U.S. patent application number 16/825097 was filed with the patent office on 2020-07-23 for isolation of extracellular vesicles (evs) from red blood cells for gene therapy.
The applicant listed for this patent is City University of Hong Kong. Invention is credited to Thi Nguyet Minh Le, Jiahai Shi, Muhammad Waqas.
Application Number | 20200230259 16/825097 |
Document ID | / |
Family ID | 65360994 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200230259 |
Kind Code |
A1 |
Le; Thi Nguyet Minh ; et
al. |
July 23, 2020 |
ISOLATION OF EXTRACELLULAR VESICLES (EVS) FROM RED BLOOD CELLS FOR
GENE THERAPY
Abstract
A method of RNA delivery using extracellular vesicles (EVs)
derived from red blood cells (RBCs). The method comprises the
purification and electroporation of the EVs and applying the
RNA-loaded EVs to target cells. The method further comprises the
treatment of cancer using the RNA-loaded EVs.
Inventors: |
Le; Thi Nguyet Minh;
(Kowloon, HK) ; Shi; Jiahai; (Kowloon, HK)
; Waqas; Muhammad; (Karachi, PK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
City University of Hong Kong |
Kowloon |
|
HK |
|
|
Family ID: |
65360994 |
Appl. No.: |
16/825097 |
Filed: |
March 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15678363 |
Aug 16, 2017 |
|
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16825097 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/0008 20130101;
C12N 2310/20 20170501; C12N 9/22 20130101; A61K 2035/128 20130101;
C12N 2310/122 20130101; C12N 15/907 20130101; C12N 2310/11
20130101; A61K 48/005 20130101; A61K 48/0091 20130101; A61K 35/00
20130101; C12N 15/88 20130101; A61K 48/0075 20130101; A61K 48/0041
20130101; C12N 5/0641 20130101; G01N 33/491 20130101; A61K 35/13
20130101; A61K 35/14 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/90 20060101 C12N015/90; C12N 9/22 20060101
C12N009/22; A61K 35/13 20060101 A61K035/13; A61K 35/14 20060101
A61K035/14; G01N 33/49 20060101 G01N033/49; C12N 15/88 20060101
C12N015/88; C12N 5/078 20060101 C12N005/078 |
Claims
1. A method for RNA delivery to target cells comprising: a)
purification of extracellular vesicles (EVs) from red blood cells
(RBCs); b) electroporation of the EVs with RNAs to form RNA-loaded
EVs; and c) applying the RNA-loaded EVs to the target cells.
2. The method of claim 1, wherein the RBCs are derived from a human
and treated with calcium ionophore.
3. The method of claim 2, wherein the EVs are purified from treated
RBCs using ultracentrifugation with a sucrose cushion.
4. The method of claim 1, wherein the RNAs comprise antisense
oligonucleotides (ASO) and mRNAs.
5. The method of claim 1, wherein the target cells comprise cancer
cells.
6. The method of claim 1, wherein the target cells comprise acute
myeloid leukemia (AML) cells, breast cancer cells, or a combination
of AML cells and breast cancer cells.
7. The method of claim 1, wherein the EVs are electroporated with
ASO antagonizing miR-125b.
8. The method of claim 1, wherein the growth of the target cells is
suppressed.
9. The method of claim 1, wherein the EVs are electroporated with
dextran.
10. The method of claim 1, comprising administering to the target
cells the RNA-loaded EVs which modulate an apoptosis-related gene
expression, thereby inducing apoptosis in the target cells.
11. A method for delivery of an antisense oligonucleotide (ASO) to
target cells to suppress gene expression, wherein the method
comprises: a) purification of extracellular vesicles (EVs) from red
blood cells (RBCs); b) electroporation of the EVs with RNAs to form
RNA-loaded EVs; and c) applying the RNA-loaded EVs to the target
cells.
12. The method of claim 11, wherein the RBCs are derived from a
human and treated with calcium ionophore.
13. The method of claim 11, wherein the RNA is an ASO antagonizing
miR-125b to inhibit the oncogenic miR-125b in the target cells.
14. The method of claim 11, wherein the target cells are acute
myeloid leukemia (AML) cells, breast cancer cells, or a combination
of AML cells and breast cancer cells.
15. A method of RNA delivery to target cells for a CRISPR genome
editing system comprising: a) purification of extracellular
vesicles (EVs) from red blood cells (RBCs); b) electroporation of
the EVs with RNAs to form RNA-loaded EVs; and c) applying the
RNA-loaded EVs to the target cells.
16. The method of claim 15, wherein the EVs are electroporated with
Cas9 mRNA and gRNA.
17. The method of claim 15, wherein the EVs are electroporated with
Cas9 and gRNA plasmids.
18. The method of claim 15, wherein the target cells are cancer
cells.
19. The method of claim 15, wherein the target cells are leukemia
cells.
20. A method of treating cancer by delivery of RNA to target cells
comprising: a) purification of extracellular vesicles (EVs) from
red blood cells (RBCs); b) electroporation of the EVs with RNAs to
form RNA-loaded EVs; and c) applying the RNA-loaded EVs to the
target cells thereby inhibiting the growth of the target cells,
wherein the target cells comprise cancer cells.
21. The method of claim 20, wherein the target cells comprises
leukemia cells, breast cancer cells, or a combination of leukemia
cells and breast cancer cells.
22. The method of claim 20, wherein the target cells comprise acute
myeloid leukemia cells.
23. The method of claim 20, wherein the step c) comprises a step of
administering the RNA-loaded EVs to a subject having the target
cells via a local or systemic administration.
24. The method of claim 20, wherein the growth of the target cells
is suppressed after the step c).
Description
SEQUENCE LISTING
[0001] The Sequence Listing file entitled "sequencelisting" having
a size of 6,395 bytes and a creation date of Aug. 16, 2017, that
was filed with the patent application is incorporated herein by
reference in its entirety.
FIELD OF INVENTION
[0002] The present invention relates to the field of molecular
biology and genome editing, more specifically the transfer of
genetic materials to recipient cells by extracellular vesicles
(EVs).
BACKGROUND
[0003] RNA therapeutics including antisense oligo nucleotides
(ASOs), small-interfering RNA (siRNAs), synthetic mRNAs and genome
editing RNA-protein complexes are emerging modalities for therapies
targeting the human genomes at high specificity and great
flexibility. ASOs and siRNAs have been widely used as the tools for
gene knockdown in biomedical research. Their ability to silence any
gene of interest offers a great potential for targeting
disease-prevalent genes. Various chemical modifications or
conjugations can be used to keep ASOs and siRNAs stable and enhance
their binding specificity. Common methods for RNA transfection
including nucleofection, lipofection and electroporation are only
suitable for ex vivo delivery. Viral transduction and nanoparticles
are often used for in vivo delivery of RNAs and DNAs however, these
methods are usually ineffective, immunogenic and toxic.
[0004] One of the most recent breakthroughs in Science is a new
technology for genome editing, the clustered regularly interspaced
short palindromic repeats (CRISPR) method that enables robust and
precise modifications of genomic DNA for a wide range of
applications in research and medicine. CRISPR is an ideal tool for
correction of genetic abnormalities in cancer as the system can be
designed to target genomic DNA directly. A CRISPR system involves
two main components: a Cas9 enzyme and a guide (gRNA). The gRNA
contains a targeting sequence for DNA binding and a scaffold
sequence for Cas9 binding. Cas9 nuclease is often used to
"knockout" target genes hence it can be applied for deletion or
suppression of oncogenes that are essential for cancer initiation
or progression. Similar to ASOs and siRNAs, the CRISPR system
offers a great flexibility in targeting any gene of interest hence,
potential CRISPR based therapies can be designed based on the
genetic mutation in individual patients. An advantage of the CRISPR
system is its ability to completely ablate the expression of
disease genes which can only be suppressed partially by RNA
interference methods with ASOs or siRNAs. Furthermore, multiple
gRNAs can be employed to suppress or activate multiple genes
simultaneously, hence increasing the treatment efficacy and
reducing resistance potentially caused by new mutations in the
target genes. The applications of CRISPR technology have evolved
very quickly from bench to bedside targeting different diseases.
Clinical trials of CRISPR-mediated modification of T cells for
cancer therapies have started in China and in the USA. Many other
CRISPR-based therapies are under development. However, most of
these therapies rely on ex vivo modification of the target cells or
systemic delivery of the CRISPR system using virus or nanoparticles
that can target very few cell types such as hepatocytes.
[0005] Acute myeloid leukemia (AML) is the most aggressive type of
blood cancer that affects nearly 352,000 people per year with the
5-year prevalence of 1.5%. AML is characterized by the increase of
myeloblasts in the peripheral blood (PB) and the bone marrow (BM).
30-40% AML patients (mostly under 60 years old) response well to
chemotherapy and hematopoietic stem cell transplantation. However,
the response rate is much lower in older patients as they cannot
tolerate the toxicity of chemotherapy. Moreover, almost all the
patients relapse after a certain time due to drug resistance.
Hence, new treatment strategies are desirable to increase the
response rate, reduce toxicity and combat drug resistance. Recent
advances in genomics have provided better understanding of the
genetic and epigenetic abnormalities in AML and suggest new
specific therapeutic targets. RNA interference and genome editing
methods are emerging as new approaches to target these
abnormalities. However, delivery of RNAs to AML cells for gene
therapies has proven challenging, especially for in vivo
treatments. Common gene therapy delivery vehicles such as
adeno-associated virus (AAV) and lipid nanoparticles (LNPs) are
mostly ineffective or toxic in AML models.
[0006] Therefore, there is a desire to improve the delivery
efficiency and reduce toxicity of gene therapies for cancer.
SUMMARY OF INVENTION
[0007] In one aspect of the invention, there is provided a method
for RNA delivery to target cells comprising the steps of: a)
purification of extracellular vesicles (EVs) from red blood cells
(RBCs); b) electroporation of the EVs with RNAs to form RNA-loaded
EVs; and c) applying the RNA-loaded EVs to the target cells.
[0008] The advantage of using EVs (including microvesicles and
exosomes) from RBCs is that the RBCs are the most abundant blood
cells hence a large amount of EVs can be obtained and purified from
RBC units that are available at any blood bank. Preferably, the
RBCs are derived from a human. They are also nontoxic, unlike
synthetic transfection reagents. RBC EVs do not contain oncogenic
DNA/RNA or growth factors that are usually abundant in EVs from
cancer cells or stem cells, hence RBC EVs do not post any
transformation risks to recipient cells.
[0009] In one embodiment, the RBCs are derived from a mammal
preferably a human and treated with ionophore in particular calcium
ionophore. The EVs are purified using ultracentrifugation with a
sucrose cushion. The term "sucrose cushion" refers to a sucrose
gradient which establishes itself during a centrifugation. In an
embodiment, the sucrose gradient is prepared by using a solution of
about 40% to about 70%, about 50% to about 60%, or about 60% of
sucrose.
[0010] In another embodiment, the electroporated EVs comprises
antisense oligonucleotides (ASO), mRNAs and plasmids. Preferably,
the ASO comprises or consists of SEQ ID NO: 1.
[0011] In a further embodiment, the target cells comprise cancer
cells, or are cancer cells. In another embodiment, the target cells
comprise leukemia cells in particular acute myeloid leukemia (AML)
cells, breast cancer cells, or a combination of AML cells and
breast cancer cells.
[0012] In another embodiment, the EVs are electroporated with ASO
antagonizing miR-125b for knockdown of miR-125b in target cells as
described above. Preferably, the ASO antagonizing miR-125b
comprises or consists of SEQ ID NO: 1.
[0013] In another embodiment, the growth of the target cells is
suppressed. In a further embodiment, the EVs are electroporated
with a small chemical such as dextran.
[0014] In another embodiment, the method comprises administering to
the target cells the RNA-loaded EVs which modulate an
apoptosis-related gene expression, thereby inducing apoptosis in
the target cells.
[0015] In a second aspect of the invention, there is provided a
method for delivery of an antisense oligonucleotide (ASO) to target
cells to suppress gene expression, comprising the steps of: a)
purification of extracellular vesicles (EVs) from red blood cells
(RBCs); b) electroporation of the EVs with RNAs to form RNA-loaded
EVs; and c) applying the RNA-loaded EVs to the target cells.
[0016] In an embodiment, as described above, the RBCs are derived
from a mammal preferably a human, and treated with ionophore in
particular calcium ionophore.
[0017] In one embodiment, the RNA is an ASO antagonizing miR-125b
to inhibit the oncogenic miR-125b in the target cells. Preferably,
the ASO antagonizing miR-125b comprises or consists of SEQ ID NO:
1.
[0018] In another embodiment, the target cells comprise cancer
cells or are cancer cells. In another embodiment, the target cells
comprise leukemia cells in particular AML, cells, breast cancer
cells, or a combination of AML cells and breast cancer cells.
[0019] In a third aspect of the invention, there is provided a
method of RNA delivery to target cells for a CRISPR genome editing
system comprising the steps of: a) purification of extracellular
vesicles (EVs) from red blood cells (RBCs), wherein the RBCs are
preferably derived from a human and treated with ionophore in
particular calcium ionophore; b) electroporation of the EVs with
RNAs which may be Cas9 mRNAs and/or gRNAs to form RNA-loaded EVs;
and c) applying the RNA-loaded EVs to the target cells. CRISPR is a
method that enables robust and precise modifications of genomic DNA
for a wide range of applications in research and medicine. The
system can be designed to target genomic DNA directly.
[0020] In one embodiment, the EVs are electroporated with Cas9 mRNA
and gRNA. Preferably, Cas9 mRNA comprises or consists of SEQ ID NO:
2. Further, the gRNA is eGFP gRNA comprising or consisting of SEQ
ID NO: 3.
[0021] In another embodiment, the EVs are electroporated with Cas9
and gRNA plasmids. In another embodiment, the target cells comprise
cancer cells or are cancer cells.
[0022] In a further embodiment, the target cells comprise leukemia
cells or are leukemia cells. In a particular embodiment, the target
cells comprise leukemia cells in particular AML cells, breast
cancer cells, or a combination of AML cells and breast cancer
cells.
[0023] In a fourth aspect of the invention, there is provided a
method of treating cancer by delivery of RNA to target cells
comprising the steps of: a) purification of extracellular vesicles
(EVs) from red blood cells (RBCs) which are preferably derived from
a mammal in particular a human and treated with ionophore in
particular calcium ionophore; b) electroporation of the EVs with
RNAs to form RNA-loaded EVs; and c) applying the RNA-loaded EVs to
the target cells thereby inhibiting the growth of the target cells,
wherein the target cells comprise cancer cells.
[0024] In one embodiment, the target cells comprise leukemia cells,
breast cancer cells, or a combination of leukemia cells and breast
cancer cells. In another embodiment, the target cells comprise
acute myeloid leukemia cells.
[0025] In another embodiment, the step c) comprises a step of
administering the RNA-loaded EVs to a subject having the target
cells via a local or systemic administration. Local administration
refers to the delivery of the RNA-loaded EVs directly to the site
of action, and includes, but not limiting to, intratumoral
administration. Systemic administration refers to the delivery of
the RNA-loaded EVs via circulatory system, and includes, but not
limiting to, intravenous injection.
[0026] In a further embodiment, the growth of the target cells is
suppressed after the step c).
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1a is a schematic diagram showing the process of
collecting the Evs from human red blood cells (RBCs). FIG. 1b is a
plot showing the concentration and the size distribution of RBC
EVs. FIG. 1c shows the expression of ALIX, TSG101, and hemoglobin A
in cell lysates and EVs, via Western blot analysis.
[0028] FIG. 2a is a schematic presentation of EV electroporation.
FIG. 2b show the results obtained from FACS analysis of AF647
fluorescence and forward scatter (FSC) of the beads that were
incubated with electroporated EVs (E-EVs) or unelectroporated EVs
(UE-EVs).
[0029] FIG. 3a is a schematic presentation of the EV uptake assay.
FIG. 3b shows the expression of HBA relative to GAPDH, via Western
blot analysis. FIG. 3c shows the uptake of RBC EVs by leukemia
MOLM13 cells, via FACS analysis.
[0030] FIG. 4a is a schematic presentation of Dextran delivery.
FIG. 4b shows RBC EVs deliver dextran to leukemia MOLM13 cells, via
FACS analysis.
[0031] FIG. 5a is a schematic presentation of ASO delivery. FIG. 5b
shows the results obtained from FACS analysis, where the MOLM13
cells were untreated or incubated with FAM ASO or with
electroporated EVs (E-EVs) or with unelectroporated EVs (UE-EVs).
FIG. 5c is a plot showing the results of FIG. 5b. FIG. 5d is a
diagram showing the average percentage of FAM-positive cells after
treatments. In particular, the results reveal that RBC EVs deliver
antisense oligonucleotides (ASO) to leukemia MOLM13 cells.
[0032] FIG. 6a shows the results obtained from FACS analysis of
AF647 fluorescence in MOLM13 cells that were untreated, incubated
with Dextran AF647 (Dex-647) alone, with Dex-647 and
unelectroporated RBC EVs (UE-EVs), with Dex-647 loaded
Lipofectamin.TM. 3000 (Lipo3000), with Dex-647 loaded INTERFERin or
with Dex-647 electroporated RBC EVs (E-EVs) for 24 hours. FIG. 6b
shows the results obtained from FACS analysis of FAM fluorescence
in MOLM13 cells that were untreated, incubated with FAM-ASO alone,
with FAM-ASO and unelectroporated RBC EVs (UE-EVs), with FAM-ASO
loaded Lipo3000, with FAM-ASO loaded INTERFERin or with FAM-ASO
electroporated RBC EVs (E-EVs) for 24 hours.
[0033] FIG. 7a shows the percentage of cell death/viability of
MOLM13 cells after treatments with Dextran AF647 (Dex-647) alone,
with Dex-647 and unelectroporated RBC EVs (UE-EVs), with Dex-647
loaded Lipofectamin.TM. 3000 (Lipo3000), with Dex-647 loaded
INTERFERin or with Dex-647 electroporated RBC EVs (E-EVs) for 24
hours. FIG. 7b shows the percentage of cell death/viability of
MOLM13 cells after treatments with FAM-ASO alone, with FAM-ASO and
unelectroporated RBC EVs (UE-EVs), with FAM-ASO loaded Lipo3000,
with FAM-ASO loaded INTERFERin or with FAM-ASO electroporated RBC
EVs (E-EVs) for 24 hours
[0034] FIG. 8a is a schematic presentation of miR-125b ASO
delivery. FIG. 8b shows the expression of miR-125b in MOLM13 cells
after treatment with unelectroporated RBC EVs (UE-EVs), with
negative control (NC)-ASO electroporated RBC EVs (E-EVs) or
anti-miR-125b ASO (125b-ASO) electroporated RBC EVs for 72 hours.
FIG. 8c shows the expression of miR-125a in MOLM13 cells after
treatment with unelectroporated RBC EVs (UE-EVs), with negative
control (NC)-ASO electroporated RBC EVs (E-EVs) or anti-miR-125b
ASO (125b-ASO) electroporated RBC EVs for 72 hours. FIG. 8d shows
the expression of BAK1 relative to GAPDH in MOLM13 cells treated
the same as in FIGS. 8b and 8c. FIG. 8e shows the number of MOLM13
cells untreated, or treated with UE-EVs or with ASO electroporated
EVs as indicated.
[0035] FIG. 9a is a schematic presentation of miR-125b ASO
delivery. FIG. 9b shows the expression of miR-125b in CA1a cells
after treatment with unelectroporated RBC EVs (UE-EVs), with
negative control (NC)-ASO electroporated RBC EVs (E-EVs) or
anti-miR-125b ASO (125b-ASO) electroporated RBC EVs for 72 hours.
FIG. 9c shows the expression of miR-125a in CA1a cells after
treatment with unelectroporated RBC EVs (UE-EVs), with negative
control (NC)-ASO electroporated RBC EVs (E-EVs) or anti-miR-125b
ASO (125b-ASO) electroporated RBC EVs for 72 hours. FIG. 9d shows
the results of crystal violet staining of CA1a cells after
treatments as indicated above.
[0036] FIG. 10a is a schematic presentation of Cas9 mRNA delivery.
FIG. 10b shows the levels of Cas9 mRNA in MOLM13 cells after
treatment with unelectroporated EVs or with EVs that were
electroporated with 5, 10 or 20 .mu.g Cas9 mRNA, determined by
qRT-PCR after 24 hours of treatment. FIG. 10c show representative
images of MOLM13 cells after treatments as indicated above. FIG.
10d shows the average percentage of MOLM13 cells stained positive
for HA-Cas9 protein as shown in FIG. 10c.
[0037] FIG. 11a is a schematic presentation of the RNA delivery.
FIG. 11b shows the results obtained from FACS analysis of GFP in
NOMO1-GFP cells after treatment with unelectroporated EVs or EVs
electroporated with Cas9 and gRNA.
[0038] FIG. 12a is a schematic presentation of plasmid delivery.
FIG. 12b shows the results obtained from FACS analysis of GFP in
293T-eGFP cells untreated, or incubated with unelectroporated EVs
(UE-EVs) or with plasmid electroporated EVs (E-EVs) as indicated.
FIG. 12c shows the plot prepared from the results of FIG. 12b.
[0039] FIG. 13a is a schematic presentation of an in vivo EV uptake
assay. FIG. 13b shows the fluorescent images of nude mice bearing
untreated tumors on the right and tumors injected with
PKH26-labeled EVs on the left. FIG. 13c shows the ex vivo
fluorescent images of the tumors at 72 hours post-treatment. FIG.
13d shows the total radiance efficiency (photons/second) of
fluorescent signals in the tumors 24-72 hours after the injection
of PKH26-labeled EVs.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to the field of molecular
biology and genome editing. More specifically the transfer of
genetic materials to recipient cells by extracellular vesicles
(EVs) and the method of purification or isolation of exosomes from
Red Blood Cells.
[0041] Cells release into the extracellular environment, diverse
types of membrane vesicles of endosomal and plasma membrane origin,
called exosomes and microvesicles, respectively. These
extracellular vesicles (EVs) represent an important mode of
intercellular communication by serving as vehicles for transfer
between cells of membrane and cytosolic proteins, lipids, and
RNA.
[0042] EVs secreted by many cell types contain RNAs that function
to alter the phenotypes of other cells. EVs contain not only RNAs
but also proteins that stabilize RNAs and facilitate the functions
of RNAs in the target cells.
[0043] EV-mediated delivery of RNAs is an attractive platform
because the natural biocompatibility of EVs is the solution to
overcome most in vivo delivery hurdles. EVs are generally nontoxic
and non-immunogenic. They are taken up readily by many cell types
but they do possess some antiphagocytic markers such as CD47 that
help them to evade the phagocytosis by macrophages of the
reticuloendothelial system. Moreover, EVs are able to extravasate
well through the interendothelial junctions and even cross the
blood-brain barrier hence, they are greatly versatile drug
carriers. Of clinical value, delivery by EVs is not hampered by the
multidrug resistance mechanism caused by overexpression of
P-glycoproteins that tumor cells often exhibit to eliminate many
chemical compounds.
[0044] For therapeutic delivery, many research groups have
attempted to produce EVs from cancer cell lines and stem cells
which are very costly due to the large-scale cell culture that
requires various supplements. Moreover, EVs from cancer and stem
cells may contain oncogenic proteins or growth factors that promote
cancer growth. EVs from plasma and blood cells are safer for cancer
therapies. RBCs EVs do not contain oncogenic DNA/RNA or growth
factors that are usually abundant in EVs from cancer cells or stem
cells, hence, RBC EVs do not post any transformation risks to
recipient cells. RBCs EVs are also nontoxic unlike synthetic
transfection reagents. A recent article by Wahlgren et al describes
a protocol for isolation of plasma exosomes, small EVs that are
derived from multivesicular bodies, and electroporation of these
exosomes with siRNAs. They demonstrated that siRNA-loaded exosomes
are taken up by monocytes and lymphocytes leading to significant
knockdown of the target genes. This method is probably applicable
to cancer therapies however, plasma exosomes are normally very
heterogeneous because they are derived from different cell types in
the circulation and the yield of exosomes from plasma is low. RBCs
on the other hand are homogenous as RBCs from each individual are
the same.
[0045] In the present invention, an RNA is selected to inhibit
expression of a target gene by binding to a miRNA or editing the
target genomic DNA. Further, there is provided a novel method for
the purification of EVs from red blood cells (RBCs) and
incorporation of RNAs in EVs for gene therapies against cancer,
including acute myeloid leukemia and breast cancer.
EXAMPLES
[0046] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner.
Example 1
Materials and Methods
[0047] Blood samples were obtained by Red Cross from healthy donors
in Hong Kong with informed consents. RBCs were separated from
plasma and white blood cells by centrifugation and treated with 10
mM calcium ionophore (Sigma) overnight. The purification of EVs
were optimized with multiple centrifugation steps including the
removal of protein contamination using a 60% sucrose cushion
(ultracentrifugation at 100,000.times.g) that yields a homogenous
population of EVs with an average diameter of .about.140 nm. Each
unit of RBCs, isolated from .about.300 ml blood, yield 7.1 mg EVs
on average. These EVs are enriched in EV markers, ALIX and TSG101,
as shown by Western blot analysis. They also contain hemoglobin A
which is a major protein from RBCs.
[0048] FIG. 1a: Culture supernatants were collected from
ionophore-treated human red blood cells and subjected to multiple
steps of centrifugation to remove dead cells and debris. EVs were
purified by ultracentrifugation with 60% sucrose cushion and washed
with phosphate buffer saline (PBS) by ultracentrifugation
(100,000.times.g). FIG. 1b: Concentrations and the size
distribution of RBC EVs were measured by a Nanosight nanoparticle
analyzer. FIG. 1c: Western blot analysis of ALIX, TSG101 (EV
markers) and Hemoglobin A (RBC marker) relative to GAPDH in the
cell lysate and EVs purified from RBCs.
[0049] Subsequently, an electroporation protocol was optimized for
the RBC EVs using Dextran conjugated with Alexa Fluor.RTM. 647
(AF647, Thermo Fisher Scientific) tested at different voltages
using a Gene Pulser Xcell electroporator (BioRad). Electroporated
EVs were added to latex beads and analyzed for AF647 using flow
cytometry. It was found that 250 V was the optimal voltage, which
resulted in 93.6% AF647 positive EV-bound beads.
[0050] FIG. 2a: Schematic presentation of EV electroporation: 50
.mu.g RBC EVs were mixed with 4 .mu.g Alexa Fluor.RTM. 647 (AF647)
labeled Dextran and electroporated at different voltages from 50 to
250 V. EVs were incubated with latex beads overnight and analyzed
by fluorescent activated cell sorting (FACS). FIG. 2b: FACS
analysis of AF647 fluorescence and forward scatter (FSC) of the
beads that were incubated with electroporated EVs (E-EVs) or
unelectroporated EVs (UE-EVs). The percentage of AF647 positive
beads are indicated above the gates.
[0051] To measure the uptake of EVs by AML cells, the RBC-derived
EVs was labelled with a fluorescent membrane dye called Bodipy.RTM.
TR (Thermo Fisher). Labeled EVs were washed extensively using the
sucrose cushion, mock electroporated and added to the AML MOLM13
cells. After 24 hours of incubation with EVs, Western blot analysis
of MOML13 cells showed a clear uptake of Hemoglobin A (HBA) protein
which was absent in the untreated cells. Importantly, treatment
with RBC EVs did not affect the viability of AML cells as shown by
FACS analysis. MOLM13 cells became 100% Bodipy positive after the
incubation with Bodipy-labeled EVs, indicating that all the cells
took up the fluorescent RBC EVs. Electroporation increased the
uptake of HBA but not Bodipy by MOLM13 cells.
[0052] FIG. 3a: Schematic presentation of the EV uptake assay: 50
.mu.g RBC EVs were labeled with Bodipy TR (a red fluorescent dye),
washed twice, mock electroporated at 250 V, and incubated with
MOLM13 cells for 24 hours. FIG. 3b: Western blot analysis of
hemoglobin A (HBA) relative to GAPDH and; FIG. 3c: FACS analysis of
live cells, gated based on size scatter (SSC) and forward scatter
(FSC), and BODIPY fluorescence in MOLM13 cells that were untreated
or incubated with electroporated EVs (E-EVs) or unelectroporated
EVs (UE-EVs).
[0053] Different amounts of EVs was further electroporated with
Dextran AF647 and it was found that the best delivery with 75 .mu.g
EVs resulted in 68.6% cells positive for AF647. Therefore, 75 .mu.g
EVs was used for subsequent experiments.
[0054] FIG. 4a: Schematic presentation of Dextran delivery: 50-100
.mu.g RBC EVs were mixed with 4 .mu.g Dextran AF647 and
electroporated at 250 V. Electroporated EVs were incubated with
MOLM13 cells for 24 hours. FIG. 4b: FACS analysis of Dextran AF647
fluorescence in MOLM13 cells that were untreated or incubated with
50-100 .mu.g Dextran AF647 electroporated EVs (E-EVs) or 100 .mu.g
unelectroporated (UE-EVs).
[0055] Testing the delivery of RNA was started with an FAM (green
fluorescent) labeled scrambled RNA oligonucleotide (Shanghai
GenePharma), about 7 kDa, that is often used as a negative control
antisense oligonucleotide (ASO). RBC EVs were electroporated with
the FAM ASO and incubated with MOLM13 cells. After 24 hours, it was
observed that .about.70% uptake of FAM ASO by MOLM13 cells. Similar
uptake was observed in NOMO-1 cells, another AML cell line (data
not shown).
[0056] FIG. 5a: Schematic presentation of ASO delivery: 75 .mu.g
RBC EVs were electroporated with 400 pmole FAM fluorescent labeled
scrambled ASO (.about.7 kDa) and incubated with MOLM13 cells for 24
hours. FIGS. 5b-5d: FACS analysis of FAM fluorescence in MOLM13
cells that were untreated or incubated with FAM ASO or with
electroporated EVs (E-EVs) or with unelectroporated EVs (UE-EVs).
The average percentage+SEM of FAM-positive cells were calculated
from 3 independent experiments as shown in FIG. 5d.
[0057] The delivery of Dextran AF647 and FAM ASO by RBC EVs was
then compared with that of two commercialized lipofection reagents,
Lipofectamine.TM. 3000 (Thermo Fisher Scientific) and
INTERFERin.TM. (Polyplus transfection) that are commonly used for
transfection of nucleic acids in mammalian cells. Consistent with
previous experiments, RBC EVs delivered Dextran AF647 and FAM ASO
to .about.75% MOLM13 cells. Lipofectamine.TM. 3000 archived only 3%
and 55% delivery of Dextran AF647 and FAM ASO whereas INTERFERin
archived only 2.7% and 38.7% delivery of Dextran AF647 and FAM ASO
respectively in MOML13 cells. The poor delivery observed with
Lipofectamine.TM. 3000 and INTERFERin was not a surprise since
blood cells including AML cells are referred to as
"difficult-to-transfect" cell types by the manufacturers. Hence,
the 75% delivery efficiency archived by RBC EVs was a great
improvement.
[0058] FIG. 6a: FACS analysis of AF647 fluorescence in MOLM13 cells
that were untreated, incubated with 4 .mu.g Dextran AF647 (Dex-647)
alone, with Dex-647 and unelectroporated RBC EVs (UE-EVs), with
Dex-647 loaded Lipofectamin.TM. 3000 (Lipo3000), with Dex-647
loaded INTERFERin or with Dex-647 electroporated RBC EVs (E-EVs)
for 24 hours. FIG. 6b: FACS analysis of FAM fluorescence in MOLM13
cells that were untreated, incubated with 2 .mu.mole FAM-ASO alone,
with FAM-ASO and unelectroporated RBC EVs (UE-EVs), with FAM-ASO
loaded Lipo3000, with FAM-ASO loaded INTERFERin or with FAM-ASO
electroporated RBC EVs (E-EVs) for 24 hours.
[0059] Moreover, RBC EVs exhibit no toxicity to the cells in
contrast to about 20-30% increase in cell death caused by the
transfection using Lipofectamine.TM. 3000 and INTERFERin.
[0060] The percentage of cell death was determined based on
Propidium iodide (PI) staining and FACS analysis as shown in FIG.
7a: MOLM13 cells that were untreated, incubated with 4 .mu.g
Dextran AF647 (Dex-647) alone, with Dex-647 and unelectroporated
RBC EVs (UE-EVs), with Dex-647 loaded Lipofectamin.TM. 3000
(Lipo3000), with Dex-647 loaded INTERFERin or with Dex-647
electroporated RBC EVs (E-EVs) for 24 hours; FIG. 7b: MOLM13 cells
that were untreated, incubated with 2 .mu.mole FAM-ASO alone, with
FAM-ASO and unelectroporated RBC EVs (UE-EVs), with FAM-ASO loaded
Lipo3000, with FAM-ASO loaded INTERFERin or with FAM-ASO
electroporated RBC EVs (E-EVs) for 24 hours. The average cell death
and SEM were calculated from three independent experiments. One-way
Anova test: ** P<0.05; ** P<0.01.
[0061] The therapeutic potential of RBC EVs to deliver an ASO that
antagonizes the oncogenic miR-125b in AML cells was further tested.
miR-125b is upregulated in different types of cancer including AML
and other leukemia. It has been shown that miR-125b suppresses
apoptosis by regulating multiple genes in the p53 network. miR-125b
also promotes proliferation of hematopoietic stem cells and
leukemia cells in both humans and mouse models. An anti-miR-125b
ASO (Shanghai Gene Pharma) comprising a sequence of SEQ ID NO: 1
was loaded into RBC EVs using electroporation and treated MOLM13
cells with these EVs. After 72 hours, it was found that the level
of miR-125b was suppressed by 80-95% in a dose-dependent manner.
miR-125a, the homologue of miR-125b, was also suppressed by 50-80%
due to the sequence similarity to miR-125b. Inhibition of miR-125
led to a significant increase in BAK1, a target of miR-125a/b which
regulates apoptosis. Treatment with miR-125b ASO loaded EVs also
dampened the growth of MOLM13 cells significantly after 3-4 days of
incubation. Hence, the inhibition of miR-125b using ASO in RBC EVs
may represent a new approach for AML treatment.
[0062] As described here, miR-125b preferably comprises or consists
of SEQ ID NO: 4 and miR-125a preferably comprises or consists of
SEQ ID NO: 5. In particular, miR-125b consists of SEQ ID NO: 4 and
miR-125a consists of SEQ ID NO: 5.
[0063] FIG. 8a: Schematic presentation of miR-125b ASO delivery:
25-100 .mu.g RBC EVs were electroporated with 2 .mu.mole
anti-miR-125b ASO and incubated with MOLM13 cells. Anti-miR-125b
ASO in this embodiment consists of SEQ ID NO: 1. FIGS. 8b-c:
Expression of miR-125b and miR-125a relative to U6b snRNA in MOLM13
cells that were untreated, incubated with 100 ug unelectroporated
RBC EVs (UE-EVs), with negative control (NC)-ASO electroporated RBC
EVs (E-EVs) or anti-miR-125b ASO (125b-ASO) electroporated RBC EVs
for 72 hours, as determined by Taqman qRT-PCR, presented as average
and SEM. FIG. 8d: Expression of BAK1 relative to GAPDH in MOLM13
cells treated the same as in FIG. 8b. FIG. 8e: Number of MOLM13
cells untreated, or treated with UE-EVs or with ASO electroporated
EVs as indicated. One-way Anova test: ** P<0.01; ***
P<0.001.
[0064] Similarly, RBC EVs were tested for the delivery of miR-125b
ASO to breast cancer MCF10aCA1a (CA1a) cells. The inventors
observed 80-90% knockdown of miR-125a and miR-125b in CA1a cells
treated with miR-125b ASO loaded EVs. As a consequence, the
knockdown of miR-125s suppressed the proliferation of CA1a
cells.
[0065] FIG. 9a: Schematic presentation of miR-125b ASO delivery:
25-50 .mu.g RBC EVs were electroporated with 2 .mu.mole
anti-miR-125b ASO and incubated with CA1a cells. FIG. 9b-c:
Expression of miR-125b and miR-125a relative to U6b snRNA in CA1a
cells that were untreated, incubated with unelectroporated RBC EVs
(UE-EVs), with negative control (NC)-ASO electroporated RBC EVs
(E-EVs) or anti-miR-125b ASO (125b-ASO) electroporated RBC EVs for
72 hours, as determined by Taqman qRT-PCR, presented as average and
SEM. FIG. 9d: Crystal violet staining of CA1a cells untreated, or
treated with UE-EVs or with ASO electroporated EVs as indicated.
Bar graph represent the average number of cells counted in
crystal-violet stained wells (n=3). One-way Anova test: **
P<0.01.
[0066] To test the feasibility of CRISPR delivery using RBC EVs,
synthetic SpCas9 mRNA (Trilink) was electroporated into RBC EVs
using the protocol that was optimized for Dextran and ASO. As the
result, a large amount of Cas9 mRNAs was detected in MOLM13 cells
after a 24-hour incubation with the electroporated EVs, using
qRT-PCR. Furthermore, using immunostaining of the HA-tag, Cas9
protein was found in the nuclei (overlapped with a nuclear stain)
of .about.50% MOLM13 cells at 48-hour post-treatment. This suggests
that RBC EVs can be used to deliver the CRISPR Cas9 system.
[0067] FIG. 10a: Schematic presentation of Cas9 mRNA delivery: RBC
EVs were electroporated with Cas9 mRNA and incubated with MOLM13
cells for 24 or 48 hours. FIG. 10b: The levels of Cas9 mRNA
relative to GAPDH mRNA in MOLM13 cells that were untreated,
incubated with unelectroporated EVs or with EVs that were
electroporated with 5, 10 or 20 .mu.g Cas9 mRNA, determined by
qRT-PCR after 24 hours of treatment. Values are presented as
mean.+-.SEM (n=3). FIG. 10c: Representative images of MOLM13 cells
that were untreated, or incubated for 48 hours with
unelectroporated EVs or with EVs that were electroporated with 10
.mu.g Cas9 mRNAs. The cells were stained for HA-Cas9 protein (using
green dye, not seen in black and white images) and nuclear DNA
(Hoechst, blue dye, not seen in black and white images). FIG. 10d:
Average percentage of MOLM13 cells stained positive for HA-Cas9
protein as shown in (c).
[0068] Subsequently, the inventors delivered Cas9 mRNA together
with an anti-eGFP gRNA in RBC EVs to AML cells, NOMO1, that are
labeled with eGFP. After one week, the inventors observed a
complete knockout of eGFP in 32.9% NOMO1 cells. Hence, the RNAs
delivered by RBC EVs were able to execute a CRISPR knockout of
eGFP. As described herein, Cas9 mRNA preferably comprises or
consists of SEQ ID NO: 2 and eGFP gRNA preferably comprises or
consists of SEQ ID NO: 3. In particular, Cas9 mRNA consists of SEQ
ID NO: 2 and eGFP gRNA consists of SEQ ID NO: 3.
[0069] FIG. 11a: Schematic presentation of the RNA delivery: RBC
EVs were electroporated with Cas9 mRNA and anti-GFP gRNA and
incubated with NOMO1-GFP cells for 7 days. FIG. 11b: FACS analysis
of GFP in NOMO1-GFP cells that were untreated, incubated with
unelectroporated EVs or EVs electroporated with Cas9 and gRNA. The
percentages of GFP-negative cells are shown above the gate.
[0070] In addition, the delivery of plasmids by RBC EVs was also
tested. RBC EVs were electroporated with two plasmids, one
expressing SpCas9 and one expressing gRNA against eGFP.
Electroporated EVs were incubated with human embryonic kidney
HEK-293T cells that homogenously express eGFP. After 96 hours, it
was found that 13.8% GFP-negative cells resulted from the EV
treatment, compared to 3.52% GFP-negative in the untreated
population. Treatment with electroporated EVs showed a distinct
peak of GFP-negative cells that suggests a homologous knockout of
eGFP by the delivery of Cas9 and gRNA plasmids. Therefore, RBC EVs
are able to deliver not only RNA but also plasmid DNA for genome
editing. Moreover, the delivery is applicable to HEK-293T solid
cancer cells.
[0071] FIG. 12a Schematic presentation of plasmid delivery: RBC EVs
were electroporated with Cas9 plasmid and eGFP gRNA plasmid and
incubated with eGFP expressing 293T cells for 96 hours. FIGS.
12b-12c FACS analysis of GFP in 293T-eGFP cells untreated, or
incubated with unelectroporated EVs (UE-EVs) or with plasmid
electroporated EVs (E-EVs) as indicated. The GFP negative cells are
indicated by the percentages in FIG. 12b and the arrow in FIG.
12c.
[0072] FIG. 13a: Schematic presentation of an in vivo EV uptake
assay. FIG. 13b: Fluorescent images of nude mice bearing untreated
tumors on the top and tumors injected with PKH26-labeled (red dye,
not seen in black and white images) EVs on the bottom. FIG. 13c: Ex
vivo fluorescent images of the tumors at 72 hours post-treatment.
FIG. 13d: Total radiance efficiency (photons/second) of fluorescent
signals in the tumors 24-72 hours after the injection of
PKH26-labeled EVs. To determine whether the RBC EVs are taken up by
tumor cells in vivo, CA1a cells were implanted in the mice, in the
flank at 2 sides (FIG. 13a). The tumor size is about 7 mm. 100
.mu.g of PKH26 labelled EVs were then injected intratumorally.
Fluorescent live imaging was done every day for 3 days (72 hours).
Images of nude mice bearing untreated tumors and tumors injected
with PKH26-labeled (red dye note seen in black and white images)
EVs were taken (FIG. 13b). With reference to FIG. 13c, it shows ex
vivo fluorescent images of the tumors at 72 hours post-treatment
and proves that PKH26-labeled EVs were taken up by tumor cells. The
total radiance efficiency (photons/second) of fluorescent signals
in the tumors decreased gradually 24 to 72 hours after the
injection of PKH26-labeled EVs as shown in FIG. 13d.
[0073] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the present
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0074] It will also be appreciated by persons skilled in the art
that the present invention may also include further additional
modifications made to the method which does not affect the overall
functioning of the method.
[0075] Any reference to prior art contained herein is not to be
taken as an admission that the information is common general
knowledge, unless otherwise indicated. It is to be understood that,
if any prior art information is referred to herein, such reference
does not constitute an admission that the information forms a part
of the common general knowledge in the art, any other country.
Sequence CWU 1
1
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uaagcguguu 3840auuuuagcag augccaauuu agauaaaguu cuuagugcau
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cauuuauuua cguugacgaa ucuuggagcu 3960cccgcugcuu uuaaauauuu
ugauacaaca auugaucgua aacgauauac gucuacaaaa 4020gaaguuuuag
augccacucu uauccaucaa uccaucacug gucuuuauga aacacgcauu
4080gauuugaguc agcuaggagg ugacuga 4107320RNAArtificial
SequenceSynthesized 3gggcacgggc agcuugccgg 20422RNAHomo sapiens
4ucccugagac ccuaacuugu ga 22524RNAHomo sapiens 5ucccugagac
ccuuuaaccu guga 24
* * * * *