U.S. patent application number 13/700067 was filed with the patent office on 2014-10-09 for microvesicles carrying small interfering rnas preparation methods and uses thereof.
This patent application is currently assigned to Micromedmark Biotech Co. Ltd.. The applicant listed for this patent is Hongwei Gu, Danqing Liu, Ke Zeng, Chenyu Zhang, Yujing Zhang. Invention is credited to Hongwei Gu, Danqing Liu, Ke Zeng, Chenyu Zhang, Yujing Zhang.
Application Number | 20140302119 13/700067 |
Document ID | / |
Family ID | 45003208 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302119 |
Kind Code |
A2 |
Zhang; Chenyu ; et
al. |
October 9, 2014 |
MICROVESICLES CARRYING SMALL INTERFERING RNAS PREPARATION METHODS
AND USES THEREOF
Abstract
Microvesicles containing interfering RNAs, preparation methods
and uses thereof are provided. Pharmaceutical compositions and kits
comprising the microvesicles containing interfering RNAs are also
provided. Microvesicles containing interfering RNAs, pharmaceutical
compositions and kits comprising such microvesicles can be used to
study the effects of interfering RNAs on receptor cells. As
microvesicles containing interfering RNAs can stably, high
efficiently and specifically deliver interfering RNAs,
microvesicles containing interfering RNAs can be used to treat
related diseases.
Inventors: |
Zhang; Chenyu; (Beijing,
CN) ; Zeng; Ke; (Beijing, CN) ; Liu;
Danqing; (Beijing, CN) ; Zhang; Yujing;
(Beijing, CN) ; Gu; Hongwei; (Beijing,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhang; Chenyu
Zeng; Ke
Liu; Danqing
Zhang; Yujing
Gu; Hongwei |
Beijing
Beijing
Beijinig
Beijing
Beijing |
|
CN
CN
CN
CN
CN |
|
|
Assignee: |
Micromedmark Biotech Co.
Ltd.
Beijing
CN
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130209544 A1 |
August 15, 2013 |
|
|
Family ID: |
45003208 |
Appl. No.: |
13/700067 |
Filed: |
May 26, 2010 |
PCT Filed: |
May 26, 2010 |
PCT NO: |
PCT/CN2010/073262 PCKC 00 |
371 Date: |
April 26, 2013 |
Current U.S.
Class: |
424/450;
435/317.1; 435/375; 435/6.13; 514/44A |
Current CPC
Class: |
A61K 9/127 20130101;
A61P 31/06 20180101; C12N 2320/32 20130101; A61P 1/00 20180101;
A61P 15/00 20180101; A61P 19/00 20180101; A61P 13/00 20180101; A61P
31/04 20180101; A61P 21/00 20180101; C12N 15/88 20130101; A61P
31/14 20180101; C12Q 1/68 20130101; A61P 31/16 20180101; C12N
15/113 20130101; A61P 5/00 20180101; A61P 11/00 20180101; A61P
25/00 20180101; A61P 31/18 20180101; A61P 3/00 20180101; A61P 35/00
20180101; A61P 31/00 20180101; A61P 7/00 20180101; C12N 2310/14
20130101; A61P 9/00 20180101; C12N 15/111 20130101; A61P 37/00
20180101 |
Class at
Publication: |
424/450;
435/317.1; 514/44.A; 435/375; 435/6.13 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12Q 1/68 20060101 C12Q001/68; A61K 9/127 20060101
A61K009/127 |
Claims
1. Cellular microvesicles comprising interfering RNAs.
2. The cellular microvesicles according to claim 1, wherein the
cellular microvesicles are obtained from donor cells of human or
animals.
3. The cellular microvesicles according to claim 2, wherein the
donor cells includes cell lines or primary cultures.
4. The cellular microvesicles according to claim 1, wherein the
interfering RNAs are enclosed in the cellular microvesicles.
5. The cellular microvesicles according to claim 1, wherein a mean
diameter of the cellular microvesicles is in the range of 10-500
nm.
6. The cellular microvesicles according to claim 1, wherein the
cellular microvesicles include at least one of an exosome, a
shedding vesicle and other biological vesicles originating from
cells.
7. A kit including the cellular microvesicles containing
interfering RNAs according to claim 1.
8. A pharmaceutical composition, comprising the cellular
microvesicles containing interfering RNAs according to claim 1.
9. A method for preparing the cellular microvesicles according to
claim 1, comprising the following steps: transferring the
interfering RNA into cells; and separating the cellular
microvesicles containing siRNA.
10. The method according to claim 9, wherein the step of separating
the cellular microvesicles comprises one or more of differential
centrifugation, immunoadsorption and ultrafiltration.
11. A research method, comprising: transferring the cellular
microvesicles containing siRNA according to claim 1 into receptors,
and studying an effect of the cellular microvesicles containing
siRNA on the functions of the receptors after transferring the
cellular microvesicles.
12. A method for preventing and/or treating diseases, comprising
transferring the cellular microvesicles containing siRNA according
to claim 1 into receptor cells.
13. The method according claim 12, wherein the diseases include
tumors, acute and chronic infectious diseases, bacterial diseases,
other acute and chronic infectious diseases caused by various
pathogenic microorganisms. respiratory system diseases, immune
system diseases, blood and hematopoietic system diseases,
circulatory system diseases, endocrine system and metabolic
diseases, digestive system diseases, nervous system diseases,
urinary system diseases, reproductive system diseases and motor
system diseases.
14. The method according to claim 12, wherein said diseases include
viral influenza, viral hepatitis, AIDS, SARS, bacterial diseases
such as tuberculosis, bacterial pneumonia, and other acute and
chronic infectious diseases caused by pathogenic
microorganisms.
15. Use of the cellular microvesicles containing siRNA according to
claim 1 in the delivery of interfering RNAs.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to cellular microvesicles
carrying interfering RNAs, preparation methods and uses thereof.
More particularly, the invention relates to provide cellular
microvesicles carrying interfering RNAs, a method of loading the
interfering RNAs on cellular microvesicles and uses in the
improvement of bio-medical experimental technique and in the
prevention/treatment of a disease thereof.
BACKGROUND ART
[0002] Cellular microvesicles (MVs) are a category of biological
vesicles with a lipid bi-layer membrane, ranging between 10-500 nm
in size. They were first reported as early as in year 1967 and
named "platelet dust" since they were derived from platelets,
contain vesicles and have a role in promoting coagulation. In vitro
studies, it has found that each of endothelial cells, vascular
smooth muscle cells, platelets, leucocytes, lymphocytes,
erythrocytes, and the like are all able to release MVs. According
to their source, MVs can be divided into two categories: exosomes
and shedding vesicles. Exosomes are secreted in the manner of
exocytosis with multi-vesicular bodies (MVBs) in the case of cells
are stimulated, and shedding vesicles are directly secreted from
the cell surface by budding. Presently, different names are given
to shedding vesicles secreted by different cells, for example,
those from neutrophil granulocytes and monocytes are called
ectosomes, and those from platelets are called microparticles.
[0003] Membrane component of cellular MVs, depending on the cells
from which they originate, is mainly composed of lipid and protein.
However, the inner component of cellular MVs is still unknown. The
plasma membrane of cellular MVs contains the features of its
original cells, i.e. contains specific molecular markers and cell
receptors/ligands on the surface of the original cells. Definite
physiological functions of cellular MVs have not been investigated
clearly up to now.
[0004] Interfering RNA (siRNA) is a kind of double-stranded RNA
molecule consisting of more than 20 nucleotides, and it plays a
role in silencing gene expression through the specific degradation
of messenger RNA (mRNA). This process is called RNA interference
(RNAi).
[0005] RNAi is a way of post-transcriptional gene silencing, and is
one of the old and evolutive highly conserved phenomena in the
living nature. Through siRNA mediated recognition and targeting
cleavage of homologous target mRNA, gene expression is suppressed
specifically and efficiently. RNA interference has the
characteristics of biocatalytic reaction, in which multiple
proteins and ATP were involved.
[0006] In recent years, study of RNA interference has made
breakthroughs, has been rated as one of the top ten most scientific
progress by the journal Science in 2001, and has been ranked the
top ten most scientific progress in 2002. By using RNA interfering
technology, the expression of the specific gene can be knocked out
or turn off. Therefore, the RNA interfering technology has been
widely used in the fields of bio-medical experiment research and
gene therapy of various diseases.
[0007] Before the RNA interfering technology appearing, gene
knockout is the major research tool in reverse genetics, but with
high difficulty of technology, complexoperation and long time of
research. RNA interference has been now an important research tool
for exploring the function of genes due to it could use siRNA or
siRNA expression vector with faster, cheaper, simply and highly
sequence-specific to silence the specific gene specifically to
obtain the mutation sequence with lost or decreased function so as
to knockout the expression of target gene specifically. In the
study of functional genomes, specific gene needed to be
functional-loss or mutation sequence reduction so as to confirm its
function. Therefore, RNAi can be used to the study of functional
genomes as a powerful study tool. Meanwhile, the establishment of
the method for construction of siRNA expression library enables the
high throughput screening using RNAi technology, it has important
significance in both clarification of signal transduction pathway
and discovery of new drug targets.
[0008] RNAi is also wildly used in the field of treatment of
diseases, In the stud of gene therapy for HIV-1, Hepatitis B and
Hepatitis C etc. using RNA interference technology, it is found
that selecting sequence in the viral genome that has no homology to
sequences in the human as the suppression sequence can void the
side effect on the normal tissues while inhibiting the replication
of virus. At the same time, choosing the suppression sequence at
the special would induce apoptosis of some malignant cells with
definite gene mutation. Moreover, tumor cells can be killed
specifically by introducing the expression of siRNA or shRNA for
some oncogenes or molecules against apoptosis using the promoters
specific for tumors.
[0009] As RNA interference is the gene silencing against
prost-transcriptional stage, corresponding to the gene knockout
genetically with traditional gene therapy, RNAi is more simply in
the whole process design, and the action is rapid and effect
obviously, which opens a new way for the gene therapy. The general
idea is that through strengthening the mechanism of RNA
interference of the key gene, to control the abnormal progress of
protein synthesis appearing in diseases and replication or
expression of exogenous pathogenic nucleic acid, especially some
nucleic acid viruses seriously harm to human health by
strengthening the mechanism of RNA interference of the key
gene.
[0010] In recently, studies have demonstrated that siRNA can
inhibit the replication of HIV in the cells cultured in vitro. HIV
infection could be prevented by siRNA through inhibit its own gene
(e.g. pie, gag, rev, tat and env) of HIV virus and its host gene
(e.g. CD4, major receptor of HIV). Meanwhile, studies have found
that siRNA inhibiting Fas is injected intravenously into the mouse
in two mouse models with autoimmune hepatitis, it is observed that
Fas mRNA and protein level in liver cells is reduced, thus
preventing liver cells from damages of apoptosis caused by
autoimmune hepatitis. Moreover, studies have found that
transformation of tumor cells from benign to malignant can be
inhibited by silencing p53 gene through RNAi.
[0011] Although RNAi has been widely used in every aspects of
bio-medical research, there are still some problems difficult to be
solved. For example, the efficiency of transferring siRNA to some
cells, e.g. immune cells, is very low using the existing
transfection method of liposome, which will affect further
application of it in this field.
[0012] Meanwhile, although many achievements were made in the
research and development siRNA drugs, it still faces many problems
for applying it into real medical treatment. Although siRNA can be
directly injected into the animals, the half life of the siRNA
without encapsulating is very short, and the therapeutic efficacy
is barely satisfactory. Presently, the carriers of delivering siRNA
drugs mainly include liposomes, nanocapsules/nanoparticles,
.beta.-cyclodextrininclusion compound (or also called
.beta.-cyclodextrin capsule) and so on. These carriers can partly
prolong the retention time of the drugs in vivo and increase the
absorption rate siRNA drug, but the targeting and high efficiency
of delivering drugs are still weak. Problems of how to effectively
administration to human while ensuring the drug release of the
efficacy at target tissues and organs as well as having higher
safety and the like are all needed to further investigation.
[0013] As an important bio-medical research tool and a potential
drug, siRNA is now facing some open problems, and the poor
specificity (targeting), less stability and lower efficiency of
delivering siRNA are the main reasons for limiting its use.
Therefore, it is an urgent need for a more stable, high effective
and specific way of delivering siRNA to deliver siRNA high
effectively and specifically.
[0014] It is unexpectedly for the applicant that cellular MV is a
vector of bio-vesicle vehicle with highly effective rate and
specificity in vivo. These cellular MVs are variable in size,
ranging between 10-500 nm. In principle, the membrane components
(including specific surface receptors and membrane lipid
structures) of MVs secreted by different cells are the same as the
plasma membrane components of the corresponding cells. Therefore,
cellular MVs carrying with receptor proteins or membrane lipid
structure from the surface of the cells, have high affinity to the
corresponding target cells. Using cellular MVs as a carrier for
delivering siRNA, siRNAs can be selectively delivered into the
target cells/tissues high efficiently and selectively, thus
enhancing the regulation of cellular functions greatly. It is
obviously that since the cellular MVs (including the membrane lipid
vesicular structures with characteristics similar to the cellular
MVs, such as exosomes and shedding vesicles as well as particular
shedding vesicles secreted by different cells) themselves have the
specificity of binding to particular tissues and cells, the siRNA
carried by cellular MV also exhibit high targeting, stability and
efficiency, they have a significant application prospect in the
study and therapy of the mechanism of diseases.
[0015] The inventors of the present invention find that using the
cellular MV as a vector to deliver the interfering RNA to the
target cells will not harm to organisms themselves, due to the
cellular MV are substances secreted by cells themselves and have
bio-affinity; meanwhile, cellular MVs can be transferred into the
target cells efficiently and selectively due to the surface of
which carry surface molecules originating from cells and have high
affinity to the target cells. The interfering RNA can functions by
combined with specific sequence of target gene mRNA to block the
translation process of protein of the target gene, thus playing a
role blocking the gene expression specifically.
[0016] The advantages of using cellular MVs as a vector to deliver
siRNA are: firstly, cellular MVs originate from cells, and is a
native existence of organism, thus it can overcome the toxicity to
cells and damage to the body of the drug carriers presently
synthesized; secondly, various technical ways used during enclosing
siRNA into cellular MVs are all easy to implement and the enclosing
efficiency is very high, which increase its application potential
in practical to a certain degree; more importantly, cellular MV are
vesicle structures with a lipid bi-layer membrane and the structure
of the outer membrane is similar to that of cytoplasm, which can
enter the cell through fusion with the cell membrane and
endocytosis. Meanwhile, cellular MVs would enter the target cells
efficiently and selectively due to its surface carrying with
molecule markers such as surface protein and various
receptors/ligands originating from the surface of the cytoplasm of
cells. If using the cellular MVs excreted from the primary culture
of tissues or cells of patients themselves to enclose siRNA, immune
rejection can be reduced and the transferring efficiency of the
cellular MVs carrying siRNA to organism can be further improved.
Based on the above-mentioned advantages, as a carrier to deliver
the siRNA as a drug, cellular MVs will play a more important role
in the development of drugs and prevention and treatment of the
clinical diseases.
SUMMARY OF THE INVENTION
[0017] The present invention provides cellular MVs containing
siRNA.
[0018] The present invention also provides a pharmaceutical
composition, which comprises cellular MVs containing siRNA and a
pharmaceutically acceptable vehicle.
[0019] The present invention further provides a kit, wherein the
kit includes cellular MVs containing siRNA or a pharmaceutical
composition comprising cellular MVs containing siRNA, and
instructions for use.
[0020] In addition, the present invention also provides a method
for preparing cellular MVs containing siRNA, comprising the
following steps: [0021] transferring interfering RNA (siRNA) into
cells using the cell transfection technology; or transferring siRNA
into cells using the viral vector method; [0022] separating
cellular MVs containing siRNA.
[0023] The present invention also provides a research method,
including: [0024] transferring siRNA and its control sequence into
donor cells, preterably by transfection or viral vector method;
[0025] separating cellular MVs containing siRNA; [0026] adding the
cellular MVs containing siRNA into a receptor, preferably receptor
cells; [0027] studying the effect of MVs containing siRNA after
they enter receptor cells.
[0028] The present invention also provides a method for preventing
and or treating diseases, including: transferring cellular MVs
containing siRNA into a receptor.
[0029] The present invention also provides the use of cellular MVs
containing siRNA in the transportation of siRNA.
DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows a transmission electron microscope (TEM)
picture of cellular MVs of healthy human plasma/serum.
[0031] FIG. 2-A shows the interference efficiency of siRNA of c-myb
gene detected by Real time-PCR.
[0032] FIG. 2-B shows the results of transfection efficiency
detected by flow cytometry.
[0033] FIG. 2-C shows the results of transfection efficiency
detected by fluorescence microscope.
[0034] FIG. 3-A shows the detection of cellular MVs by flow
cytometry.
[0035] FIG. 3-B shows the detection of cellular MVs containing
siRNA by flow cytometry.
[0036] FIG. 3-C shows transferring siRNA into the target cell using
the cellular microvesicle as a carrier.
[0037] FIG. 4-A shows the specific down-regulation of the
expression of target protein in the target cell with siRNA.
[0038] FIG. 4-B shows the effect of cellular MVs which do not
comprise siRNA on the migration ability of the target cell.
[0039] FIG. 4-C shows the effect of cellular MVs containing siRNA
on the migration ability of the target cell.
[0040] FIG. 4-D shows the statistical results of the effect of
cellular MVs containing siRNA on the migration ability of the
target cell.
[0041] FIG. 5 shows the inhibitory effect of cellular MVs
containing siRNA on the HIV.
DETAILED DESCRIPTION
[0042] Cellular MVs Containing Interfering RNA
[0043] Cellular microvesicles (MVs) are a category of natural
biologic vesicles with a lipid bi-layer membrane, which is excreted
from cells, ranging between 10-500 nm in size, including Exosome
excreted from Multivesicutar bodies (MVBs), shedding vesicles
excreted by cell budding and particular shedding vesicles secreted
by different cells.
[0044] Cellular MVs include any MVs produced by various cells
obtained from human or animals, especially including cells of
healthy or diseased human or animals which may be primary cultures
or subcultures (cell lines), such as endothelial cells, vascular
smooth muscle cells, platelets, leucocytes, lymphocytes and
erythrocytes.
[0045] SiRNA includes all the siRNA sequences designed for receptor
genes, which will degrade the target genes specifically through the
mechanism of RNAi.
[0046] The present invention provides a pharmaceutical composition
and a kit that can be used for the treatment of a disease.
[0047] According to one embodiment of the present invention, there
is provided a pharmaceutical composition, comprising cellular MVs
containing siRNA and pharmaceutically acceptable vehicles. The
pharmaceutically acceptable vehicles include, for example normal
saline, serum, cell culture medium, phosphate buffer solution
(PBS), etc.
[0048] According to one embodiment of the present invention, there
is provided a kit, wherein the kit includes cellular MVs containing
siRNA or a pharmaceutical composition comprising cellular MVs
containing siRNA, and instructions for use.
[0049] Diseases that can be prevented or treated with cellular MVs
containing siRNA or a pharmaceutical composition comprising
cellular MVs containing siRNA or a kit include: various tumors;
various acute and chronic infectious diseases, for example viral
diseases such as viral influenza, viral hepatitis, AIDS, SARS,
bacterial diseases such as tuberculosis, bacterial pneumonia, and
other acute and chronic infectious diseases caused by various
pathogenic microorganisms; other acute and chronic diseases, such
as respiratory system diseases, immune system diseases, blood and
hematopoietic system diseases, circulatory system diseases,
endocrine system and metabolic diseases, digestive system diseases,
nervous system diseases, urinary system diseases, reproductive
system diseases and motor system diseases.
[0050] Methods
[0051] In addition, the present invention also provides a method
for preparing cellular MVs containing siRNA, including the
following steps: [0052] transferring interfering RNA (siRNA) into
cells using the cell transfection technology; or transferring siRNA
into cells using the viral vector method; [0053] separating
cellular MVs containing siRNA.
[0054] The method for preparing cellular MVs includes, e.g.,
differential centrifugation, immune adsorption and
ultrafiltration.
[0055] Preferably, the cellular MVs are prepared with differential
centrifugation which comprises, for example, the following steps:
firstly, centrifuging the body fluid, blood, cells, tissues, cells
or tissues cultured in vitro to remove all kinds of cells and
fragments; then ultracentifugating the supernatant to get the
precipitates which are cellular MVs.
[0056] Or alternatively, preferably the cellular MVs are prepared
with immune-adsorption which comprises, for example, the following
steps: (1) firstly, centrifuging the body fluid, blood, cells,
tissues, cells or tissues cultured in vitro to remove all kinds of
cells and fragments to get the supernatant; (2) incubating
cell-specific antibodies or immunemagnetic beads (Invitrogen, US)
absorbed on the tissue culture dish with the supernatant (e.g., for
30-60 min) to achieve the absorbed cellular MVs.
[0057] Or alternatively, preferably the cellular MVs are prepared
with for example ultrafiltration including following steps:
[0058] (1) firstly, centrifuging the body fluid, blood, cells,
tissues, cells or tissues cultured in vitro to remove all kinds of
cells and fragments to get the supernatant; (2) centrifuging the
supernatant in an ultrafiltration centrifuge tube with a 100 KDa
MWCO (Millipore, US) at 4000 rpm, concentrating to achieve cellular
MVs.
[0059] Preferably, the present invention provides a method for
preparing cellular MVs containing siRNA. The MVs can be transferred
into the receptor cells or organisms efficiently and specifically,
and interfere with the expression of protein of the target genes by
siRNA.
[0060] According to one embodiment of the invention, the method
includes: [0061] 1) enclosing siRNA in microvesicles of the donor
cells; [0062] 2) separating the cellular MVs excreted by donor
cells; [0063] 3) detecting the loading efficiency of siRNA in the
cellular IVs; [0064] 4) transferring the cellular MVs containing
siRNA into receptor, for example receptor cells, preferably by
injecting into organisms.
[0065] According to another embodiment of the invention, the method
for preparing cellular MVs containing siRNA comprises: [0066] 1)
designing siRNA sequence for target gene; [0067] 2) synthesizing
mature siRNA or constructing siRNA expression vector chemically.
[0068] 3) transferring siRNA or siRNA expression vector into cells
through using cell transfection technology.
[0069] Preferably, the siRNA sequence of target gene is designed
with the following principles: [0070] (1) siRNA fragment meets
AAN19TT, NAN19NN, NARN17YNN and NANN17YNN (N represents any base, R
and Y represent purine and pyrimidine respectively); [0071] (2)
selecting complementary DNA exon sequences without repeated
sequences and antisense strand with balanced content of A, G, C, T
(or the content of GC is between 30% and 70%); [0072] (3) avoiding
the sequences with clustered single bases, especially G base;
[0073] (4) avoiding untranslated regions of 3' and 5' ends (5'-UTR,
3'-UTR) which usually are binding sites of mRNA binding protein.
[0074] (5) avoiding the start codon or exon-exon boundaries.
[0075] The designed siRNA sequence is researched for BLAST in EST
or Unigene data base of NCBI (National Center for Biotechnology
Information) to ensure the specificity of the siRNA sequence to the
target gene.
[0076] More than 4 of siRNA sequences are designed and synthesized,
and then siRNA sequences with best silence effect are screened by
experiment for further gene function study.
[0077] Preferably, method for the construction of siRNA expression
vector includes: inserting DNA molecules with about 70 bp bases in
length comprising a specific stem-loop and termination signal into
a certain vector.
[0078] Preferably, method for transfecting siRNA is performed by
liposome method (Lipofectamine 2000, Invitrogen).
[0079] The method for preparing cellular MVs is selected from one
or more of differential centritfugation, immune-adsorption and
ultrafiltration.
[0080] The method for detecting the loading efficiency of siRNA in
cellular MVs is selected from one or more of RT-PCR, Real time-PCR,
Northern blot, immunofluorescence and flow cytometry.
[0081] RT-PCR, for example, includes the following steps: [0082]
(1) extracting total RNA of cells or tissues after RNA
interference, and cDNA samples are achieved by reverse
transcription; [0083] (2) performing PCR reactions with target gene
specific primers; [0084] (3) performing agarose gel electrophoresis
of the PCR product; [0085] (4) observing the results under UV lamp
after EB staining.
[0086] Real time PCR, for example, includes the following steps:
[0087] (1) extracting total RNA of cells or tissues after RNA
interference, and cDNA samples are achieved by reverse
transcription; [0088] (2) designing target gene specific primers;
[0089] (3) performing PCR reactions by adding fluorescent
probes.
[0090] Northern blotting, for example, includes the following
steps: [0091] (1) collecting serum/plasma, and cell, tissue
samples; [0092] (2) extracting total RNA by Trizol reagent; [0093]
(3) performing denature PAGE and trarsmembrane; [0094] (4)
preparing target gene probe labeled with isotope; [0095] (5)
performing membrane hybridization; [0096] (6) detecting the results
by isotopic signals, e.g., Phosphor Scanning.
[0097] Immunofluorescence, for example, includes the following
steps: [0098] (1) attaching cells on the support; [0099] (2) fixing
cells with cell fixative, e.g., paraformaldehyde; [0100] (3)
blocking cells with skimmed milk or bovine serum albumin; [0101]
(4) labeling protein specific for target gene with fluorescent
labeled antibodies; [0102] (5) observing the fluorescence intensity
of the cells under fluorescence microscope.
[0103] The receptor cells include all existing cell lines, cell
strain and primary cultures of cells or tissues of healthy human or
patients with diseases.
[0104] Organisms into which cellular MVs can enter include human,
various animals and various pathogenic microorganisms. The animals
include chondrichthyes, teleost, amphibians, reptiles, birds and
mammals. The pathogenic microorganisms include bacterium,
spirochetes, mycoplasma, rickettsia, chlamydia and actinomycetes.
Specially, the pathogenic microorganisms include various kinds of
DNA and RNA viruses, such as hepatitis B virus, smallpox virus,
AIDS virus, SARS, influenza virus, etc.
[0105] The present invention also provides a research method,
including:
[0106] transferring siRNA and its control sequence into donor
cells;
[0107] separating cellular MVs containing siRNA;
[0108] adding the cellular MVs containing siRNA into receptor
cells;
[0109] studying the effect of MVs containing siRNA after they enter
receptor cells.
[0110] According to one embodiment of the invention, the method for
studying the gene function using cellular MVs carrying siRNA
includes: [0111] 1) transferring siRNA and its control sequence
into the donor cells by transfection; [0112] 2) separating and
preparing microvesicles of the donor cells containing siRNA; [0113]
3) adding the cellular MVs into receptor cells: [0114] 4) studying
the effect of cellular MVs carrying siRNA on the function of
receptor cells after they enter receptor cells to investigate the
effect of their target genes on the function of cells,
[0115] The method for studying effect of cellular MVs carrying
siRNA on the function of receptor cells after entering receptors
includes one or more of confocal fluorescence microscopy, Western
bloting and method of cell migration.
[0116] For example, Western blotting includes the following steps:
[0117] (1) extracting total proteins of cells or tissues with the
protein lysis solution. [0118] (2) performing SDS-PAGE and
trarsmembrane. [0119] (3) blocking cells with skimmed milk or
bovine serum albumin; [0120] (4) labeling protein specific for
target gene on the membrane with HRP labeled antibodies; [0121] (5)
adding HRP substrate to produce luminescence reaction; [0122] (6)
radioautographing.
[0123] The present invention also provides a method for preventing
and or treating diseases, including: transferring cellular MVs
containing siRNA into receptor.
[0124] According to one of the embodiment of the invention, method
for preventing/treating diseases using cellular MVs carrying siRNA
includes: [0125] 1) transferring siRNA into donor cells by
transfection; [0126] 2) separating and preparing microvesicles of
the donor cells containing siRNA; [0127] 3) adding the cellular MVs
into receptor cells or injecting the cellular MVs into patients.
[0128] 4) cellular MVs carrying siRNA enter receptor cells or
tissues of patients and change the content of protein of the target
gene through interfering the expression of target genes of the
receptor cells or tissues of patients. [0129] 5) cellular MVs
carrying siRNA play a role in preventing/treating diseases by
changing the proteins in cells to affect cellular function.
[0130] Diseases include: various tumors; various acute and chronic
infectious diseases, for example viral diseases such as viral
influenza, viral hepatitis, AIDS, SARS, bacterial diseases such as
tuberculosis, bacterial pneumonia, and other acute and chronic
infectious diseases caused by various pathogenic microorganisms;
other acute and chronic diseases, such as respiratory system
diseases, immune system diseases, blood and hematopoietic system
diseases, circulatory system diseases, endocrine system and
metabolic diseases, digestive system diseases, nervous system
diseases, urinary system diseases, reproductive system diseases and
motor system diseases.
[0131] SiRNA includes all the siRNA sequences designed for receptor
genes, which will degrade the target genes specifically through the
mechanism of RNAi.
[0132] Genes include all gene fragments which can be transcribed
into molecules with function, such as protein gene, microRNA gene
and so on. Disease-causing genes include various genes of organisms
including human and various animals including chondrichthyes,
teleost, amphibians, reptiles, birds and mammals, etc. which
themselves participate in the occurrence and development of
diseases.
[0133] The above-mentioned pathogenic microorganisms include
bacterium, spirochetes, mycoplasma, rickettsia, chlamydia and
actinomycetes. Specially, the pathogenic microorganisms include
various kinds of DNA and RNA viruses, such as hepatitis B virus,
smallpox virus, AIDS virus. SARS, influenza virus, etc.
[0134] The present invention also provides the use of cellular MVs
containing siRNA in delivering siRNA.
EXAMPLES
[0135] It can be understood that the specific embodiments described
herein are illustrated by way of examples and does not as a
limitation of the invention. The main features of the present
invention can be applied in various embodiments without departing
from the scope of the invention. It will be realized or can be
confirmed by a person skilled in the art that many equivalents can
be applied to the specific steps described herein using
conventional experiments. These equivalents are considered to be
within the scope of the invention and covered by the appended
claims.
Example 1
Separation and Detection of the Cellular MVs in the Serum/Plasma
and Cell Culture Medium
[0136] Differential centrifugation is used in this example to
separate cellular MVs from serum/plasma or cell culture medium:
[0137] Specifically, serum/plasma or cultured cells is centrifuged
under 300 g for 5 min to get the supernatant; (2) the supernatant
is centrifuged under 1500 g for 20 min to get the supernatant; (3)
the supernatant is centrifuged under 10000 g for 30 min to get the
supernatant; (4) the supernatant is centrifuged under 110000 g for
70 min to get the precipitates which are cellular MVs.
[0138] The separated cellular MVs are observed under transmission
electron microscope (TEM).
[0139] Cellular MVs precipitates are fixed in 2.5% of glutaral at
4.degree. C., overnight rinsed three times with PBS for 10 min
each, then fixed in 1% of osmium tetroxide at room temperature for
60 min. The fixed samples are embedded with 10% of gelatin and then
refixed with glutaral at 4.degree. C. After that, the samples are
cut into small pieces (with a volume of less than 1 mm.sup.3). The
samples are dehydrated with ethanol solutions of increasing
concentration successively (30%, 50%, 70%, 90%, 95% and
100%.times.3). After embedding with epoxy resin, the samples are
sliced with Leica UC6 microtome and finally observed under FEI
Tecnai T20 transmission electron microscope at 120 kV.
[0140] The transmission electron microscope (TEM) picture of
cellular MVs created by differential centrifugation is shown in
FIG. 1. FIG. 1 shows that cellular MVs separated from healthy human
serum/plasma are different in size, ranging between 10-500 nm.
Example 2
Transfection of siRNA into Donor Cells
[0141] In this example, fluorescence labeled siRNA is transfected
into cells according to the following steps and the transfection
efficiency is detected.
[0142] Firstly, siRNA sequence is designed for different sites of
human c-myb gene sequence:
TABLE-US-00001 (sense strand + loop + antisense strand):
5'-GGTGGAACAGAATGGAACATTGAAGAAGTGTTCCATTCTGTTCCACC TT-3';
[0143] Meanwhile, a random sequence is designed as the negative
control:
TABLE-US-00002 (sense strand + loop + antisense strand):
5'-GACTTCATAAGGCGCATGC TTGAAGAAG GCATGCGCCTTATGAAG TC TT-3'.
[0144] Furthermore, the above-mentioned designed siRNA is
synthesized commercially, siRNA against c-myb gene is labeled with
green fluorescent dye FITC.
[0145] The siRNA is transfected into human monocytes/macrophages
cell line THP-1 cells (Type Culture Collection of Chinese Academy
of Sciences, Shanghai, China) with liposome Lipofectamine 2000
(Invutrigen, US), the detailed method is as follows: [0146] (1)
THP-1 cells are cultured in the RPMI 1640 medium (Gibco, US)
supplemented with 10% FBS (Gibco, US), 5% CO.sub.2, at 37.degree.
C. [0147] (2) 30 .mu.l lipofectamine 2000 and 600 pmol negative
control of siRNA is mixed with 1 ml OPTI-MEM (Gibco, US)
respectively to form mixture A and B, then kept at room temperature
for 5 min. [0148] (3) 30 .mu.l lipofectamine 2000 and 600 pmol
c-myb siRNA is mixed with 1 ml OPTI-MEM (Gibco, US) respectively to
form mixture C and D, then kept at room temperature for 5 min.
[0149] (4) mixture A is mixed with mixture B to form mixture E,
kept for 20 min. [0150] (5) mixture C is mixed with mixture D to
form mixture F, kept for 20 min. [0151] (6) mixture E and mixture F
are added into the cells in control group and experimental group
respectively, OPTI-MEM is added to 15 ml. Cultured with 5% CO.sub.2
at 37.degree. C. [0152] (7) normal culture medium is refreshed
after 6 h. [0153] (8) the transfection is completed after 24-48 h,
and samples can be collected.
[0154] Real time-PCR was used to detect mRNA level of c-myb gene so
as to detect interference efficiency, the method includes: [0155]
(1) collecting the transfected THP-1 cells [0156] (2) preparing
cDNA sample: the total RNA is extracted with Trizol reagent
(Invitrogen, US) and the cDNA samples was achieved through reverse
transcription of the total RNA. The reaction system of reverse
transcription contains 4 .mu.l 5.times.AMV buffer solution, 2 .mu.l
10 mM each dNTP mixture (Takara, Japan), 0.5 .mu.l RNase Inhibitor
(Takara, Japan), 2 .mu.l AMV (Takara, Japan) and 0.5 .mu.l OligodT
(Takara, Japan). The reaction steps are that incubated at
16.degree. C. for 15 min, reacted at 42.degree. C. for 1 h, and
incubated at 85.degree. C. for 5 min. [0157] (3) Real-time PCR
reaction: 0.3 .mu.l Taq enzyme (Takara, Japan), 0.5 .mu.l 10 .mu.m
forward and reverse primers, 1.2 .mu.l 25 mM MgCl.sub.2, 1.6 .mu.l
2.5 mM each dNTP mixture (Takara, Japan), 1 .mu.l 20.times. EVA
GREEN, 2 .mu.l 10.times.PCR buffer solution and 12.4 .mu.l H.sub.2O
are added to 1 .mu.l cDNA. The PCR system is 20 .mu.l. The
instrument used is ABI Prism 7300 fluorescence ration PCR
instrument. The reaction condition is: 95.degree. C., 5 min for one
cycle.fwdarw.95.degree. C. 15 s, 60.degree. C. 1 min for 40 cycles.
[0158] (4) Data processing: the data processing method is
.DELTA.C.sub.T method. .DELTA.C.sub.T is set as the cycle number
when the reaction reached threshold. Therefore, the comparison of
siRNAs in the two groups of samples can be represented by equation
2.sup.-.sup..DELTA..sup.CT, wherein .DELTA.C.sub.T=C.sub.T
group1-C.sub.T group2. The data processing method of the cells and
tissues is that U6 is used as an internal standard. Thus the
comparison of mRNA expression in the two groups of samples can be
represented by equation 2.sup.-.sup..DELTA..sup.CT, wherein
.DELTA.C.sub.T=[C.sub.TmiRNA-C.sub.T
U6].sub.group1-[C.sub.TmiRNA-C.sub.T U6].sub.group2.
[0159] The result is shown in FIG. 2-A, compared with the negative
control group transfected with random sequence (left column), mRNA
expression of c-myb gene in cells transfected with c-mybsiRNA
decreases significantly, suggesting the feasibility and efficiency
of the transfection method used in this experiment.
[0160] Meanwhile, flow cytometry is used to detect the efficiency
of siRNA transferred into cells. The results is shown in FIG. 2-B.
The flow cytometry includes the following steps: collecting THP-1
cells after interference and adjusting the cell concentration to
10.sup.6/ml: detecting the fluorescence intensity of the cells by
flow cytometry (BD FACS. Calibur) with the zoom modes of voltage
being lin at both forward and lateral when detection, and
fluorescence intensity is detected with FL1-H with the zoom mode of
voltage being log. It can be seen from the results that, compared
with the control group (fine line), the fluorescence intensity of
the experiment group (thick line) transfected with c-mybsiRNA
increase, indicating that the transfection efficiency of siRNA is
high, and such method is an intuitive and efficient one in
detection of transfection efficiency of siRNA.
[0161] In addition, fluorescence microscopy method can also be used
to detect the efficiency of siRNA transferring into cells, the
method includes: THP-1 cells that have been transfected with
c-mybsiRNA are placed on the object stage of fluorescence invert
microscope (OLYMPUS) and detected with a excitation wavelength of
488 nm.
[0162] The result is shown in FIG. 2-C, after transfection, the
cells show green fluorescence (as shown in the bright), suggesting
that the efficiency of the siRNA transferring into the cells is
high.
Example 3
Transferring of Cellular MVs Carrying with siRNA into Receptor
Cells
[0163] In this example, cellular MVs of THP-1 cells transfected
with c-mybsiRNA are collected to detect their loading efficiency.
Meanwhile, the cellular MVs are added to target cells to detect
their efficiency of transferring into the target cells.
[0164] Human monocytes/macrophages cell line THP-1, which plays an
important role in the inflammatory response is selected as the
study object. THP-1 cells are incubated in the 1640 medium (Gibco,
US) supplemented with 10% fetal calf serum (FBS)(Gibco, US) at
37.degree. C., 5% CO.sub.2. First, the THP-1 cells transfected with
c-mybsiRNA are prepared according to the method of siRNA
transtection in example 2.
[0165] Next, according to the method of separating the cellular MVs
in example 1, THP-1 cells transfected with c-mybsiRNA are
separated.
[0166] Then, the loading efficiency of siRNA in cellular MVs
separated is detected by flow cytometry.
[0167] The result is shown in FIG. 3-A. During detection, zoom
modes of voltage are lin at both forward and lateral, and
fluorescence intensity is detected with FL1-H and the zoom mode of
voltage is log. It can be observed seen from the results that part
of cellular MVs excreted by THP-1 (right part of the vertical lines
in FIG. 3-B) is labeled with fluorescence. As c-myb siRNA is
labeled with fluorescence, the fluorescence intensity is detected
by flow cytometry could reflect the loading efficiency of siRNA in
cellular MVs.
[0168] Finally, microvesicles secreted by THP-1 cells carrying
c-mybsiRNA are added to the cell culture medium of human venule
endothelial cells HMEC-1 (Georgia CDC, US). HMEC-1 cells are
incubated in the MCDB-31 medium (Gibco, US) supplemented with 10
ng/mL epidermal growth factor (Becton-Dickinson, US), 10 ng/mL
hydrocortisone (Sigma) and 10% FBS (Gibco, US) at 37.degree. C., 5%
CO.sub.2.
[0169] Under the physiological condition, monocytes/macrophages
cells can interact with vascular endothelial cells. Molecules on
the surface of monocytes can specifically bind to the
receptors/ligands on the surface of endothelial cell, thus inducing
a series of signal transduction as well as activity of cell
physiology. Therefore, monocytes/macrophages cell line THP-1 and
human venule endothelial cells HMEC-1, the interaction of these two
kinds of cells in vivo can be simulated.
[0170] The transferring efficiency of THP-1 microvesicle carrying
siRNA into HMEC-1 cells is detected. Due to the cellular
microvesicles are labeled with green fluorescence, the fluorescent
result of HMEC-1 is detected by fluorescence microscopy, which
would reflect the transferring efficiency of MVs into HMEC-1. The
result is shown in FIG. 3-C.
[0171] From the result, it can be seen that cellular MVs carrying
siRNA could enter target cells HMEC-1 (bright point in FIG. 3-C)
efficiently and specifically. As all cells could excrete cellular
MVs like hemocyte; and all cells could receive cellular MVs
excreted by the cells which could specifically act on them.
Consequently, the action mode of THP-1 cellular MVs entering into
HMEC-1 cells can also simulate the one all cellular MVs entering
into their target cells on the organism.
[0172] According to the above-mentioned results, it is not
difficult to find that cellular MVs are ideal vectors that transfer
siRNA stablly, efficiently and specifically, which may transfer
siRNA to its receptor cells through cellular MVs. It is suggested
that siRNA can be high-affinity and specifically delivered as a
drug to target cells through microvesicles, and the purpose of drug
prevention/treatment can be achived by affecting the function of
target cells involving in developing disease.
Example 4
Study of the Gene Function with Cellular MVs Carrying siRNA
[0173] In this example, cellular MVs were used to transfer siRNA
against target genes to receptor cells efficiently and
specifically, which, by specifically reducing the expression of
target gene in receptor cells, simulate the pathological conditions
or plays an action of gene knockout, so aso to study the
physiological functions of target genes in cells.
[0174] In this example, c-myb gene is selected as the study object,
which encodes a cell transcription factor and plays an important
role in cell differentiation, proliferation, migration and survival
of hemocyte. C-myb has been proven to be an important
proto-oncogene and has a close relation to the occurrence and
development of a series of cancers.
[0175] In order to study the function of c-myb gene in endothelial
cells, the following experiment is designed: [0176] 1) preparing
the THP-1 cells transfected with c-myb siRNA according to the
transfection method of siRNA in example 2. [0177] 2) separating the
THP-1 cells transfected with c-myb siRNA according to the
separation method of cellular MVs in example 1. [0178] 3) Adding
the cellular MVs carrying c-mybsiRNA to the culture medium of
HMEC-1 cells and collecting cells after 6 h to perform Western
blot. The detailed steps include: [0179] (1) extracting total
protein with the protein lysis solution; [0180] (2) performing
SDS-PAGE electrophoresis under constant pressure of 90V; [0181] (3)
carrying out membrane-transferring experiment under constant
current of 160 Ma; [0182] (4) blocking the membrane with 5% skim
milk; [0183] (5) labeling c-myb and GAPDH with monoclonal
antibodies against anti-c-myb (Santa Cluz Co., LTD) and monoclonal
antibodies against anti-GAPDH (Santa Cluz Co., LTD) respectively;
[0184] (6) adding the corresponding HRP to label the secondary
antibody; [0185] (7) adding the substrate of HRP to trigger
luminescence reaction. [0186] (8) autoradiographing.
[0187] By combining with the target gene mRNA of siRNA and
recruiting the silencing complex in the cells, the target gene mRNA
of siRNA are degraded, thus leading to the decrease of the
expression level of its target protein. Therefore, the efficiency
of siRNA entering the receptor cells can be detected by Western
blot which detect the expression level of the specific protein.
[0188] The result is shown in FIG. 4-A. It can be seen from the
result that, as an internal reference standard, GAPHD has
demonstrated that the amounts of the total proteins added during
Western blot are the same in all of the bands. Meanwhile, compared
with the cells transferred with negative control (band 2), the
expression level of c-myb protein have decreased significantly in
the cells transferred with c-myb siRNA (band 3). Therefore, it has
been proven by the inventors that: c-myb siRNA carrying cellular
MVs is delivered to HMEC-1 cells and has an interference effect on
the expression of the c-myb protein in HMEC-1 cells. It is further
demonstrated that siRNA can be delivered to the target cells
efficiently and specifically, which, as a experimental tool, study
the function of the genes in specific cells.
[0189] At the same time, effect of siRNA carried by MVs on the
migration ability of its target cell HMEC-1 is also detected in
example 1.
[0190] As an important transcription factor, c-myb gene plays an
important role in cell growth, migration, and differentiation.
Though researches have proven that c-myb has significant regulatory
effect on the migration of various kinds of cells, which it plays a
role in the migration of endothelial cells still needs further
investigation.
[0191] So in this example, the expression of c-myb protein in
endothelial cell line HMEC-1 cell is reduced specifically by using
siRNA carried by cellular MVs in this example to detect the
migration function of cells under this condition, thus studying
whether the c-myb gene has an effect on migration function of
endothelial cells
[0192] The detailed experimental steps include: [0193] (1)
preparing the THP-1 cells transfected with c-myb siRNA according to
the transfection method of siRNA in example 2. [0194] (2)
separating the THP-1 cells transfected with c-myb siRNA according
to the separation method of cellular MVs in example 1. [0195] (3)
incubating the HMEC-1 cells for 2 h with THP-1 cellular MVs
carrying with c-mybsiRNA. [0196] (4) detecting the migration
ability of HMEC-1.
[0197] The detection method of the cell migration experiment
include: covering polycarbonate membrane (8 .mu.m in pore diameter)
at the bottom of the upper small chamber on Transwell Boyden
Chamber (6.5 mm, Costar, Cambridge, Mass., US) with 0.1% gelatin;
suspending the HMEC-1 cells in medium without serum at a
concentration of (1-10).times.10.sup.5 cells/mL; incubating the
cells with or without the cellular MVs containing siRNA derived
from THP-1 for 2 h, then adding the HMCE-1 cells on the upper small
chamber, while adding 0.5 mL of medium with 10% FBS to the lower
small chamber; incubating in incubator with 5% CO.sub.2 for 4 h;
fixing the cells migrated to the lower layer with 90% ethanol at
room temperate for 15 min; washing; staining with 0.1% crystal
violet at room temperate for 15 min; scraping down the cells
remained on the filter membrane; photographing (Olympus, BX51,
Japan); count the cells.
[0198] The microscopic picture of cells after migration is shown in
FIG. 4-B\C\D. It can be seen from the results that, compared with
the negative control (FIG. 4-B), the migration ability of HMCE-1
cells treated with cellular MVs carrying c-myb siRNA increases
significantly. Number of cells is counted in 5 random visual
fields, the result is shown in FIG. 4-D. The number of migrated
cells of HMEC-1 cells which are treated with cellular MVs carrying
c-myb siRNA increases significantly in compared with the
control.
[0199] So, migration ability of HMEC-1 cells can be enhanced
significantly by c-myb genes with lower expression. Instead, it is
therefore suggested that c-myb gene has inhibitory effect on the
migration of endothelial cells.
[0200] Thus in this example, it has been proven that preparing
cellular MVs carrying siRNA and relative method can be used as a
study tool of medical biology, to study the function of the genes
by selectively decrease the expression of certain genes in the
cells.
Example 5
Prevention/Treatment of Diseases with Cellular MVs Carrying
siRNA
[0201] In this example, cellular MVs carrying siRNA against HIV
gene are used to inhibit the survival and reproduction of HIV in
its host cells.
[0202] The detailed method includes the following steps: [0203] 1)
designing siRNA sequence for HIV genomic sequence; [0204] 2)
inserting siRNA sequence into a vector; [0205] 3) transfecting the
vector carrying HIV siRNA into the donor cell 293T cell; [0206] 4)
collecting the cellular MVs excreted by donor cells; [0207] 5)
adding the cellular MVs containing HIV siRNA excreted by donor
cells to the HIV host cells; [0208] 6) detecting the content of
viruses in host cells.
[0209] The result is shown in FIG. 5. The ordinate represents the
content of HIV in their host cells. If the blank cells completely
freed of viruses are used as the control (horizontal axis 1
represents the column), its value is set to be 1; The content of
viruses in cells only with viruses but without any therapeutic
measures (horizontal axis 2 represents column) will be more than 16
times of the control cells. However, if the cellular MVs carrying
viruses siRNA are used as the treatment tool, the content of
viruses in the host cells will dramatically decreased. It can be
seen from the result that, after the siRNA carried by cellular MVs
is added (horizontal axis 5 and 6 represent columns), the content
of viruses in host cells reduces to about 40% (horizontal axis 6
represents column). More importantly, if the content of siRNA
carried by MVs (horizontal axis 5 represents column), the HIV in
host cells can even be totally inhibited, and the content of HIV
can decreases to level equal to that of the non-viruses group
(horizontal axis 1 represents column).
[0210] Furthermore, in order to exclude the inhibitory effect of
the cellular lMVs carrying siRNA on HIV is caused by the vector
itself and not siRNA, we have also transferred the blank control
without siRNA as another control (horizontal axis 3 represents
column). It can be seen from the result that only the blank vector
can not have inhibitory effect on the HIV, which has also
demonstrated that it is siRNA itself and not the vector that have
inhibitory effect on the viruses.
[0211] Meanwhile, we have also added a anti-HIV drug with short
peptides as the positive control (horizontal axis 4 represents
column). It is observed from the result that the content of HIV
content can only be reduced to about 50% by the drug. Therefore, in
compared with the conventional drugs for treating AIDS, the
cellular MVs carrying HIV siRNA has higher efficiency and better
effect of inhibiting viruses. It is further suggested that using
the cellular MVs carrying siRNA to treat diseases has great
development potential.
Example 6
Detection of Pharmaceutical Composition Consisting of Cellular MVs
and siRNA Carried Thereof
[0212] In this example, a series of methods are used to detect the
existence of cellular MVs and pharmaceutical composition composed
of siRNA carried by cellular MVs. [0213] (1) The
fluorescence-labeled siRNAs are transfected into donor cells
according to the method described in example 2. The result is shown
in FIG. 2-C, it is observed under fluorescence microscope that
fluorescence-labeled siRNA had been transfected into cells. [0214]
(2) Cellular MVs, excreted by donor cells transfected with
fluorescence-labeled siRNA, are separated and identified according
to the method described in example 1. The result is shown in FIG.
1. It is observed that the separated and obtained cellular MVs
comply with the characteristics of cellular MVs from the shape,
size and membrane structure, etc. [0215] (3) Flow cytometry is used
to detect if there are any siRNA enclosed in the microvesicles
which had already been separated and purified as well as identified
as cellular MVs, i.e. if they make up complex of cellular MVs and
siRNA, the result is shown in FIG. 3-B. Due to siRNA is labeled
with fluorescence, if siRNA is contained in cellular MVs, the
cellular MVs must be labeled with fluorescence. Therefore, we use
the flow cytometry to detect the content of fluorescence carried by
cellular MVs. As it is shown in FIG. 3-B that, most of the cellular
MVs carry with fluorescence (right part of the vertical lines in
FIG. 3-B), which proves siRNAs are enclosed in cellular MVs, i.e.
proves the existence of cellular MVs-siRNA drug complex.
[0216] The present invention provides, including: (1) cellular MVs
carrying siRNA; (2) the treatment of various clinical diseases
(including: various tumors; acute and chronic infectious diseases
and other acute and chronic infectious diseases caused by various
pathogenic microorganisms; other acute and chronic diseases, such
as respiratory system diseases, immune system diseases, blood and
hematopoietic system diseases, e.g., circulatory system diseases of
cardiovascular and cerebrovascular diseases, endocrine and
metabolic system diseases, digestive system diseases, nervous
system diseases, urinary system diseases, reproductive system
diseases and motor system diseases) are researched using siRNA
carried by cellular MVs; (3) functions of specific genes are
researched using the cellular MVs which highly and specifically
deliver the siRNA as a experimental tool.
[0217] According to a series of studies mentioned above, it is
clearly that the present invention provides a method for preparing
cellular MVs carrying siRNA, which has a highly targeting property,
stability and high efficiency.
[0218] According to the above-mentioned method, the present
inventor has confirmed that siRNA could be delivered to target
cells by cellular MVs stably, efficiently and specifically, and
influence the functions of the target cells by acting on their
target genes. Therefore, the cellular MVs carrying siRNA, can not
only act as a bio-medical research tool, playing a role in the
study of the gene function; also act as a drug, entering the
organisms efficiently and specifically, playing a role of changing
the gene expression and influencing the cell functions, thus
treating/preventing diseases.
* * * * *