, or twenty-eight or more, or twenty-nine or
more, or thirty or more, or thirty-one or more, or thirty-two or
more, or thirty-three or more, or thirty-four or more, or all of
miR-7a, -21, -22, -24, -34a, -155, -195, -27a, -192, -214, -377,
-455, -23b, -106a, -200b, -532, -34c, -26b, -122, -9, -196b, -151,
-653, -687, -483, -1906, -677, -541, -125b, -500, -466j, -544,
-1a-2, -1194, and/or -700, in a sample isolated from the patient to
monitor the therapy.
14. The composition of claim 1, wherein the subject is a
mammal.
15. The composition of claim 1, wherein the subject is a human.
16. The composition of claim 1, wherein an effective amount
comprises from 0.1 mg to about 1,000 mg per kg of body weight of
the subject to be treated, in a single or multiple doses.
17. The method of claim 10, wherein the exosomes are allogeneic or
autologous to the subject receiving the exosomes.
18. A purified or isolated population of exosomes isolated from a
body fluid of a non-diseased subject, wherein optionally: (a) the
microRNA (miR) profile of the exosomes comprises lack up-regulation
of one or both of the exosome markers miR-26b and/or -122, as
compared to the exosome miR profile of a subject that is suffering
from a liver or fibrotic disease or an associated disorder and/or
comprise lack of down-regulation of one or both of miR-9and/or
-196b, as compared to the exosome miR profile of a subject that is
suffering from a liver or fibrotic disease or an associated
disorder; or (b) the microRNA (miR) profile of the exosomes
comprises lack of up-regulation of one or both of miR-26b or -122
or lack of down-regulation of miR-9, or -196b, as compared to the
exosome miR profile of a subject that is suffering from a liver or
fibrotic disease or an associated disorder.
19.-20. (canceled)
21. The purified or isolated population of exosomes of claim 18,
wherein the exosomes further comprise a miR profile comprising lack
of up regulation one or more of miR-7a, -21, -22, -24, -34a, -155,
-26b, -122, -1906, -195, -677, -541, -125b, -500, -466j, -544,
-1a-2, -1194, and/or -700, and/or lack of down-regulation of
miR-27a, -192, -214, -377, -196b, -9, -455, -23b, -106a, -200b,
-532, -34c, -151, -653, -687, and/or -483, as compared to the
exosome miR profile of a subject that is suffering from a liver or
fibrotic disease or an associated disorder.
22. A purified or isolated population of exosomes of claim 18,
wherein the fluid is selected from the group of urine, saliva,
lymphatic fluid, breast fluid, blood, serum or plasma.
23. A purified or isolated population of exosomes of claim 18,
further comprising a pharmaceutically acceptable carrier.
24. A purified or isolated population of exosomes of claim 23,
formulated for low temperature storage below about -70.degree. C.,
freeze-drying or lyophilisation.
25. A kit for one or more of: a) inhibiting the progression of, b)
preventing or c) treating, a fibrotic disease or an associated
disorder in a subject in need thereof, comprising an effective
amount of the pharmaceutical composition of claim 1, and reagents
and instructions for use.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 62/057971, filed
Sep. 30, 2014, the content of which is hereby incorporated by
reference in its entirety.
BACKGROUND
[0003] Chronic scarring, or "fibrosis", of the liver, lung, kidney,
heart and other vital organ systems has no effective treatment, and
is estimated to account for up to 45% of all deaths in the
industrialized world. There are currently no FDA or EMEA-approved
antifibrotic therapies.
[0004] Fibrosis is a common and debilitating pathology in many
chronic liver diseases that hinders effective treatment and
heightens the need for liver transplantation. Hepatic fibrosis is a
common response to chronic injury and inflammation in the liver,
resulting in excessive production and deposition of insoluble
collagen and extracellular matrix components.
[0005] Affecting 5.5 million American adults (i.e. 2-3% of the
adult US population), hepatic fibrosis is a serious medical problem
because it compromises normal hepatic structure and function and is
a harbinger of cirrhosis, hepatocarcinoma, and end-stage liver
disease. Liver fibrosis is most commonly seen in patients with
chronic liver injury (hepatitis and alcohol abuse) although the
pediatric population can be affected as well (biliary atresia,
congenital). With the current explosion of obesity-related health
problems such as fatty livers, a huge increase in the number of
patients at risk for or who have developed liver fibrosis is to be
expected. There are many other types of scarring that lack
effective treatment. For example, dermal scarring and abdominal
adhesions occur in a high proportion of the >42 million
surgeries performed each year in the United States but, again there
is no FDA-approved medication to prevent or reduce scars. This
market alone is estimated at $4 billion.
[0006] Thus, a need exists for effective and safe treatments for
hepatic fibrosis. This disclosure satisfies these needs and
provides related advantages as well.
SUMMARY
[0007] This disclosure provides pharmaceutical compositions and
purified or isolated products that have therapeutic use for
treating an unmet medical need. As disclosed in more detail herein,
this disclosure provides a pharmaceutical comprising, or
alternatively consisting essentially of, or yet further consisting
of, a pharmaceutically acceptable carrier and an effective amount
of miRNA and/or exosomes isolated from a body fluid of a
non-diseased subject. In one aspect, the disease is a fibrotic
disease or liver disease, e.g., liver fibrosis and the exosomes
have a unique molecular profile in that the microRNA (miR or miRNA)
and/or the miRNA profile of the exosomes comprise the lack of
up-regulation of one or both of the markers miR-26b and/or -122, as
compared to the exosome miR profile of a subject that is suffering
from a liver or a fibrotic disease or an associated disorder. In
one aspect the compositions comprise, or alternatively consist
essentially of, or yet further consist of, one or more, two or
more, three or more, four or more, five or more, six or more, seven
or more, eight or more, nine or more, ten or more, eleven or more,
twelve or more, thirteen or more, fourteen or more, fifteen or
more, sixteen or more, or alternatively all of miR-9, -196b,
miR-27a, -192, -214, -377, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483. These compositions are useful for
the treatment of disease, such as fibrotic disease, hepatic liver
disease and associated disorders.
[0008] This disclosure also provides a pharmaceutical compositions
comprising, or alternatively consisting essentially of, or yet
further consisting of, a pharmaceutically acceptable carrier and an
effective amount of exosomes isolated from a body fluid of a
non-diseased subject. In one aspect, the exosomes have a unique
molecular profile in that the microRNA (miR) comprises the lack of
down-regulation of one or both of the markers miR-9 and/or -196b,
as compared to the exosome miR profile of a subject that is
suffering from a liver or fibrotic disease or an associated
disorder.
[0009] In further aspect, the exosomes of the above-noted
compositions are identified by the microRNA (miR) profile by the
lack of up-regulation of miR-26b and/or -122 and/or lack of
down-regulation of miR-9 and/or -196b in the exosomes, as compared
to the exosome miR profile of a subject that is suffering from a
fibrotic disease, a liver disease, or an associated disorder.
[0010] In further aspect, the exosomes of the above-noted
compositions are further identified by the microRNA (miR) exosome
profile by the lack of up-regulation of one or more, two or more,
three or more, four or more, and all of (and all integers there
between) of miR-7a, -21, -22, -24, -26b, -34a, -155, -122, -1906,
-195, 677, -541, -125b, -500, -466j, -544, -1a-2, -1194, and/or
-700- and/or lack of down-regulation of one or more, two or more,
three or more, four or more, and all of (and all integers there
between) miR-27a, -192, -9, -196b, -214, -377, -455, -23b, -106a,
-200b, -532, -34c, -151, -653, -687, and/or -483 in the exosomes,
as compared to the exosome miR profile of a subject that is
suffering from a liver or fibrotic disease or an associated
disorder.
[0011] For each of the above compositions, the fluid from which the
exosomes are isolated or purified is selected from the group of
blood, serum, urine, lymphatic fluid, saliva, breast milk and/or
plasma. The fluids can be maintained separately or combined from
the same or multiple donors.
[0012] The compositions can then be administered to subjects
identified as likely to have liver or hepatic disease or an
associated disorder.
[0013] The compositions are useful for the preparation of a
medicament and/or to perform methods for one or more of: a)
inhibiting the progression of, b) preventing or c) treating, liver
fibrosis or an associated disorder in a subject in need thereof.
The methods comprise, or alternatively consist essentially of, or
yet further consist of, administering to the subject an effective
amount of the pharmaceutical composition described above.
Non-limiting examples of an associated disorder is selected from
the group of: inflammation of the liver, high deposition of lipids,
high deposition of insoluble collagen, high deposition of
extracellular matrix components, cirrhosis, hepatocarcinoma, and
end-stage liver disease. These conditions are well known in the art
and can be diagnosed by a treating physician.
[0014] The therapy and patient's health can be monitored by
determining the level of one or more, two or more, three or more,
or all of miR-26b, -122, miR-9 and/or -196b in a sample isolated
from the patient prior to, during and after the therapy. In a
further aspect, the therapy and patient's health and therapy
response can be monitored by determining the level of one or more,
two or more, three or more, or four or more, or five or more, or
six or more, or seven or more, or eight or more, or nine or more,
or ten or more, or eleven or more, or twelve or more, or thirteen
or more, or fourteen or more, or fifteen or more, or sixteen or
more, or seventeen or more, or eighteen or more, or nineteen or
more, or twenty or more, or twenty-one or more, or twenty-two or
more, or twenty-three or more, or twenty-four or more, or
twenty-five or more, or twenty-six, or twenty-seven or more, or
twenty-eight or more, or twenty-nine or more, or thirty or more, or
thirty-one or more, or thirty-two or more, or thirty-three or more,
or thirty-four or more, or all of miR -21, -22, -7a, -24, -155,
-195, -34a, -26b, -122, -9, -196b, -677, -541, -125b, -500, -466j,
-544, -1a-2, -1194, -700, -27a, -192, -214, -377, -1906, -455,
-23b, -106a, -200b, -532, -34c, -151, -653, -687, and/or -483, in a
sample isolated from the patient prior to, during and after the
therapy and wherein high levels of one or more of miR -21, -22,
-7a, -24, -155, -195, -34a, -1906, 26b, -122, -677, -541, -125b,
-500, -466, -544, -1a-2, -1194 and/or -700, and one or more of low
levels of miR-9, -377, -27a, -192, -214, -196b, -455, -23b, -106a,
-200b, -532, -34c, -151, -653, -687, and/or -483, identifies the
patient as likely to have hepatic disease.
[0015] In one aspect, this disclosure also provides determining the
hepatic health status of a subject comprising determining the level
of one or more, two or more, three or more, or four or more, or
five or more, or six or more, or seven or more, or eight or more,
or nine or more, or ten or more, or eleven or more, or twelve or
more, or thirteen or more, or fourteen or more, or fifteen or more,
or sixteen or more, or seventeen or more, or eighteen or more, or
nineteen or more, or twenty or more, or twenty-one or more, or
twenty-two or more, or twenty-three or more, or twenty-four or
more, or twenty-five or more, or twenty-six, or twenty-seven or
more, or all of miR-26b, -122, 9, -196b, -677, -541, -125b, -500,
-466j, -544, -1a-2, -1194, -700, -27a, -192, -214, -377, -1906,
-455, -23b, -106a, -200b, -532, -34c, -151, -653, -687, and/or
-483, in a sample isolated from the patient wherein up-regulation
of one or more of miR -21, -22, -7a, -24, -155, -195, -34a, -1906,
26b, -122, -677, -541, -125b, -500, -466, -544, -1a-2, -1194 and/or
-700 and/or one or more of down-regulation of miR-9, -377, -27a,
-192, -214, -196b, -455, -23b, -106a, -200b, -532, -34c, -151,
-653, -687, and/or -483, identifies the patient as likely to have
hepatic disease.
[0016] Also provided herein are isolated or purified exosomes
isolated from a body fluid (e.g., urine, saliva, lymphatic fluid,
breast milk, blood, serum and/or plasma) of a non-diseased subject.
In one aspect, the exosomes have a unique molecular profile in that
the microRNA (miR) profile of the exosomes comprise the lack of
up-regulation of one or both of the markers miR26b and/or -122
and/or the lack of down-regulation of one or more of miR-9 and/or
-196b, as compared to the exosome miR profile of a subject that is
suffering from a liver or fibrotic disease or an associated
disorder.
[0017] In a further aspect, the exosomes are further identified by
the microRNA (miR) profile by the lack of up-regulation of one or
more of miR-7a, -21, -22, -24, -34a, -155, -195, -677, -541, -125b,
-122, -155, -195, -1906, -500, -466j, -544, -1a-2, -1194, and/or
-700, and/or the lack of down-regulation of miR-27a, -196b, -9,
-192, -214, -377, -455, -23b, -106a, -200b, -532, -34c, -151, -653,
-687, and/or -483 as compared to the exosome miR profile of a
subject that is suffering from a liver or fibrotic disease or an
associated disorder.
[0018] A kit also is provided for one or more of: a) inhibiting the
progression of, b) preventing or c) treating, liver fibrosis or an
associated disorder in a subject in need thereof, comprising an
effective amount of the isolated or purified exosomes and/or the
pharmaceutical composition as described above and/or reagents
and/or instructions for use.
[0019] Yet further provided is a kit comprising one or more probes
and/or primers to determine the expression profile of one or more,
two or more, three or more or all four of miR26b, miR-122 miR-9;
and/or miR-196b. In a further aspect, the kit independently or in
addition also comprises, consists essentially of, or yet further
consists of, one or more, two or more, three or more, four or more,
five or more, six or more, seven or more, eight or more, nine or
more, ten or more, eleven or more, twelve or more, thirteen or
more, fourteen or more, fifteen or more, or all (including all
integers there between) of miR-7a, -26b, -122, -196b, -9, -21, -22,
-24, -34a, -155, -195, -27a, -192, -214, -377, -455, -23b, -106a,
-200b, -532, -34c, -151, -653, -687, -483, -1906, -677, -541,
-125b, -500, -466j, -544, -1a-2, -1194, and/or -700. In a further
aspect, the kit further comprises detectable labels that in one
aspect are attached to the probes and/or primers, and wherein in
one aspect, wherein the detectable label is not a
polynucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A & 1B show that mice with liver fibrosis have a
circulating exosomal miR content that is distinct from that of
normal mice. (A) MiRs in circulating exosomes from Balb/c mice
treated intraperitoneal (i.p.) with corn oil (30 .mu.l) or carbon
tetrachloride (CCl.sub.4; 4 .mu.l CCl.sub.4 diluted in 26 .mu.l
corn oil)) for 1 week or 5 weeks. Profiling was performed on
exosomes from 1 ml of pooled serum (5 mice; 200 .mu.l/mouse) using
a mouse miRnome miR PCR Array. Left: MiRs outside the outer
diagonal lines were up-regulated (red) or down-regulated (green)
more than 2-fold in response to CCl.sub.4. Right: Sirius
red-stained liver sections. (B) RT-PCR analysis of circulating
exosomes collected at 1-, 3- or 4 weeks in which miRs were selected
based on their expression in (A) and compared to published studies
reporting their up--(dark gray; " ") or down--(light gray; " ")
regulation in fibrotic liver tissues. Exosomal miRs termed "novel"
are examples of miRs not previously reported in liver fibrosis but
which can have serum exosome biomarker utility in early or late
fibrotic injury.
[0021] FIG. 2 shows fibrogenic gene expression in cultured hepatic
stellate cell (HSC). Connective Tissue Growth Factor (CTGF), alpha
smooth muscle actin (.alpha.SMA) or collagen .alpha.1 (I)
expression were assessed after 48-hr treatment of Day 9 primary HSC
with exosomes purified from serum of mice with carbon
tetrachloride-(CCl.sub.4)-induced liver fibrosis (upper) or that
were healthy (lower). Exosomes from fibrotic mice either did not
alter (CTGF, collagen) or exacerbated (.alpha.SMA) gene expression
(upper) whereas exosomes from normal mice reduced expression of the
same genes (lower).
[0022] FIGS. 3A & 3B show effect on CTGF, .alpha.SMA or miR-214
expression by activated HSC in vivo after concurrent treatment with
CCl4 and serum exosomes. Mice received CCl.sub.4 or oil (i.m) over
a 7 day period, followed a few hours later by administration of
exosomes (300 .mu.l) i.p. On Day 9, HSC were harvested from each
liver and briefly placed in culture for 24 hrs. The isolated HSC
were then analyzed by (A) RT-PCR for mRNA expression or (B)
immuno-cytochemistry (ICC) for protein production of key fibrogenic
markers. CCl.sub.4-induced expression of CTGF or .alpha.SMA was
reduced by exosomes (A, B). Expression of miR-214, which is
decreased during fibrosis, was increased to control levels by
exosomes (A).
[0023] FIGS. 4A-4E show in vivo effects of exosomes on
CCl.sub.4-induced fibrotic signals in the liver (short-term
injury). Transgenic (TG), Connective Tissue Growth Factor--Green
Fluorescent Protein (CTGF-GFP) mice received CCl.sub.4
intramuscular (i.m.) (4 .mu.l CCl.sub.4 diluted in 26 .mu.l corn
oil) over a 7 day period, followed a few hours later by
administration of exosomes (100-300 .mu.l; 3.mu.l) i.p. On Day 9,
livers were examined macroscopically (A) followed by resection and
analysis for direct green fluorescent protein (GFP) expression (a
measure of CTGF promoter activity) (B, C). Total liver RNA was
subject to RT-PCR with the finding that CCl.sub.4-induced changes
in CTGF, .alpha.SMA or miR-214 were reversed by exosomes. (E)
Immunostaining for .alpha.SMA. Oil-treated (control) animals show
positive staining in smooth muscle cells of vasculature only.
CCl.sub.4-treated animals show massive activation of HSC, the level
of which was strongly attenuated in exosome-treated mice.
[0024] FIGS. 5A-5D show in vivo effects of exosomes on CCl4-induced
fibrosis in the liver (long-term injury). TG CTGF-GFP mice received
CCl.sub.4 i.m. over a 5 week period. Some mice also received
exosomes by i.p. administration (100-300 .mu.l) on the same day as
and for the last 2 weeks of CCl.sub.4 (carbon tetrachloride)
treatment. Livers were assessed for (A) direct GFP expression
(=CTGF promoter activity) (B) expression of CTGF, .alpha.SMA,
collagen or miR-214 by RT-PCR of total hepatic RNA; and (C, D)
Immunostaining for .alpha.SMA, CTGF or collagen I. All outcome
measures affected by CCl.sub.4 were reversed by exosomes.
[0025] FIG. 6 shows differential expression of miRs in healthy and
fibrotic mice. Exosomes from 1 ml of pooled serum (5 mice; 200
.mu.l/mouse) were analyzed by miRnome miR PCR Array. The array
compares exosomal miR expression in healthy mice relative to
fibrotic mice to identify the principal components in serum that
are expressed at relatively higher (Healthy.sub.HIGH, darker
shading) or lower (Healthy.sub.LOW, lighter shading) levels in
normal versus fibrotic mice. MiRs lying outside the diagonal line
were expressed >.+-.2-fold in healthy mice as compared to
fibrotic mice. The "Top 10" Healthy.sub.HIGH and Healthy.sub.LOW
miRs (highest differential expression).
[0026] FIGS. 7A-7I show RT-PCR analysis of exosomal
Healthy.sub.HIGH mIRs in circulating exosomes from control or
fibrotic (CCL4-treated) mice. This experiment looked specifically
at transcripts of (A) 200b-3p, (B) 23b-3p, (C) 34c-3p, (D) 455-3p,
(E) 483-5p, (F) 532-5p, (G) 687, (H) 106a, and (I) 151-30
[0027] FIGS. 8A and 8B show quantitiative real-time polymerase
chain reaction (qRT-PCR) for collagen .alpha. (I) or .alpha.SMA or
CTGF mRNA relative to GAPDH mRNA in (FIG. 8A) mouse primary HSC or
(FIG. 8B) human LX-2 HSC cells (B) transfected for 24 hrs with
microRNA mimics (miR-455, -23b, -106a, -200b, -532, -34c, -151,
-653, -687, or -483). A scramble microRNA mimic was used as a
control (n=3 independent experiments performed in triplicate;
*P<0.001 vs control, +P<0.01 vs control).
[0028] FIG. 9 is a table identifying some major fibrotic diseases
that are provided as examples of "associated disorders."
DETAILED DESCRIPTION
[0029] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure to more fully describe the
state of the art to which this disclosure pertains.
[0030] The practice of the present disclosure employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature for example
in the following publications. See, e.g., Sambrook and Russell eds.
MOLECULAR CLONING: A LABORATORY MANUAL, 3.sup.rd edition (2001);
the series CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et
al. eds. (2007)); the series METHODS IN ENZYMOLOGY (Academic Press,
Inc., N.Y.); PCR 1: A PRACTICAL APPROACH (M. MacPherson et al. IRL
Press at Oxford University Press (1991)); PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane eds.
(1999)); CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (R.
I. Freshney 5.sup.th edition (2005)); OLIGONUCLEOTIDE SYNTHESIS (M.
J. Gait ed. (1984)); Mullis et al. U.S. Pat. No. 4,683,195; NUCLEIC
ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds. (1984));
NUCLEIC ACID HYBRIDIZATION (M. L. M. Anderson (1999));
TRANSCRIPTION AND TRANSLATION (B. D. Hames & S. J. Higgins eds.
(1984)); IMMOBILIZED CELLS AND ENZYMES (IRL Press (1986)); B.
Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); GENE
TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos
eds. (1987) Cold Spring Harbor Laboratory); GENE TRANSFER AND
EXPRESSION IN MAMMALIAN CELLS (S.C. Makrides ed. (2003))
IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and
Walker, eds., Academic Press, London (1987)); WEIR'S HANDBOOK OF
EXPERIMENTAL IMMUNOLOGY (L. A. Herzenberg et al. eds (1996)).
Definitions
[0031] As used herein, certain terms may have the following defined
meanings. As used in the specification and claims, the singular
form "a," "an" and "the" include singular and plural references
unless the context clearly dictates otherwise. For example, the
term "a cell" includes a single cell as well as a plurality of
cells, including mixtures thereof.
[0032] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
not excluding others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the composition or
method. "Consisting of" shall mean excluding more than trace
elements of other ingredients for claimed compositions and
substantial method steps. Embodiments defined by each of these
transition terms are within the scope of this disclosure.
Accordingly, it is intended that the methods and compositions can
include additional steps and components (comprising) or
alternatively including steps and compositions of no significance
(consisting essentially of) or alternatively, intending only the
stated method steps or compositions (consisting of).
[0033] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 0.1. It
is to be understood, although not always explicitly stated that all
numerical designations are preceded by the term "about". The term
"about" also includes the exact value "X" in addition to minor
increments of "X" such as "X+0.1" or "X-0.1." It also is to be
understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
[0034] As used herein, the term "exosome" intends a membrane body
having an average diameter of from about 10 nm to about 2,000 nm.
The term includes microvesicles and exosomes. Microvesicles are
also known as circulating microvesicles or microparticles and are
fragments of plasma membrane ranging from 100 nm to 1000 nm in
approximate diameter shed from almost all cell types. For the
purpose of this disclosure and unless specifically noted, the term
exosome also includes smaller intracellularly generated
extracellular vesicles formed by inward budding of the limiting
membranes of multivesicular bodies (MVB) which, upon fusion with
the plasma membrane, result in their secretion and deposition into
body fluids (e.g., blood, urine). Exosomes contain a complex
mixture of microRNAs (miRs), mRNAs and proteins that reflect the
transcriptional and translational status of the producer cell.
Exosomes are from about 10 to about 250 nm, or alternatively from
about 10 to about 200 nm, or alternatively from about 10 to about
175 nm, or alternatively from about 25 to 175 nm, or alternatively
from about 40 to about 250 nm, or alternatively from about 40 to
about 200 nm, or alternatively from about 50 to 250 nm, or
alternatively from about 50 to 200 nm, or alternatively from about
50-150 nm in average diameter. The exosome membranous vesicles
arise by inward budding from the limiting membranes of MVB. Upon
fusion of MVBs with the plasma membrane, exosomes are liberated
from the cells, traverse intercellular spaces, and may be taken up
by neighboring cells (Johnstone, R. M. (2006) Blood Cells Mol. Dis.
36(2):315-321; Thery, C. (2011) F1000 Biol. Rep. 3:15; Thery, C. et
al. (2002) Nat. Rev. Immunol. 2(8):569-579). Exosomes contain a
complex mixture of miRs, mRNAs and proteins and can be isolated
from a variety of body fluids as described herein and known in the
art.
[0035] As used herein, the term "fibrosis" intends the formation of
an abnormal amount of insoluble scar tissue as the result of
inflammation, irritation, or healing. It is a common and highly
debilitating pathology and an end-stage manifestation of diseases
such as systemic sclerosis, renal, pulmonary, or cardiac
hypertension, myocardial infarction, and chronic liver disease
(e.g., hepatitis, alcoholic liver disease, or non-alcoholic
steatohepatitis). Numerous studies suggest that fibrosis in
different organ systems share common mechanisms including
inflammation, a prolonged wound healing response, activation of
pro-fibrotic signals, increased matrix deposition and decreased
matrix degradation, increased tissue stiffness, and loss of tissue
elasticity. Collectively these changes in tissue architecture
conspire to cause a loss of normal cell function and viability.
Thus anti-fibrotic interventions will likely have utility for
antagonizing fibrogenic pathways in a broad variety of fibrotic
organ systems, or in tissues undergoing other types of scarring
responses.
[0036] As used herein, the term "fibrotic disease or condition"
intends a pathological condition having symptoms and clinical
markers of fibrotic tissue, e.g., systemic sclerosis, renal,
pulmonary, or cardiac hypertension, myocardial infarction, and
chronic liver disease (e.g., hepatitis, alcoholic liver disease, or
non-alcoholic steatohepatitis) and/or in various organs or tissues,
e.g., liver, heart, kidney, lung, pancreas, the joints and the eye.
Non-limiting examples of fibrotic conditions and associated
disorders are provided in FIG. 9 and include without limitation,
scleroderma, keloids and rheumatoid arthritis.
[0037] As used herein, "a non-diseased subject" intends a subject
not diagnosed with a fibrotic disease. In one aspect, the
non-diseased subject is one that does not have a clinical diagnosis
of hepatic fibrosis and/or liver disease and/or has normal liver
function. Clinical parameters for determining if a subject is
suffering from a fibrotic disease are known in the art and briefly
described herein. Without being limited, exemplary clinical tests
for assessing liver function include: serum bilirubin test, serum
alkaline phosphatase test, prothrombin time test, alanine
transaminase test, aspartate transaminase test, gamma glutamyl
transpeptidase test, lactate dehydrogenase test, alpha fetoprotein
test, mitochondrial antibody test, and serum .alpha.-1 antitrypsin
test. Clinical tests for detecting and diagnosing liver fibrosis
include without limitation: PGA index, FIB-4 index, Fibrometer,
FibroSure, Act-test, SAFE, Heapscore, FibroQ, AAR, APRI, CDS, API,
Pohls score, Loks model, liver biopsy, ultrasonography, computed
tomography, ultrasound elastography, and magnetic resonance
elastography. For example, when the fibrotic condition is liver
fibrosis, the above measurements can be combined with approved
clinical tests for liver function and/or liver fibrosis.
[0038] The term "lack of up-regulation" intends and lack of
"down-regulation" intends that the microRNA marker was not
determined to be over- or under-expressed as compared to a
predetermined value. In one aspect, the predetermined value is a
preliminary value from the subject prior to the subsequent
measurement (as in prior to therapy) or is a value from a
population of subjects that do or do not have clinical
manifestation of the related disorder. For example, when the
disclosure relates to the treatment of liver fibrosis, a
predetermined value can be the average or median exosome miRNA
value as measured from a population of subjects that do or do not
have a fibrotic or hepatic disease or an associated disorder.
[0039] The term "identify" or "identifying" is to associate or
affiliate a patient closely to a group or population of patients
who likely experience the same or a similar clinical response to
treatment.
[0040] The terms "protein," "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0041] The term "marker" refers to a clinical or sub-clinical
expression of a gene or miRNA of interest.
[0042] "Expression" as applied to a gene, refers to the
differential production of the miR or mRNA transcribed from the
gene or the protein product encoded by the gene. A differentially
expressed gene may be over expressed (high expression) or under
expressed (low expression) as compared to the expression level of a
normal or control cell, a given patient population or with an
internal control gene (housekeeping gene). In one aspect, it refers
to a differential that is about 1.5 times, or alternatively, about
2.0 times, alternatively, about 2.0 times, alternatively, about 3.0
times, or alternatively, about 5 times, or alternatively, about 10
times, alternatively about 50 times, or yet further alternatively
more than about 100 times higher or lower than the expression level
detected in a control sample.
[0043] In one aspect of the disclosure, a "predetermined threshold
level" or "threshold value" is used to categorize expression as
high or low. As a non-limiting example of the disclosure, the
threshold level of the miR of the exosome is a level of miR
expression found in subjects that have been diagnosed with a
fibrotic or hepatic disease or an associate disorder. Alternatively
or in addition, the predetermined threshold level is the measured
miRNA expression level for that individual subject prior to a
subsequent measurement, e.g., prior to therapy or prior to an
additional dose of the therapy.
[0044] In one aspect of the disclosure, miR expression can be
provided as a ratio above the threshold level and therefore can be
categorized as high expression or up-regulated, whereas a ratio
below the threshold level is categorized as down-regulated or low
expression.
[0045] In another aspect, "expression" level is determined by
measuring the expression level of a gene of interest for a given
patient population, determining the median expression level of that
gene for the population, and comparing the expression level of the
same gene for a single patient to the median expression level for
the given patient population. For example, if the expression level
of a gene of interest for the single patient is determined to be
above the median expression level of the patient population, that
patient is determined to have high expression (up-regulated) of the
gene of interest. Alternatively, if the expression level of a gene
of interest for the single patient is determined to be below the
median expression level (down-regulated) of the patient population,
that patient is determined to have low expression of the gene of
interest.
[0046] A "internal control" or "housekeeping" gene refers to any
constitutively or globally expressed gene whose presence enables an
assessment of the expression level of a gene or genes of interest.
Such an assessment comprises a determination of the overall
constitutive level of gene transcription and a control for
variation in sampling error. Examples of such genes include, but
are not limited to, RNU6-2, cel-miR-39, SNORD61, SNORD68, SNORD72,
SNORD95, SNORD96A, GADPH and/or .beta.-actin.
[0047] "Cells," "host cells" or "recombinant host cells" are terms
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0048] The phrase "amplification of polynucleotides" includes
methods such as PCR, ligation amplification (or ligase chain
reaction, LCR) and amplification methods. These methods are known
and widely practiced in the art. See, e.g., U.S. Pat. Nos.
4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu,
D. Y. et al. (1989) Genomics 4:560-569 (for LCR). In general, the
PCR procedure describes a method of gene amplification which is
comprised of (i) sequence-specific hybridization of primers to
specific genes within a DNA sample (or library), (ii) subsequent
amplification involving multiple rounds of annealing, elongation,
and denaturation using a DNA polymerase, and (iii) screening the
PCR products for a band of the correct size. The primers used are
oligonucleotides of sufficient length and appropriate sequence to
provide initiation of polymerization, i.e., each primer is
specifically designed to be complementary to each strand of the
genomic locus to be amplified.
[0049] Reagents and hardware for conducting PCR are commercially
available. Primers useful to amplify sequences from a particular
gene region are preferably complementary to, and hybridize
specifically to sequences in the target region or in its flanking
regions. Nucleic acid sequences generated by amplification may be
sequenced directly. Alternatively the amplified sequence(s) may be
cloned prior to sequence analysis. A method for the direct cloning
and sequence analysis of enzymatically amplified genomic segments
is known in the art.
[0050] The term "encode" as it is applied to polynucleotides refers
to a polynucleotide which is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed from its gene and/or
translated from its mRNA to produce the polypeptide and/or a
fragment thereof. The antisense strand is the complement of such a
nucleic acid, and the encoding sequence can be deduced
therefrom.
[0051] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, though preferably less than 25% identity, with one of the
sequences of the present disclosure.
[0052] The term "interact" as used herein is meant to include
detectable interactions between molecules, such as can be detected
using, for example, a hybridization assay. The term interact is
also meant to include "binding" interactions between molecules.
Interactions may be, for example, protein-protein, protein-nucleic
acid, protein-small molecule or small molecule-nucleic acid in
nature.
[0053] The term "isolated" as used herein refers to molecules or
biological or cellular materials being substantially free from
other materials. In one aspect, the term "isolated" refers to
nucleic acid, such as DNA or RNA, or protein or polypeptide, or
cell or cellular organelle, or tissue or organ, separated from
other DNAs or RNAs, or proteins or polypeptides, or cells or
cellular organelles, or tissues or organs, respectively, that are
present in the natural source. The term "isolated" also refers to a
nucleic acid or peptide that is substantially free of cellular
material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Moreover, an "isolated
nucleic acid" is meant to include nucleic acid fragments which are
not naturally occurring as fragments and would not be found in the
natural state. The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides. The
term "isolated" is also used herein to refer to cells or tissues
that are isolated from other cells or tissues and is meant to
encompass both cultured and engineered cells or tissues.
[0054] A "blood cell" refers to any of the cells contained in
blood. A blood cell is also referred to as an erythrocyte or
leukocyte, or a blood corpuscle. Non-limiting examples of blood
cells include white blood cells, red blood cells, and
platelets.
[0055] "Expression" as applied to a gene, refers to the production
of the miR or mRNA transcribed from the gene, or the protein
product encoded by the mRNA. The expression level of a gene may be
determined by measuring the amount of miR or mRNA or protein in a
cell or tissue sample. In one aspect, the expression level of a
gene is represented by a relative level as compared to a
housekeeping gene as an internal control. In another aspect, the
expression level of a gene from one sample may be directly compared
to the expression level of that gene from a different sample using
an internal control to remove the sampling error.
[0056] "Overexpression" or "underexpression" refers to increased or
decreased expression, or alternatively a differential expression,
of a gene in a test sample as compared to the expression level of
that gene in the control sample. In one aspect, the test sample is
a diseased cell, and the control sample is a normal cell. In
another aspect, the test sample is an experimentally manipulated or
biologically altered cell, and the control sample is the cell prior
to the experimental manipulation or biological alteration. In yet
another aspect, the test sample is a sample from a patient, and the
control sample is a similar sample from a healthy individual. In a
yet further aspect, the test sample is a sample from a patient and
the control sample is a similar sample from patient not having the
desired clinical outcome. In one aspect, the differential
expression is about 1.5 times, or alternatively, about 2.0 times,
or alternatively, about 2.0 times, or alternatively, about 3.0
times, or alternatively, about 5 times, or alternatively, about 10
times, or alternatively about 50 times, or yet further
alternatively more than about 100 times higher or lower than the
expression level detected in the control sample. Alternatively, the
gene is referred to as "over expressed" or "under expressed".
Alternatively, the gene may also be referred to as "up regulated"
or "down regulated".
[0057] A "predetermined value" for a gene as used herein, is so
chosen that a patient with an expression level of that gene higher
than the predetermined value is likely to experience a more or less
desirable clinical outcome than patients with expression levels of
the same gene lower than the predetermined value, or vice-versa.
Expression levels of genes, such as those disclosed in the present
disclosure, are associated with clinical outcomes. One of skill in
the art can determine a predetermined value for a gene by comparing
expression levels of a gene in patients with more desirable
clinical outcomes to those with less desirable clinical outcomes.
In one aspect, a predetermined value is a gene expression value
that best separates patients into a group with more desirable
clinical outcomes and a group with less desirable clinical
outcomes. Such a gene expression value can be mathematically or
statistically determined with methods well known in the art.
[0058] Alternatively, a gene expression that is higher than the
predetermined value is simply referred to as a "high expression",
or a gene expression that is lower than the predetermined value is
simply referred to as a "low expression".
[0059] Briefly and for the purpose of illustration only, one of
skill in the art can determine a predetermined values by comparing
expression values of a gene in patients with more desirable
clinical parameters to those with less desirable clinical
parameters. In one aspect, a predetermined value is a gene
expression value that best separates patients into a group with
more desirable clinical parameter and a group with less desirable
clinical parameter. Such a gene expression value can be
mathematically or statistically determined with methods well known
in the art.
[0060] As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA). The term should also be
understood to include, as equivalents, derivatives, variants and
analogs of either RNA or DNA made from nucleotide analogs, and, as
applicable to the embodiment being described, single (sense or
antisense) and double-stranded polynucleotides.
Deoxyribonucleotides include deoxyadenosine, deoxycytidine,
deoxyguanosine, and deoxythymidine. For purposes of clarity, when
referring herein to a nucleotide of a nucleic acid, which can be
DNA or an RNA, the terms "adenosine," "cytidine," "guanosine," and
"thymidine" are used. It is understood that if the nucleic acid is
RNA, a nucleotide having a uracil base is uridine.
[0061] The terms "oligonucleotide" or "polynucleotide," or
"portion," or "segment" thereof refer to a stretch of
polynucleotide residues which is long enough to use in PCR or
various hybridization procedures to identify or amplify identical
or related parts of miR or mRNA or DNA molecules. The
polynucleotide compositions of this disclosure include miR, RNA,
cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense
and antisense strands, and may be chemically or biochemically
modified or may contain non-natural or derivatized nucleotide
bases, as will be readily appreciated by those skilled in the art.
Such modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.). Also included are synthetic
molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical
interactions. Such molecules are known in the art and include, for
example, those in which peptide linkages substitute for phosphate
linkages in the backbone of the molecule.
[0062] microRNAs, miRNAs, or miRs are single-stranded RNA molecules
of that are of various lengths, e.g., from about 15 to about 50
nucleotides, or alternatively from about 19 to about 25 nucleotides
in length, which regulate gene expression. miRNAs are encoded by
genes from whose DNA they are transcribed but miRNAs are not
translated into protein (non-coding RNA); instead each primary
transcript (a pre-miRNA) is processed into a short stem-loop
structure called a pre-miRNA and finally into a functional miRNA.
Mature miRNA molecules are partially complementary to one or more
messenger RNA (mRNA) molecules, and their main function is to
down-regulate gene expression.
[0063] As used herein, a "pre-miRNA" molecule is an RNA molecule of
about 100 to about 200 nucleotides, or alternatively from about 100
to about 130 nucleotides which can adopt a secondary structure
comprising a double stranded RNA stem and a single stranded RNA
loop and further comprises the nucleotide sequence of the miRNA
(and its complement sequence) in the double stranded RNA stem. The
miRNA and its complement can be located about 10 to about 20
nucleotides from the free ends of the miRNA double stranded RNA
stem.
[0064] "RNA interference" (RNAi) refers to sequence-specific or
gene specific suppression of gene expression (protein synthesis)
that is mediated by short interfering RNA (siRNA).
[0065] "Short interfering RNA" (siRNA) refers to double-stranded
RNA molecules (dsRNA), generally, from about 10 to about 30
nucleotides in length that are capable of mediating RNA
interference (RNAi), or 11 nucleotides in length, 12 nucleotides in
length, 13 nucleotides in length, 14 nucleotides in length, 15
nucleotides in length, 16 nucleotides in length, 17 nucleotides in
length, 18 nucleotides in length, 19 nucleotides in length, 20
nucleotides in length, 21 nucleotides in length, 22 nucleotides in
length, 23 nucleotides in length, 24 nucleotides in length, 25
nucleotides in length, 26 nucleotides in length, 27 nucleotides in
length, 28 nucleotides in length, or 29 nucleotides in length. As
used herein, the term siRNA includes short hairpin RNAs (shRNAs). A
siRNA directed to a gene or the mRNA of a gene may be a siRNA that
recognizes the mRNA of the gene and directs a RNA-induced silencing
complex (RISC) to the mRNA, leading to degradation of the mRNA. A
siRNA directed to a gene or the mRNA of a gene may also be a siRNA
that recognizes the mRNA and inhibits translation of the mRNA.
[0066] "Double stranded RNA" (dsRNA) refer to double stranded RNA
molecules that may be of any length and may be cleaved
intracellularly into smaller RNA molecules, such as siRNA. In cells
that have a competent interferon response, longer dsRNA, such as
those longer than about 30 base pair in length, may trigger the
interferon response. In other cells that do not have a competent
interferon response, dsRNA may be used to trigger specific
RNAi.
[0067] An "antagomir" is a polynucleotide that is complementary to
the miR sequence and, once it binds to and engages the miR, it will
prevent the miR from binding its mRNA target.
[0068] An mRNA "protector" is a molecule that competes with miR for
its binding site on a specific target mRNA moiety. Binding of the
protector prevents subsequent binding to the target by the mIR. The
activity of the mIR on the specific target gene is thus prevented
by the protector which itself is inactive.
[0069] When a marker is used as a basis for selecting a patient for
a treatment described herein, the marker is measured before and/or
during treatment, and the values obtained are used by a clinician
in assessing any of the following: (a) probable or likely
suitability of an individual to initially receive treatment(s); (b)
probable or likely unsuitability of an individual to initially
receive treatment(s); (c) responsiveness to treatment; (d) probable
or likely suitability of an individual to continue to receive
treatment(s); (e) probable or likely unsuitability of an individual
to continue to receive treatment(s); (f) adjusting dosage; (g)
predicting likelihood of clinical benefits; or (h) toxicity. As
would be well understood by one in the art, measurement of the
genetic marker or polymorphism in a clinical setting is a clear
indication that this parameter was used as a basis for initiating,
continuing, adjusting and/or ceasing administration of the
treatments described herein.
[0070] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of the
condition or disease. For example, in the case of liver fibrosis,
the term "treatment" intends a more favorable clinical assessment
by a treating physician or assistant and/or reduced expression of
fibrosis markers, e.g., .alpha.SMA, CTGF, collagen, matrix
molecules and/or a shift toward normal read-outs in tests that
diagnose liver function and/or liver fibrosis.
[0071] "An effective amount" intends to indicated the amount of a
composition, compound or agent (exosomes) administered or delivered
to the subject that is most likely to result in the desired
response to treatment. The amount is empirically determined by the
patient's clinical parameters including, but not limited to the
stage of disease, age, gender and histology.
[0072] The term "suitable for a therapy" or "suitably treated with
a therapy" shall mean that the patient is likely to exhibit one or
more more desirable clinical outcome as compared to patients having
the same disease and receiving the same therapy but possessing a
different characteristic that is under consideration for the
purpose of the comparison.
[0073] The term "blood" refers to blood which includes all
components of blood circulating in a subject including, but not
limited to, red blood cells, white blood cells, plasma, clotting
factors, small proteins, platelets and/or cryoprecipitate. This is
typically the type of blood which is donated when a human patent
gives blood.
[0074] A "composition" is intended to mean a combination of active
exosome or population of exosomes and another compound or
composition, inert (e.g., a detectable label or saline) or active
(e.g., a therapeutic compound or composition) alone or in
combination with a carrier which can in one embodiment be a simple
carrier like saline or pharmaceutically acceptable or a solid
support as defined below.
[0075] A "pharmaceutical composition" is intended to include the
combination of an active exosome or population of exosomes with a
carrier, inert or active such as a solid support, making the
composition suitable for diagnostic or therapeutic use in vitro, in
vivo or ex vivo.
[0076] As used herein, the term "pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutical carriers,
such as a phosphate buffered saline solution, water, and emulsions,
such as an oil/water or water/oil emulsion, and various types of
wetting agents. The compositions also can include stabilizers and
preservatives. For examples of carriers, stabilizers and adjuvants,
see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ.
Co., Easton).
[0077] A "subject," "individual" or "patient" is used
interchangeably herein, and refers to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, rats, rabbits, simians, bovines, ovines,
porcines, canines, felines, farm animals, sport animals, pets,
equines, and primates, particularly humans.
[0078] "Administration" can be effected in one dose, continuously
or intermittently throughout the course of treatment. Methods of
determining the most effective means and dosage of administration
are known to those of skill in the art and will vary with the
composition used for therapy, the purpose of the therapy, the
target cell being treated, the disease being treated and the
subject being treated. Single or multiple administrations can be
carried out with the dose level and pattern being selected by the
treating physician. Suitable dosage formulations and methods of
administering the agents are known in the art. Route of
administration can also be determined and method of determining the
most effective route of administration are known to those of skill
in the art and will vary with the composition used for treatment,
the purpose of the treatment, the health condition or disease stage
of the subject being treated, and target cell or tissue.
Non-limiting examples of route of administration include oral
administration, nasal administration, inhalation, injection, and
topical application.
[0079] An agent of the present disclosure can be administered for
therapy by any suitable route of administration. It will also be
appreciated that the preferred route will vary with the condition
and age of the recipient, and the disease being treated.
Modes for Carrying Out the Disclosure
Diagnostic Methods
[0080] This disclosure provides diagnostic methods. In one aspect,
therapy and a subject's health can be monitored by determining the
expression level of one or more, two or more, three or more, or all
four of of miR-26b, miR-122, miR-9 and/or miR-196b in a sample of
exosomes isolated from the subject prior to, during, and/or after
the therapy. The method can further comprise, or alternatively
consist essentially of, or yet further consist of, determining the
expression level of one or more, two or more, three or more, four
or more, five or more, six or more, seven or more, eight or more,
nine or more, ten or more, eleven or more, twelve or more, thirteen
or more, fourteen or more, fifteen or more, or all (including all
integers there between) of miR-7a, -21, -26b, -122, -196b, -9, -22,
-24, -34a, -155, -195, -27a, -192, -214, -377, -455, -23b, -106a,
-200b, -532, -34c, -151, -653, -687, -483, -1906, -677, -541,
-125b, -500, -466j, -544, -1a-2, -1194, and/or -700 in a sample of
exosomes isolated from the subject.
[0081] In one aspect, the exosomes have an average diameter from
about 10 to about 250 nm, or alternatively from about 10 to about
200 nm, or alternatively from about 10 to about 175 nm, or
alternatively from about 25 to 175 nm, or alternatively from about
40 to about 250 nm, or alternatively from about 40 to about 200 nm,
or alternatively from about 50 to 250 nm, or alternatively from
about 50 to 200 nm, or alternatively from about 50-150 nm in
average diameter. In another aspect, the term exosome also includes
microvesicles that range from 100 nm to 1000 nm in approximate
diameter.
[0082] The measurement of the above-noted miRNA markers can be
combined with clinical parameters. Without being limited, exemplary
clinical tests for assessing liver function include: serum
bilirubin test, serum alkaline phosphatase test, prothrombin time
test, alanine transaminase test, aspartate transaminase test, gamma
glutamyl transpeptidase test, lactate dehydrogenase test, alpha
fetoprotein test, mitochondrial antibody test, and serum .alpha.-1
antitrypsin test. Clinical tests for detecting and diagnosing liver
fibrosis include without limitation: PGA index, FIB-4 index,
Fibrometer, FibroSure, Act-test, SAFE, Heapscore, FibroQ, AAR,
APRI, CDS, API, Pohls score, Loks model, liver biopsy,
ultrasonography, computed tomography, ultrasound elastography, and
magnetic resonance elastography. For example, when the fibrotic
condition is liver fibrosis, the above measurements can be combined
with approved clinical tests for liver function and/or liver
fibrosis.
[0083] The method also can be used to determine if a subject is a
suitable for the therapy as described herein, by performing the
above diagnostic method. When the exosome expression level is
up-regulated (high expression) for mi-26b and/or -122 and/or
down-regulated for miR-9 and/or -196b, the subject is likely to
have a fibrotic disease, a liver or a hepatic disease or an
associated disorder, and if the exosome expression is normal or
reversed from the above, the subject is not likely to have a
fibrotic disease, a liver disease, or hepatic disease or an
associated disorder, and therefore therapy is not needed. The
method can further comprise determining and correlating the
expression level of one or more, two or more, three or more, four
or more, five or more, six or more, seven or more, eight or more,
nine or more, ten or more, eleven or more, twelve or more, thirteen
or more, fourteen or more, fifteen or more, or all (including all
integers there between) of miR-7a, -21, -22, -24, -26b, -122,
-196b, -9, -34a, -155, -195, -27a, -192, -214, -377, -455, -23b,
-106a, -200b, -532, -34c, -151, -653, -687, -483, -1906, -677,
-541, -125b, -500, -466j, -544, -1a-2, -1194, and/or -700 in a
sample of exosomes isolated from the subject, and wherein high
levels of one or more of miR-26b, -21, -22, -7a, -24, -155, -195,
-34a, -1906, -122, -677, -541, -125b, -500, -466, -544, -1a-2,
-1194 and/or-700, and one or more of low levels of miR-9, -377,
-27a, -192, -214, -196b, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483, identifies the patient as likely to
have fibrotic disease, a liver or a hepatic disease and in need of
therapy. If the subject is in need of therapy, the compositions of
this disclosure, alone or in combination with other known
therapies, can then be administered to the subject in need of
treatment. The diagnostic methods can be repeated throughout and
after therapy to monitor the subject's health status and the
efficacy of the therapy.
[0084] The therapy and patient's health can be monitored by
determining the level of one or more, two or more, three or more,
or all of miR-26b, -122, miR-9 and/or -196b in a sample isolated
from the patient prior to, during and after the therapy. In a
further aspect, the therapy and patient's health and therapy
response can be monitored by determining the level of one or more,
two or more, three or more, or four or more, or five or more, or
six or more, or seven or more, or eight or more, or nine or more,
or ten or more, or eleven or more, or twelve or more, or thirteen
or more, or fourteen or more, or fifteen or more, or sixteen or
more, or seventeen or more, or eighteen or more, or nineteen or
more, or twenty or more, or twenty-one or more, or twenty-two or
more, or twenty-three or more, or twenty-four or more, or
twenty-five or more, or twenty-six, or twenty-seven or more, or all
of (and integers there between) of miR-26b, -122, -9, -196b, -677,
-541, -125b, -500, -466j, -544, -1a-2, -1194, -700, -27a, -192,
-214, -377, -1906, -455, -23b, -106a, -200b, -532, -34c, -151,
-653, -687, -21, -22, -7a, -24, -155, -195, -34a, and/or -483, in a
sample isolated from the patient prior to, during and after the
therapy and wherein high levels of one or more of miR-26b, -21,
-22, -7a, -24, -155, -95, -34a, -1906, -122, -677, -541, -125b,
-500, -466, -544, -1a-2, -1194 and/or-700, and one or more of low
levels of miR-9, -196b, -455, -23b, -106a, -200b, -532, -34c, -151,
-653, -687, -377, -27a, -192, -214, and/or -483, identifies the
patient as likely to have fibrotic disease, a liver or a hepatic
disease.
[0085] In one aspect, this disclosure also provides determining the
hepatic health status of a subject comprising determining the level
of one or more, two or more, three or more, or four or more, or
five or more, or six or more, or seven or more, or eight or more,
or nine or more, or ten or more, or eleven or more, or twelve or
more, or thirteen or more, or fourteen or more, or fifteen or more,
or sixteen or more, or seventeen or more, or eighteen or more, or
nineteen or more, or twenty or more, or twenty-one or more, or
twenty-two or more, or twenty-three or more, or twenty-four or
more, or twenty-five or more, or twenty-six, or twenty-seven or
more, or all of (an integers there between) of miR-26b, -122, -9,
-196b, -677, -541, -125b, -500, -466j, -544, -1a-2, -1194, -700,
-27a, -192, -214, -377, -1906, -455, -23b, -106a, -200b, -532,
-34c, -151, -653, -687, -21, -22, -7a, -24, -155, -195, -34a and/or
-483, in a sample isolated from the patient wherein up-regulation
of one or more of miR-26b, -122, -677, -541, -125b, -500, -466,
-544, -1a-2, -1194, -21, -22, -7a, -24, -155, -195, -34a, -1906,
and/or -700, and/or one or more of down-regulation of miR-9, -377,
-27a, -192, -214, -196b, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483, identifies the patient as likely to
have fibrotic disease, a liver or a hepatic disease.
[0086] Measurement of expression level or activity level can be
accomplished by methods known in the art and briefly described
herein, e.g., by PCR, qPCR, miRNA arrays, RNA-seq, multiplex miRNA
profiling. The tools and methodologies are known in the art and
commercially available, from Abcam (see abcam.com, last accessed
Sep. 25, 2015). The measurement can be compared to suitable
controls, e.g., a prior measurement for that subject or a suitable
internal control.
[0087] Collection of samples of exosomes from body fluid, e.g.,
urine, blood, saliva, breast milk, lymphatic fluid, serum or plasma
can be done with methods known in the art and described briefly
herein. The exosomes can be purified from the fluid using the
methods disclosed herein in art-recognized methods, such as by
ultracentrifugation as described by Thery et al. (2006) "Isolation
and characterization of exosomes from cell culture supernatants and
biological fluids" Curr. Protoc. Cell Biol., Chapter 3, or as
disclosed in Hong et al. (2014) PLoS One 9(8):e103310, doc:10,1371
and Jayachandran et al. (2012) J. Immun. Methods, 375:207-214.
Commercial kits also are available, e.g., PureExo (101BIO, Palo
Alto Calif., for serum and plasma), Exo MIR Plus (Bioo Scientific,
Austin Tex., USA), ExoQuick (SBI, Mountain View, Calif., USA, for
tissue culture) and Exo-Spin Kit (Cell Guidance Systems, Carlsbad
Calif., USA). As apparent to the skilled artisan, the isolation
method will depend on the size and composition of the exosome to be
isolated As an example, ultracentrifugation can be used but for
larger microvesicles the speed shall not exceed about 70,000 g or
alternatively about 60,000 g. Alternatively, ultracentrifugation is
used for smaller exosomes, but being much smaller, speeds of 90,000
or alternatively of 100,000g or more are needed.
[0088] The methods are useful in the diagnosis of a subject, e.g.,
a mammal, an animal, or yet further a human patient. For the
purpose of illustration only, a mammal includes but is not limited
to a human, a simian, a murine, a rat, a bovine, a canine, a
feline, an equine, a porcine or an ovine.
Compositions
[0089] Provided herein is a purified or isolated population of
exosomes isolated from a body fluid of a non-diseased subject. In
one aspect, the exosomes have an average diameter from about 10 to
about 250 nm, or alternatively from about 10 to about 200 nm, or
alternatively from about 10 to about 175 nm, or alternatively from
about 25 to 175 nm, or alternatively from about 40 to about 250 nm,
or alternatively from about 40 to about 200 nm, or alternatively
from about 50 to 250 nm, or alternatively from about 50 to 200 nm,
or alternatively from about 50-150 nm in average diameter. In
another aspect, the term exosome also includes microvesicles that
range from 100 nm to 1000 nm in approximate diameter. Methods to
isolated and characterize the exosomes are known in the art (see
Rekker et al. (2014) "Comparison of serum exosome isolation methods
for miroRNA profiling" Clin. Biochem. 47(102):135-138) and briefly
described herein.
[0090] In an alternate aspect, the compositions can comprise, or
alternatively consist essentially of, or yet further consist of,
one or more, two or more, three or more, four or more, five or
more, six or more, seven or more, eight or more, nine or more, ten
or more, eleven or more, twelve or more, thirteen or more, fourteen
or more, fifteen or more, sixteen or more, or alternatively all of
miR-9, -196b, -27a, -192, -214, -377, -455, -23b, -106a, -200b,
-532, -34c, -151, -653, -687, and/or -483 and/or polynucleotides
that encode them. The polynucleotides can be inserted into an
appropriate expression vector and delivered using techniques known
in the art.
[0091] Also provided herein are compositions that interfere with or
inhibit the fibrotic activity of miRNA, e.g., one or more two or
more, three or more, four or more, five or more, six or more, seven
or more, eight or more, nine or more, ten or more, eleven or more,
twelve or more, thirteen or more, fourteen or more, fifteen or
more, sixteen or more, or alternatively all of (and integers there
between) of miRs -21, -22, -7a, -24, -155, -195, -34a, -26b, -122,
-1906, -677, -541, -125b, -500, -466j, -544, -1a-2, -1194, and -700
because the exosome levels of these are elevated in fibrosis and
they thus could contribute to driving the fibrotic response. These
agents include, for example inhibitory RNA, an antagomir, and/or a
protectors, and/or the polynucleotides that encode them, that are
specific to the listed miRs. These can be combined with one or
more, two or more, three or more, four or more, five or more, six
or more, seven or more, eight or more, nine or more, ten or more,
eleven or more, twelve or more, thirteen or more, fourteen or more,
fifteen or more, sixteen or more, or alternatively all of miR-9,
-196b, miR-27a, -192, -214, -377, -455, -23b, -106a, -200b, -532,
-34c, -151, -653, -687, and/or -483 and/or polynucleotides that
encode them. The polynucleotides can be inserted into an
appropriate expression vector and delivered using techniques known
in the art.
[0092] These compositions are useful for the treatment of disease,
such as fibrotic disease, a liver or a hepatic disease and
associated disorders.
[0093] miRNA, inhibitory RNA, antagomirs, and protectors can be
prepared by any appropriate method, e.g., by isolation form natural
products such as exosomes or recombinantly produced, for example,
by a chemical synthetic method or a method using genetic
recombination technique. When the production is carried out by a
method using genetic recombination technique, miRNA can, for
example, be produced through a transcription reaction with use of a
DNA template and a RNA polymerase obtained by means of gene
recombination. Examples of suitable RNA polymerase include a T7 RNA
polymerase, a T3 RNA polymerase, and a SP6 RNA polymerase. They can
be produced in a eukaryotic or prokaryotic cells, e.g., E. coli or
other bacteria, yeast, mammalian, human, murine or simian for
example.
[0094] In some aspects, the miRNAs are contained in or encoded by
other nucleic acid molecules, and it is these nucleic acids that
are isolated and purified for use in the described methods. The
miRNAs can be contained within larger RNA molecules which, when
processed, produce the miRNAs described herein. In another example,
the miRNAs are encoded by nucleic acid molecules, which may be
contained, for example, in vectors. Thus, also provided herein are
vectors that contain nucleic acid that encodes the miRNAs.
[0095] In some instances, the miRNAs or nucleic acids encoding the
miRNA are produced synthetically using well-known methods or are
isolated from cells or tissues. Typically, the miRNAs or nucleic
acid molecules containing or encoding the miRNAs are obtained using
genetic engineering techniques to produce a recombinant nucleic
acid molecule, which can then be isolated or purified by techniques
well known to one of ordinary skill in the art. In these
recombinant methods, nucleic acid encoding the miRNA is cloned into
an appropriate expression vector. It is well within the skill of a
skilled artisan to design DNA that encodes a miRNA provided
herein.
[0096] Any suitable host/vector system can be used to express one
or more of the miRNAs described herein. It is well with the skill
of those in the art to select an appropriate system based on, for
example, whether the miRNA or nucleic acid molecule encoding the
miRNA is being isolated and purified for subsequent use, and/or
whether the miRNA will be expressed in vivo following
administration to a subject.
[0097] In particular examples, the miRNAs described herein
(including precursor miRNAs) are encoded by vectors for expression
of the miRNA in vivo following administration of the vector to a
subject. The choice of vector, including the particular regulatory
elements contained in the vector for expression of heterologous
nucleic acid, can be influenced by the cell type to which the
vector is being targeted, and such selection is well within the
level of skill of the skilled artisan. For example, the nucleic
acid encoding the miRNA can be under the control of a tissue- or
cell-specific promoter, such that the miRNA is only expressed in
that particular tissue or cell type. Tissue- or cell-specific
promoters are well known in the art.
[0098] In further examples, the nucleic acid encoding the miRNA is
cloned into a viral vector, including, but not limited to,
retroviral, adenoviral, lentiviral and adeno-associated viral
vectors. Although viral vectors can be replication incompetent or
replication competent, for subsequent use in therapeutic
applications, typically replication incompetent vectors are
selected.
[0099] The activity of the miRNAs can be assessed using in vitro
assays and animal models well known to those skilled in the art.
The miRNAs also can be assessed in human clinical trials under
appropriate supervision.
[0100] In one aspect, the non-diseased subject is one that is not
suffering from liver or a fibrotic disease or an associated
disorder. In another aspect, the exosomes have a microRNA (miR)
profile comprising, or alternatively consisting essentially of, or
yet further consisting of, lack of up-regulation of one or both of
the markers miR-26b and/or -122, as compared to the miR profile of
a subject that is suffering from liver or a fibrotic disease or an
associated disorder.
[0101] Also provided is a purified or isolated population of
exosomes isolated from a body fluid of a non-diseased subject,
wherein the microRNA (miR) profile of the exosomes comprises, or
alternatively consist essentially of, or yet further consist of,
lack of down-regulation of one or both of miR-9 and/or -196b, as
compared to the miR profile of a subject that is suffering from
liver or a fibrotic disease or an associated disorder.
[0102] Further provided is a purified or isolated population of
exosomes isolated from a body fluid of a non-diseased subject,
wherein the microRNA (miR) profile of the exosomes comprises lack
of up-regulation of one or both of the markers miR-26b and/or -122
and/or lack of down-regulation of one or both of miR-9 and/or
-196b, as compared to the miR profile of a subject that is
suffering from liver or a fibrotic disease or an associated
disorder.
[0103] In one aspect, the purified or isolated population of
exosomes further comprise lack of up-regulation or low expression
of one or more, two or more, three or more, four or more, five or
more, six or more, seven or more, eight or more, nine or more, ten
or more, eleven or more, twelve or more, thirteen or more, fourteen
or more, fifteen or more, or all (including all integers there
between) of miR-7a, -21, -22, -24, -34a, -155, -195, -677, -541,
-125b, -500, -466j, -544, -1a-2, -1194, -1906, -26b, -122 and/or
-700 and/or lack of down-regulation or high expression of one or
more, two or more, three or more, four or more, five or more, six
or more, seven or more, eight or more, nine or more, ten or more,
eleven or more, twelve or more, or all of miR -27a, -192, -214,
-377, -455, -23b, -9, -106a, -200b, -196b, -532, -34c, -151, -653,
-687, and/or -483.
[0104] The purified or isolated population of exosomes are isolated
or purified from a body fluid selected from the group of urine,
lymphatic fluid, breast milk, saliva, blood, serum and/or plasma.
The exosomes can be isolated from more than one source and combined
or alternatively maintained as a tissue-specific sample.
[0105] This disclosure also provides pharmaceutical compositions
comprising, or consisting essentially of, or yet further consisting
of, purified or isolated exosomes and/or miRNA as described above.
In one aspect, the pharmaceutical composition comprises, or
alternatively consists essentially of, or yet further consists of,
a pharmaceutically acceptable carrier and an effective amount of
these exosomes isolated from a body fluid of a non-diseased
subject. Non-limiting examples of carriers include phosphate
buffered saline (PBS), saline or a biocompatible matrix material
such as a collagen matrix or a decellularized liver matrix (DCM as
disclosed in Wang et al. (2014) J. Biomed. Mater Res. A.
102(4):1017-1025) for topical or local administration. The
compositions can optionally contain a protease inhibitor, glycerol
and/or dimethyl sulfoxide (DMSO). They can be further formulated in
liposomes or micelles, using methods known in the art.
[0106] For each of the above compositions, the fluid from which the
exosomes are isolated or purified is selected from the group of
urine, breast milk, lymphatic fluid, saliva, blood, serum or plasma
and can be present in a variety of concentrations.
[0107] The pharmaceutically acceptable carrier comprises one or
more of a biocompatible matrix or a liquid carrier,
[0108] The pharmaceutical compositions of this disclosure can be
formulated for freeze-drying or lyophilisation using methods known
in the art.
[0109] The pharmaceutical composition are intended for in vitro and
in vivo use. The compositions can comprise a concentration of
exosomes and/or miRNA and/or inhibitory molecules (as measured by
exosomal protein content (measured by Bicinchoninic protein assay
(BCA), commercially available from Bio-Rad or Pierce Biotechnology,
Inc., for example) from about 1 mg/ml to about 10 mg/ml, or
alternatively from about 1 to about 8 mg/ml, or alternatively from
2 to about 8 mg/ml, or alternatively from 2 to about 5 mg/ml, or
about 2 to 4 mg/ml, or alternatively from 3 mg/ml to 20 mg/ml When
administered to the subject, an effective amount of the exosomes
are administered to the subject, to cause at least about 75%, or
alternatively at least about 80%, or alternatively at least about
85%, or alternatively at least about 90%, or alternatively at least
about 95%, or alternatively at least about 99% effectiveness in the
methods provided herein as compared to a control that does not
receive the composition. Comparative effectiveness can be
determined by suitable in vitro or in vivo methods as known in the
art and briefly exemplified herein.
[0110] In one aspect, the compositions are pharmaceutical
formulations for use in the therapeutic methods of this disclosure
and for the treatment of the appropriate or relevant disease. While
the examples are noted for the treatment of hepatic disease, the
principles can be applied to other disease conditions, including
fibrotic disease in other organ or tissue as noted above. In the
context of this disclosure when the exosomes and/or miRNA are
isolated from a subject that is not suffering from a fibrotic or
hepatic disease or an associated disorder, the exosomes are useful
for treating a subject having the disease. When the exosomes and/or
miRNA are isolated from a subject that is not suffering from a
cardiac fibrotic disease or an associated disorder, the exosomes
are useful for treating a subject having the cardiac fibrotic
disease or an associated disorder.
[0111] In a further aspect, the disclosure provides a
pharmaceutical composition comprising, or alternatively consisting
essentially of, or yet further consisting of, the isolated or
purified exosomes in a concentration such that composition
comprises at least 75%, or alternatively at least 80%, or
alternatively at least 85%, or alternatively at least 90%, or
alternatively at least 95% or alternatively at least 97%, or
alternatively at least 98%, or alternatively, at least 99% of
exosomes (% noted as mg of exosomes and/or miRNA per mg of total
proteins) in the total composition.
Therapeutic Methods
[0112] The compositions are useful for the preparation of a
medicament and/or to perform methods for one or more of: a)
inhibiting the progression of, b) preventing or c) treating, a
disease, e.g., a fibrotic disease or an associated disorder. As
noted above, the exosomes are selected and purified from the body
fluid of a patient that does not have the disease or disorder to be
treated. In addition or alternatively, RNA polynucleotides and/or
DNA that encodes them are isolated or chemically or recombinantly
produced. In one aspect, the exosomes previously have been isolated
from the subject prior to disease diagnosis. For example, a patient
may be at risk for a disease, e.g., liver disease, and the exosomes
are isolated and processed for storage (at for example, in an
appropriate solvent such as glycerol and DMSO, at from about
-70.degree. C. to -90.degree. C. or alternatively about -80.degree.
C.)
[0113] In one aspect, the compositions are useful for the
preparation of a medicament and/or to perform methods for one or
more of: a) inhibiting the progression of, b) preventing or c)
treating, liver fibrosis or an associated disorder in a subject in
need thereof. The methods comprise, or alternatively consist
essentially of, or yet further consist of, administering to the
subject an effective amount of the pharmaceutical composition
described above. Non-limiting examples of "an associated disorder"
include the group of: inflammation of the liver, high deposition of
lipids, high deposition of insoluble collagen, high deposition of
extracellular matrix components, cirrhosis, hepatocarcinoma,
end-stage liver disease. These conditions are well known in the art
and can be diagnosed by a treating physician.
[0114] The therapy and patient's health can be monitored using the
diagnostic methods disclosed herein. A non-limiting example of such
is by determining the level of one, or two or more, three or more,
or all of miR-26b or -122, miR-9, or -196b in a sample isolated
from the patient prior to, during and after the therapy. In one
aspect, this disclosure also provides determining the liver or
hepatic health status of a subject by determining the level of one
or more or two or more of miR-26b or -122, miR-9, and/or -196b in a
sample isolated from the subject, lack of down-regulation (e.g.,
normal or up-regulation) of one or more of these markers identifies
the patient as not likely to have hepatic disease and
down-regulation of these markers identifies the subject as likely
to have liver fibrosis or an associated disease.
[0115] The therapy and patient's health can be monitored by
determining the level of one or more, two or more, three or more,
or all of miR-26b, -122, miR-9 and/or -196b in a sample isolated
from the patient prior to, during and after the therapy. In a
further aspect, the therapy and patient's health and therapy
response can be monitored by determining the level of one or more,
two or more, three or more, or four or more, or five or more, or
six or more, or seven or more, or eight or more, or nine or more,
or ten or more, or eleven or more, or twelve or more, or thirteen
or more, or fourteen or more, or fifteen or more, or sixteen or
more, or seventeen or more, or eighteen or more, or nineteen or
more, or twenty or more, or twenty-one or more, or twenty-two or
more, or twenty-three or more, or twenty-four or more, or
twenty-five or more, or twenty-six, or twenty-seven or more, or all
of (and integers there between) of miR-26b, -122, -9, -196b, -677,
-541, -125b, -500, -466j, -544, -1a-2, -1194, -700, -27a, -21, -22,
-7a, -24, -155, -195, -34a, -192, -214, -377, -1906, -455, -23b,
-106a, -200b, -532, -34c, -151, -653, -687, and/or -483, in a
sample isolated from the patient prior to, during and after the
therapy and wherein high levels of one or more of miR-26b, -122,
-677, -541, -125b, -500, -466, -21, -22, -7a, -24, -155, -195,
-34a, -1906, -544, -1a-2, -1194 and/or -700, and one or more of low
levels of miR-9, -377, -27a, -192, -214, -196b, -455, -23b, -106a,
-200b, -532, -34c, -151, -653, -687, and/or -483, identifies the
patient as likely to have fibrotic or hepatic disease.
[0116] In one aspect, this disclosure also provides determining the
health status, e.g., hepatic health status of a subject comprising
determining the level of one or more, two or more, three or more,
or four or more, or five or more, or six or more, or seven or more,
or eight or more, or nine or more, or ten or more, or eleven or
more, or twelve or more, or thirteen or more, or fourteen or more,
or fifteen or more, or sixteen or more, or seventeen or more, or
eighteen or more, or nineteen or more, or twenty or more, or
twenty-one or more, or twenty-two or more, or twenty-three or more,
or twenty-four or more, or twenty-five or more, or twenty-six, or
twenty-seven or more, or all of (and integers there between) of
miR-26b, -122, -9, -196b, -677, -541, -125b, -500, -466j, -544,
-1a-2, -1194, -700, -27a, -21, -22, -7a, -24, -155, -195, -34a,
-192, -214, -377, -1906, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483, in a sample isolated from the
patient prior to, during and after the therapy and wherein high
levels of one or more of miR-26b, -122, -677, -541, -125b, -500,
-466, -21, -22, -7a, -24, -155, -195, -34a, -1906, -544, -1a-2,
-1194 and/or-700, and one or more of low levels of miR-9, -377,
-27a, -192, -214, -196b, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483, identifies the patient as likely to
have fibrotic or hepatic disease.
[0117] The compositions can then be administered to subjects
identified as likely to have fibrosis, liver fibrosis, hepatic
disease or an associated disorder. In these methods, the exosomes
are allogeneic or autologous to the subject receiving the
exosomes.
[0118] As noted, the composition is administered in an effective
amount. For example, an effective amount comprises from about 1 to
about 1,000 mg/kg, or alternatively from about 1 to about 500
mg/kg, or alternatively from about 5 to about 500 mg/kg, or
alternatively from about 10 to about 100 mg/kg, or alternatively
from about 5 mg/kg to about 100 mg/kg, or alternatively from about
10 mg/kg to about 80 mg/kg, or alternatively from about 10 mg/kg to
about 50 mg/kg, or alternatively from about 15 mg/kg to about 50
mg/kg, or alternatively more than 5 mg/kg, or alternatively more
than about 10 mg/kg, or alternatively more than about 15 mg/kg, or
alternatively more than about 20 mg/kg, or alternatively more than
25 mg/kg, or alternatively more than 30 mg/kg, each as measured per
kg of body weight of the subject. The effective amount is in one
aspect, per dose, or as a daily dose, or alternatively the total
over the course of treatment.
[0119] The compositions can be administered by oral, parenteral
(e.g., intramuscular, intraperitoneal, intravenous, ICV,
intracisternal injection or infusion, subcutaneous injection, or
implant), by inhalation spray nasal, vaginal, rectal, sublingual,
urethral (e.g., urethral suppository) or topical routes of
administration (e.g., gel, ointment, cream, aerosol, etc.) and can
be formulated, alone or together, in suitable dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants, excipients, and vehicles
appropriate for each route of administration. Non-limiting examples
of carriers include phosphate buffered saline (PBS), saline or a
biocompatible matrix material such as a decellularized liver matrix
(DCM as disclosed in Wang et al. (2014) J. Biomed. Mater Res. A.
102(4):1017-1025) for topical or local administration. The
compositions can optionally contain a protease inhibitor, glycerol
and/or dimethyl sulfoxide (DMSO).
[0120] The pharmaceutical compositions can be conveniently
presented in dosage unit form and can be prepared by any of the
methods well known in the art of pharmacy. The pharmaceutical
compositions can be, for example, prepared by uniformly and
intimately bringing the active ingredient into association with a
liquid carrier, a finely divided solid carrier or both, and then,
if necessary, shaping the product into the desired formulation. In
the pharmaceutical composition the active object compound is
included in an amount sufficient to produce the desired therapeutic
effect. For example, pharmaceutical compositions of the disclosure
may take a form suitable for virtually any mode of administration,
including, for example, topical, ocular, oral, buccal, systemic,
nasal, injection, transdermal, rectal, and vaginal, or a form
suitable for administration by inhalation or insufflation.
[0121] Systemic formulations include those designed for
administration by injection (e.g., subcutaneous, intravenous,
intramuscular, intrathecal, or intraperitoneal injection) as well
as those designed for transdermal, transmucosal, oral, or pulmonary
administration.
[0122] Useful injectable preparations include sterile suspensions,
solutions, or emulsions of the active compound(s) in aqueous or
oily vehicles. The compositions may also contain formulating
agents, such as suspending, stabilizing, and/or dispersing agents.
The formulations for injection can be presented in unit dosage
form, e.g., in ampules or in multidose containers, and may contain
added preservatives.
[0123] Alternatively, the injectable formulation can be provided in
powder form for reconstitution with a suitable vehicle, including
but not limited to sterile pyrogen free water, buffer, and dextrose
solution, before use. To this end, the active compound(s) can be
dried by any art-known technique, such as lyophilisation, and
reconstituted prior to use.
[0124] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are known in the art.
[0125] For oral administration, the pharmaceutical compositions may
take the form of, for example, lozenges, tablets, or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinised maize
starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose);
fillers (e.g., lactose, microcrystalline cellulose, or calcium
hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or
silica); disintegrants (e.g., potato starch or sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulfate). The
tablets can be coated by methods well known in the art with, for
example, sugars, films, or enteric coatings. Additionally, the
pharmaceutical compositions containing the 2,4-substituted
pyrmidinediamine as active ingredient or prodrug thereof in a form
suitable for oral use may also include, for example, troches,
lozenges, aqueous, or oily suspensions, dispersible powders or
granules, emulsions, hard or soft capsules, or syrups or
elixirs.
[0126] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions, and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient (including drug
and/or prodrug) in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of
tablets. These excipients can be for example, inert diluents, such
as calcium carbonate, sodium carbonate, lactose, calcium phosphate
or sodium phosphate; granulating and disintegrating agents (e.g.,
corn starch or alginic acid); binding agents (e.g., starch,
gelatin, or acacia); and lubricating agents (e.g., magnesium
stearate, stearic acid, or talc). The tablets can be left uncoated
or they can be coated by known techniques to delay disintegration
and absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate can
be employed. They may also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form
osmotic therapeutic tablets for control release. The pharmaceutical
compositions of the disclosure may also be in the form of
oil-in-water emulsions.
[0127] Liquid preparations for oral administration may take the
form of, for example, elixirs, solutions, syrups, or suspensions,
or they can be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid
preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives, or hydrogenated
edible fats); emulsifying agents (e.g., lecithin, or acacia);
non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol,
cremophore.TM., or fractionated vegetable oils); and preservatives
(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, preservatives,
flavoring, coloring, and sweetening agents as appropriate.
[0128] Preparations for oral administration can be suitably
formulated to give controlled release or sustained release of the
active compound, as is well known. The sustained release
formulations of this disclosure are preferably in the form of a
compressed tablet comprising an intimate mixture of compound of the
disclosure and a partially neutralized pH-dependent binder that
controls the rate of compound dissolution in aqueous media across
the range of pH in the stomach (typically approximately 2) and in
the intestine (typically approximately about 5.5).
[0129] To provide for a sustained release of the exosomes, one or
more pH-dependent binders can be chosen to control the dissolution
profile of the sustained release formulation so that the
formulation releases compound slowly and continuously as the
formulation is passed through the stomach and gastrointestinal
tract. Accordingly, the pH-dependent binders suitable for use in
this disclosure are those which inhibit exosome breakdown and/or
release of its contents during its residence in the stomach (where
the pH is-below about 4.5), and which promotes the release of a
therapeutic amount of the compound of the disclosure from the
dosage form in the lower gastrointestinal tract (where the pH is
generally greater than about 4.5). Many materials known in the
pharmaceutical art as "enteric" binders and coating agents have a
desired pH dissolution properties. The examples include phthalic
acid derivatives such as the phthalic acid derivatives of vinyl
polymers and copolymers, hydroxyalkylcelluloses, alkylcelluloses,
cellulose acetates, hydroxyalkylcellulose acetates, cellulose
ethers, alkylcellulose acetates, and the partial esters thereof,
and polymers and copolymers of lower alkyl acrylic acids and lower
alkyl acrylates, and the partial esters thereof. One or more
pH-dependent binders present in the sustained release formulation
of the disclosure are in an amount ranging from about 1 to about 20
wt %, more preferably from about 5 to about 12 wt % and most
preferably about 10 wt %.
[0130] One or more pH-independent binders may be in used in oral
sustained release formulation of the disclosure. The pH-independent
binders can be present in the formulation of this disclosure in an
amount ranging from about 1 to about 10 wt %, and preferably in
amount ranging from about 1 to about 3 wt % and most preferably
about 2.0 wt %.
[0131] The sustained release formulation of the disclosure may also
contain pharmaceutical excipients intimately admixed with the
compound and the pH-dependent binder. Pharmaceutically acceptable
excipients may include, for example, pH-independent binders or
film-forming agents such as hydroxypropyl methylcellulose,
hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone,
neutral poly(meth)acrylate esters, starch, gelatin, sugars,
carboxymethylcellulose, and the like. Other useful pharmaceutical
excipients include diluents such as lactose, mannitol, dry starch,
microcrystalline cellulose and the like; surface active agents such
as polyoxyethylene sorbitan esters, sorbitan esters and the like;
and coloring agents and flavoring agents. Lubricants (such as talc
and magnesium stearate) and other tableting aids can also be
optionally present.
[0132] The pharmaceutical compositions can be in the form of a
sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution, and isotonic sodium chloride solution. The compositions
may also be administered in the form of suppositories for rectal or
urethral administration of the drug.
[0133] For topical use, creams, ointments, jellies, gels,
solutions, suspensions, etc., containing the compounds of the
disclosure, can be employed. In some embodiments, the compounds of
the disclosure can be formulated for topical administration with
polyethylene glycol (PEG). These formulations may optionally
comprise additional pharmaceutically acceptable ingredients such as
diluents, stabilizers, and/or adjuvants.
[0134] Included among the devices which can be used to administer
compounds of the disclosure, are those well-known in the art, such
as metered dose inhalers, liquid nebulizers, dry powder inhalers,
sprayers, thermal vaporizers, and the like. Other suitable
technology for administration of particular compounds of the
disclosure, includes electrohydrodynamic aerosolizers. As those
skilled in the art will recognize, the formulation of compounds,
the quantity of the formulation delivered, and the duration of
administration of a single dose depend on the type of inhalation
device employed as well as other factors. For some aerosol delivery
systems, such as nebulizers, the frequency of administration and
length of time for which the system is activated will depend mainly
on the concentration of compounds in the aerosol. For example,
shorter periods of administration can be used at higher
concentrations of compounds in the nebulizer solution. Devices such
as metered dose inhalers can produce higher aerosol concentrations
and can be operated for shorter periods to deliver the desired
amount of compounds in some embodiments. Devices such as dry powder
inhalers deliver active agent until a given charge of agent is
expelled from the device. In this type of inhaler, the amount of
compounds in a given quantity of the powder determines the dose
delivered in a single administration.
[0135] Formulations of compounds of the disclosure for
administration from a dry powder inhaler may typically include a
finely divided dry powder containing compounds, but the powder can
also include a bulking agent, buffer, carrier, excipient, another
additive, or the like. Additives can be included in a dry powder
formulation of compounds of the disclosure, for example, to dilute
the powder as required for delivery from the particular powder
inhaler, to facilitate processing of the formulation, to provide
advantageous powder properties to the formulation, to facilitate
dispersion of the powder from the inhalation device, to stabilize
to the formulation (e.g., antioxidants or buffers), to provide
taste to the formulation, or the like. Typical additives include
mono-, di-, and polysaccharides; sugar alcohols and other polyols,
such as, for example, lactose, glucose, raffinose, melezitose,
lactitol, maltitol, trehalose, sucrose, mannitol, starch, or
combinations thereof; surfactants, such as sorbitols,
diphosphatidyl choline, or lecithin; and the like.
[0136] For prolonged delivery, the exosome compositions can be
formulated as a depot preparation for administration by
implantation or intramuscular injection. The active ingredient can
be formulated with suitable polymeric or hydrophobic materials
(e.g., as an emulsion in an acceptable oil) or ion exchange resins,
or as sparingly soluble derivatives (e.g., as a sparingly soluble
salt). Alternatively, transdermal delivery systems manufactured as
an adhesive disc or patch which slowly releases the active
compound(s) for percutaneous absorption can be used. To this end,
permeation enhancers can be used to facilitate transdermal
penetration of the active compound(s). Suitable transdermal patches
are described in, for example, U.S. Pat. No. 5,407,713; U.S. Pat.
No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168;
U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No.
5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S.
Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No.
4,921,475.
[0137] Alternatively, other pharmaceutical delivery systems can be
employed. Liposomes and emulsions are well-known examples of
delivery vehicles that can be used to deliver active compound(s) or
prodrug(s). Certain organic solvents such as dimethylsulfoxide
(DMSO) may also be employed, although usually at the cost of
greater toxicity.
[0138] The pharmaceutical compositions may, if desired, be
presented in a pack or dispenser device which may contain one or
more unit dosage forms containing the active compound(s). The pack
may, for example, comprise metal or plastic foil, such as a blister
pack. The pack or dispenser device can be accompanied by
instructions for administration.
[0139] The compositions will generally be used in an amount
effective to achieve the intended result, for example, in an amount
effective to treat or prevent the particular condition being
treated. The compound(s) can be administered therapeutically to
achieve therapeutic benefit or prophylactically to achieve
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated and/or
eradication or amelioration of one or more of the symptoms
associated with the underlying disorder such that the patient
reports an improvement in feeling or condition, notwithstanding
that the patient may still be afflicted with the underlying
disorder. Therapeutic benefit also includes halting or slowing the
progression of the disease, regardless of whether improvement is
realized.
[0140] The amount of compound administered will depend upon a
variety of factors, including, for example, the particular
condition being treated, the mode of administration, the severity
of the condition being treated, the age and weight of the patient,
the bioavailability of the particular active compound.
Determination of an effective dosage is well within the
capabilities of those skilled in the art. As known by those of
skill in the art, the preferred dosage of compounds of the
disclosure will also depend on the age, weight, general health, and
severity of the condition of the individual being treated. Dosage
may also need to be tailored to the sex of the individual and/or
the lung capacity of the individual, where administered by
inhalation. Dosage, and frequency of administration of the
compositions will also depend on whether the compositions are
formulated for treatment of acute episodes of a condition or for
the prophylactic treatment of a disorder. A skilled practitioner
will be able to determine the optimal dose for a particular
individual.
[0141] For prophylactic administration, the compound can be
administered to a patient at risk of developing one of the
previously described conditions. For example, if it is unknown
whether a patient is allergic to a particular drug, the compound
can be administered prior to administration of the drug to avoid or
ameliorate an allergic response to the drug. Alternatively,
prophylactic administration can be applied to avoid the onset of
symptoms in a patient diagnosed with the underlying disorder.
[0142] Effective dosages can be estimated initially from in vitro
assays. For example, an initial dosage for use in animals can be
formulated to achieve a therapeutic concentration and/or dosage of
the exosome composition, as measured in an in vitro assay.
Calculating dosages to achieve such effective dosages for other
animal models or human patients is well within the capabilities of
skilled artisans. For guidance, the reader is referred to Fingl
& Woodbury, "General Principles," In: Goodman and Gilman's The
Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latest
edition, Pergamagon Press, and the references cited therein.
[0143] Initial dosages can also be estimated from in vivo data,
such as animal models. Animal models useful for testing the
efficacy of compounds to treat or prevent the various diseases
described above are well-known in the art. Ordinarily skilled
artisans can routinely adapt such information to determine dosages
suitable for human administration.
[0144] Dosage amounts will typically be in the range of from about
0.0001 or 0.001 or 0.01 mg/kg/day to about 1000 mg/kg/day, but can
be higher or lower, depending upon, among other factors, the
activity of the composition, its bioavailability, the mode of
administration, and various factors discussed above. Dosage amount
and interval can be adjusted individually to provide local and/or
systemic concentration of the exosomes that are sufficient to
maintain therapeutic or prophylactic effect. For example, the
compositions can be administered once per week, several times per
week (e.g., every other day), once per day, or multiple times per
day, depending upon, among other things, the mode of
administration, the specific indication being treated, and the
judgment of the prescribing physician. Skilled artisans will be
able to optimize effective local dosages without undue
experimentation.
[0145] Preferably, the compound(s) will provide therapeutic or
prophylactic benefit without causing substantial toxicity. Toxicity
of the compound(s) can be determined using standard pharmaceutical
procedures. The dose ratio between toxic and therapeutic (or
prophylactic) effect is the therapeutic index. Compounds(s) that
exhibit high therapeutic indices are preferred.
Kits
[0146] Also provided are kits for administration of the
compositions and carrying out the diagnostic methods comprising the
composition that may include an appropriate dosage amount. Kits may
further comprise suitable packaging and/or instructions for use of
the compositions and/or diagnostic methods. Kits may also comprise
a means for the delivery of the at least one compositions and a
device such as an inhaler, spray dispenser (e.g., nasal spray),
syringe for injection, or pressure pack for capsules, tables,
suppositories, or other device as described herein.
[0147] In one aspect, a kit comprising one or more probes and/or
primers to determine the expression profile of one or more, two or
more, three or more or all four of miR26b, miR-122 miR-9; and/or
miR-196b. In a further aspect, the kit also comprises one or more,
two or more, three or more, four or more, five or more, six or
more, seven or more, eight or more, nine or more, ten or more,
eleven or more, twelve or more, thirteen or more, fourteen or more,
fifteen or more, or all (including all integers there between) of
miR-26b, -122, -9, -196b, -677, -541, -125b, -500, -466j, -544,
-1a-2, -1194, -700, -27a, -21, -22, -7a, -24, -155, -195, -34a,
-192, -214, -377, -1906, -455, -23b, -106a, -200b, -532, -34c,
-151, -653, -687, and/or -483.
[0148] In a further aspect, the kit further comprises detectable
labels that in one aspect are attached to the probes and/or
primers, wherein in one aspect, the detectable label is not a
polynucleotide.
[0149] Additionally, the kits can contain the composition and
reagents to prepare a composition for administration. The
composition can be in a dry or lyophilized form or in a solution,
particularly a sterile solution. When the composition is in a dry
form, the reagent may comprise a pharmaceutically acceptable
diluent for preparing a liquid formulation. The kit may contain a
device for administration or for dispensing the compositions,
including, but not limited to, syringe, pipette, transdermal patch,
or inhalant.
[0150] The kits may include other therapeutic compounds for use in
conjunction with the compounds described herein and as such, the
methods as disclosed herein can contain other appropriate
therapeutic compounds or agents. These compounds can be provided in
a separate form or mixed with the compositions of the present
disclosure. The kits will include appropriate instructions for
preparation and administration of the composition, side effects of
the compositions, and any other relevant information. The
instructions can be in any suitable format, including, but not
limited to, printed matter, videotape, computer readable disk, or
optical disc.
[0151] Kits may also be provided that contain sufficient dosages of
the compounds or composition to provide effective treatment for an
individual for an extended period, such as a week, 2 weeks, 3,
weeks, 4 weeks, 6 weeks, or 8 weeks or more.
[0152] The following examples are intended to illustrate, and not
limit, the disclosed herein. For example, while the examples are
noted to be for the isolation, purification and use of exosome
compositions for the treatment of a fibrotic or liver disease or an
associated disorder, the methods and compositions can be modified
for the treatment of other fibrotic diseases as noted herein.
EXAMPLES
Example 1
Isolation of Exosomes for MiR Profiling.
[0153] PureExo Exosome Isolation Kits were used to isolate serum
exosomes. MiR profiling was performed on exosomal RNA from 1 ml of
pooled serum (5 mice; 200 .mu.l/mouse) using a mouse miRnome miR
PCR Array. miR profiling was performed for the 940 best
characterized miRs in the mouse miRnome on exosomes isolated from
the circulation of mice after 1 or 5 weeks of treatment with
CCl.sub.4, as compared to oil-treated controls, with liver
injury/fibrosis confirmed histologically. Balb/c mice were treated
i.p. with corn oil (30 .mu.l) or carbon tetrachloride (CCl.sub.4; 4
.mu.l CCl.sub.4 diluted in 26 .mu.l corn oil)) for up to 5 weeks.
Differentially expressed miRs were confirmed and/or further
evaluated by qRT-PCR of exosomal RNA independently obtained at 1-,
4- or 5-weeks of CCl.sub.4 administration (n=5).
[0154] Isolated exosomes from mice serum were bi-membrane vesicles,
50-200 nm in diameter, and positive for the exosome markers, CD9
and flotillin-1. Microarray analysis revealed significant
alterations in the expression of many hundreds of miRs after either
1- or 5-wks of CCl.sub.4 treatment as compared to their respective
oil controls. Applicants then focused on selected miRs previously
reported to be altered in fibrotic liver, and confirmed the data by
RT-PCR. The exosomal levels of these miRs after 5 weeks of
CCl.sub.4 (including up-regulation of miR-7a, -21, -22, -24, -34a,
-155, -195, -455, -23b, -106a, -200b, -532, -34c, -151, -653, -687,
and/or -483, and down-regulation of miR-27a, -192, -214, -377,
-1906, -677, -541, -125b, -500, -466j, -544, -1a-2, -1194, and/or
-700) reflected their previously documented changes at the tissue
level in fibrotic liver. In addition, several exosomal miRs that
have not yet to be reported in the literature as being altered
during liver fibrosis emerged as potentially novel fibrosis markers
(e.g. up-regulation of miR-26b or -122; down-regulation of miR-9 or
-196b). As compared to their levels at 5 weeks, many of these miRs
exhibited individually distinct patterns of expression during the
course of fibrosis progression.
[0155] Dynamic changes occur in the miR content of circulating
exosomes during experimental hepatic fibrosis supporting the
concept that fibrosis progression and severity is amenable to
minimally-invasive assessment through determination of signature
exosomal miRs.
Example 2
MircoRNA (miR) Profiles of Circulating Exosomes from Healthy Mice
are Distinct from Those of Mice with Hepatic Fibrosis.
[0156] MiR profiling was performed for the 940 best characterized
miRs in the mouse miRnome on exosomes isolated from the circulation
of mice after 1 or 5 weeks of treatment with CCl.sub.4, as compared
to oil-treated controls, with liver injury/fibrosis confirmed
histologically; mice were treated i.p. with corn oil (30 .mu.l) or
carbon tetrachloride (CCl.sub.4; 4 .mu.l CCl.sub.4 diluted in 26
.mu.l corn oil) for 1 week or 5 weeks. (FIG. 1A). Of the several
hundred miRs that were significantly up- or down-regulated in
circulating exosomes as a function of CCl.sub.4 versus oil and/or
duration of CCl.sub.4 exposure (FIG. 1A), Applicants then focused
on selected miRs previously reported to be altered in fibrotic
liver, and confirmed the data by RT-PCR. Chen, L., Charrier, A.,
Zhou, Y., Yu, B., Agarwal, K., Tsukamoto, H., Lee, L. J.,
Paulaitis, M. E., Brigstock, D. R. (2014) Epigenetic regulation of
connective tissue growth factor by delivery of microRNA-214 in
exosomes from mouse or human hepatic stellate cells Hepatology
DOI:10.1002/hep.26768. PMID: 24122827.
[0157] As shown in FIG. 1B, the exosomal levels of these miRs often
reflected their previously documented changes at the tissue level
(including Applicants' own miR-214 data; Chen et al. (2014),
supra). In addition, several exosomal miRs were validated by RT-PCR
as potentially novel fibrosis markers even though they have not yet
been reported to be altered in fibrotic livers. Many of these miRs
exhibited individually distinct patterns of expression over time,
highlighting that appropriately selected "slates" of exosomal miRs
have biomarker potential for discriminating different stages of
liver injury and/or fibrosis. This aside, the data show that the
miR payload in circulating exosomes is altered during fibrosing
liver injury.
[0158] Without being bound by theory, these data show that the
molecular cargo in exosomes from healthy individuals reflects a
non-disease status is that actually of potential therapeutic
benefit when delivered in a setting of liver fibrosis.
Example 3
Exosomes from Serum of Normal Mice Reduce Expression of
Pro-Fibrogenic Signals in in vitro-Activated Hepatic Stellate
Cells, the Principal Fibrosis-Producing Cells of the Liver
[0159] Based on the significant differences between the payloads of
exosomes from serum of normal versus fibrotic mice (FIGS. 1A &
1B), Applicants next tested whether these exosomes differentially
regulated function of hepatic stellate cells (HSC). Although HSC
are an usually quiescent cell type in the liver, during injury they
become `activated` and produce molecules that are pro-fibrogenic
including connective tissue growth factor (CTGF), alpha smooth
muscle actin (.alpha.SMA; a contractile protein that facilitates
wound contraction/closure), and collagen (which is deposited as
insoluble scar material). While HSC can be activated in vivo by
many injurious agents, simply isolating the cells from normal
non-injured liver and growing them in vitro causes them to undergo
autonomous activation because they perceive the in vitro culture
conditions as a wound environment. This process of culture-induced
activation was used in this experiment. Culture-induced expression
of CTGF, .alpha.SMA or collagen .alpha.1 (I) in HSC on Day 9 of
culture was reduced >90% following 48-hr incubation of the cells
with exosomes isolated from the circulation of healthy mice (FIG.
2, lower) whereas circulating exosomes from fibrotic mice caused an
8-fold increase in .alpha.SMA expression and essentially no change
in expression of CTGF or collagen .alpha.1 (I) (FIG. 2, upper).
Thus exosomes from the circulation of healthy, but not of fibrotic
mice, significantly attenuated activation/fibrosis-related gene
expression in HSC.
Example 4
Exosomes from Serum of Normal Mice Reduce Short-Term Expression of
Pro-Fibrogenic Signals in Hepatic Stellate Cells in vitro Following
Activation of the Cells in vivo with a Fibrotic Stimulus
[0160] Applicants then tested whether serum exosomes could reverse
fibrogenic gene expression in HSC after they had been activated in
vivo. Mice received intramuscular injections (i.m.) of CCl.sub.4
(in oil) or oil (carrier control) every other day for 1 week. On
the same day, mice also received intraperitoneal (i.p.) injection
of exosomes that had been purified from the serum of normal mice.
On day 10, HSC were isolated, placed in culture for 1 day, and then
analyzed. CCl.sub.4-induced fibrogenic gene expression (FIG. 3A)
and protein production (FIG. 3B) in HSC was attenuated by serum
exosomes.
Example 5
Effect of Exosomes on CCl.sub.4-Induced Fibrotic Signaling in Mouse
Livers
[0161] Transgenic CTGF-EGFP mice that express enhanced green
fluorescent protein (EGFP) under the control of the CTGF promoter
were used in this experiment. In response to CCl.sub.4
administration, these mice show liver-specific GFP induction. This
response is principally attributed to CTGF induction in HSC during
injury but other cells such as hepatocytes or Kupffer cells may
contribute to the GFP-positive staining since CTGF may be
upregulated in these cells during liver injury. Thus, examination
of liver GFP fluorescence allowed Applicants to globally assess
total CTGF production by all cell types in the liver. The CTGF-EGFP
TG mouse is an extremely powerful and unique model in view of the
very large dynamic changes that occur in GFP expression in a
liver-specific manner, which is reflective of a massive induction
in hepatic CTGF expression against a background CTGF expression
which is normally very low in healthy mature mice.
[0162] Male or female mice received i.m. injections of CCl.sub.4 (4
CCl.sub.4 diluted in 26 .mu.l corn oil) every other day for 1 week.
On the same day, mice also received i.p injection of exosomes
(100-300 .mu.l; 3 .mu.g/ul) that had been purified from the serum
of normal mice. On day 10, livers were resected and examined.
[0163] Mice receiving exosome therapy had an overall more healthy
appearance (better coat quality, etc., not shown) and livers from
CCl.sub.4-treated mice had surface nodules and were pale in
appearance whereas those receiving exosomes were vascularized and
smooth (=normal) (FIG. 4A). CCl.sub.4 treatment caused a robust
induction of GFP production which was dose-dependently inhibited by
exosomes (FIGS. 4B & 4C). RT-PCR of total hepatic RNA showed
that CCl.sub.4-induced increases in CTGF and .alpha.SMA expression
were reduced to baseline levels by exosomes (FIG. 4D).
[0164] Immunostaining for .alpha.SMA (marker of activated HSC)
showed a substantial reduction in CCl.sub.4-treated animals
receiving exosomes as compared to those receiving CCl4 alone (FIG.
4E).
Example 6
Effect of Exosomes on CCl.sub.4-Induced Fibrosis in Mice
[0165] The purpose of this experiment is to expose mice to 5 weeks
of CCl.sub.4 to allow fibrosis to develop.
[0166] Male TG CTGF-EGFP mice were administered i.m. injection of
CCl.sub.4 (4 .mu.l CCl.sub.4 diluted in 26 .mu.l corn oil) three
times a week for 5 weeks. Starting at the beginning of week 4, some
mice also received exosomes (from the circulation of healthy mice)
by i.p. injection several hours after each CCl.sub.4 injection
during weeks 4 and 5 (100-300 .mu.l; 3 .mu.g/ul).
[0167] Induction of hepatic GFP (a surrogate of CTGF promoter
activity) by GFP was attenuated by exosomes (FIG. 5A). RT-PCR
showed that CCl.sub.4-induced increase in CTGF, collagen, or
.alpha.SMA expression and decreased in miR-214 expression trended
to baseline in mice treated with exosomes (FIG. 5B) Immunostaining
for .alpha.SMA, CTGF and collagen was strongly increased in
CCl.sub.4-treated animals but this was significantly reduced in
mice on exosome therapy. (FIGS. 5C & 5D).
Example 7
MircoRNA (miR) Profiles of Circulating Exosomes from Healthy Mice
are Distinct from Those of Mice with Hepatic Fibrosis Regardless of
Selection Bias.
[0168] The data presented in Example 1 and FIGS. 1A & 1B
compares progressive exosomal expression of selected miRs over time
during fibrosis; however, this selection was inherently "biased" in
the sense that the data was searched for miRs that had been
previously implicated in fibrosis, or which were novel. Thus, the
data was also analyzed in an unbiased method.
[0169] Since exosomes from normal mice were shown to be are
anti-fibrotic in mice with 5-week liver fibrosis, as shown above,
the 5-week data shown in FIG. 1 was reanalyzed and ranked in an
unbiased manner to identify the mIRs in the circulating exosomes
that were most different between the groups. To facilitate this,
the data were replotted using expression in normal animals as
relative to that in fibrotic animals (this comparison to the prior
examples). Data were ranked in a non-biased manner to identify
exosomal miRs in serum from healthy mice that were expressed
significantly higher ("Healthy.sub.HIGH") or lower
("Healthy.sub.LOW") than those in serum from fibrotic mice. (FIG.
6).
[0170] It was surmised that the anti-fibrotic activity of exosomes
from healthy individuals was due, at least in part, to their high
expression of Healthy.sub.HIGH miRs and low expression of
Healthy.sub.LOW miRs.
[0171] Next, RT-PCR was used to independently verify the
differential array data by confirming reduced levels of
Healthy.sub.HIGH miRs in fibrotic mice, as shown in FIG. 7.
Analysis of miR 653-5p was also conducted, yielding similar
results, but the data is not shown in FIG. 7.
Example 8
Effect of miRs on Fibrosis in vitro.
[0172] To determine whether the presence of Healthy.sub.HIGH miRs
was causal of fibrosis, the ability for the miRs to individually
attenuate the activated function in cultured mouse activated
hepatic stellate cells, which are an in vitro test of fibrogenesis,
was determined. This cell model was chosen because these cells
drive the activated phenotype in fibrosing liver injury. Mimics of
each mIR individually showed very significant anti-fibrotic
activity by inhibiting expression of CTGF, .alpha.SMA, and/or
collagen .alpha.(I), as shown in FIG. 8.
[0173] Rapid advances in the understanding of fibrogenesis and
fibrosis have resulted in identification of a plethora of targets
for anti-fibrotic therapy (Cohen-Naftaly, M. et al. (2011) Therap.
Adv. Gastroenterol. 4(6):391-417; Ghiassi-Nejad, Z. et al. (2008)
Expert Rev. Gastroenterol. Hepatol. 2(6):803-816) but the most
appropriate targets, type of therapeutic agent (e.g., cytotoxin,
siRNA, miR, antibody, etc.), and mode of in vivo administration
(carriers, delivery route, etc.) remains unclear. Since exosomes
have evolved to protect their cargo from extracellular degradation
and to deliver it into target cells, this function is exploitable
as a novel therapeutic approach for liver fibrosis.
[0174] Applicants' disclosure is a novel application, namely to
treat disease using exosomes that originate from the body fluids
(urine saliva, lymphatic fluid, breast milk, blood, serum, and/or
plasma) of healthy non-diseased subjects. Applicants' disclosure
supports the concept of "banking" exosomes from healthy subjects
for subsequent transfer back to the same subject when diseased, or
alternatively to transfer to other diseased subjects. The
disclosure has broad implications for treating a myriad of medical
conditions extending far beyond those of the liver.
[0175] The methods as disclosed herein involve the transfer into
subjects having fibrosis or fibrotic or hepatic disease or
associated disorder of a composition comprising a naturally
occurring organelle which, unlike other organelles, is released
from the cells in which it is produced. These organelles are called
"exosomes" and are tiny vesicles. During their production in their
cells of origin, exosomes are packaged with a complex mixture of
microRNA, mRNA, proteins and other molecules that are present in
the producer cell and reflect its current biosynthetic and
homeostatic state. Exosomes from cells in a healthy environment
have a different molecular "payload" than those from an injury
environment. Exosomes are released by the producer cell and
shuttled to neighboring cells which take up the exosomal contents
whereupon the recipient cell is reprogrammed according to the
nature of the information received.
[0176] It is to be understood that while the disclosure has been
described in conjunction with the above embodiments, that the
foregoing description and examples are intended to illustrate and
not limit the scope of the disclosure. Other aspects, advantages
and modifications within the scope of the disclosure will be
apparent to those skilled in the art to which the disclosure
pertains.
[0177] The disclosures illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the disclosure claimed.
[0178] Thus, it should be understood that although the present
disclosure has been specifically disclosed by preferred embodiments
and optional features, modification, improvement and variation of
the disclosures embodied therein herein disclosed may be resorted
to by those skilled in the art, and that such modifications,
improvements and variations are considered to be within the scope
of this disclosure. The materials, methods, and examples provided
here are representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the
disclosure.
[0179] The disclosure has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
disclosure. This includes the generic description of the disclosure
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0180] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0181] All publications, patent applications, patents, and other
references mentioned herein are expressly incorporated by reference
in their entirety, to the same extent as if each were incorporated
by reference individually. In case of conflict, the present
specification, including definitions, will control.
* * * * *