U.S. patent application number 16/905719 was filed with the patent office on 2021-01-07 for exosome transfer of nucleic acids to cells.
This patent application is currently assigned to Codiak BioSciences, Inc.. The applicant listed for this patent is Codiak BioSciences, Inc.. Invention is credited to Jan LOTVALL, Hadi VALADI.
Application Number | 20210000976 16/905719 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
United States Patent
Application |
20210000976 |
Kind Code |
A1 |
LOTVALL; Jan ; et
al. |
January 7, 2021 |
EXOSOME TRANSFER OF NUCLEIC ACIDS TO CELLS
Abstract
Methods for introducing nucleic acids to cells via exosomes for
use in gene modulation and therapy, such as for gene silencing and
to introduce genetic material into cells to compensate for abnormal
genes or to induce or repress a process in the recipient cell.
Inventors: |
LOTVALL; Jan; (Boston,
MA) ; VALADI; Hadi; (Goteborg, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Codiak BioSciences, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Codiak BioSciences, Inc.
Cambridge
MA
|
Appl. No.: |
16/905719 |
Filed: |
June 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16201937 |
Nov 27, 2018 |
10695443 |
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16905719 |
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15857539 |
Dec 28, 2017 |
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16201937 |
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15476844 |
Mar 31, 2017 |
9889210 |
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15857539 |
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14750457 |
Jun 25, 2015 |
9629929 |
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15476844 |
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11799148 |
Apr 30, 2007 |
9085778 |
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14750457 |
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60797149 |
May 3, 2006 |
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Current U.S.
Class: |
1/1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/87 20060101 C12N015/87; C12N 15/113 20060101
C12N015/113 |
Claims
1. A method of transferring genetic material, comprising: a)
providing exosomes containing selected genetic material; and b)
transferring the selected genetic material from the exosomes to
recipient cells.
2. The method of claim 1, wherein the selected genetic material is
selected from the group consisting of mRNA, tRNA, rRNA, siRNA,
regulating RNA, non-coding and coding RNA, DNA fragments, and DNA
plasmids.
3. The method of claim 1, wherein use of recipient cells are
selected from the group consisting of cells from recipients having
inherited diseases in hematopoietic, non-hematopoietic, stem cells,
and organs.
4. The method of claim 1, wherein the selected material comprises
nucleic acids and the recipient cells comprise CD4 T-cells for
treatment of HIV infected T-cells.
5. The method of claim 1, wherein the selected genetic material is
transferred to treat diseases, induce or repress cell death, change
cellular ageing, induce tolerance, re-direct existing immune
responses, or change intracellular activity or cellular
behaviour.
6. The method of claim 1, wherein providing the exosomes further
comprises a) producing exosomes lacking genetic material; and b)
adding selected genetic material to the exosomes lacking genetic
material.
7. The method of claim 6, wherein the selected genetic material is
selected from the group consisting of mRNA, tRNA, rRNA, siRNA,
regulating RNA, non-coding and coding RNA, DNA fragments, and DNA
plasmids.
8. The method of claim 7, wherein the selected genetic material is
added to the exosomes lacking genetic material using a method
selected from the group consisting of transformation, transfection
and microinjection of the selected genetic material into
exosomes.
9. The method of claim 1, wherein the exosomes are produced by a
method selected from the group consisting of isolation from a
selected type of donor cell; isolation from a person with a
particular disease or condition; and isolation from a genetically
modified donor cell.
10. The method of claim 9, further comprising genetically modifying
the donor cell to erase production of a nucleic acid, or to
up-regulate or down-regulate production of a nucleic acid.
11. The method of claim 1, further comprising introducing the
selected genetic material into the exosomes by introducing the
selected genetic material into donor cells followed by
intracellular translocation of the genetic material into the
exosomes.
12. The method of claim 1, further comprising introducing the
selected genetic material into the exosomes using a method selected
from the group consisting of transformation, transfection and
microinjection of the selected genetic material into the
exosomes.
13. The method of claim 1, wherein the genetic material is
transferred to the recipient cells by a method selected from the
group consisting of addition of the exosomes to cell cultures in
vitro, intravenous injection of the exosomes, in vivo
administration, and administration targeted to particular cells in
the body.
14. The method of claim 1, wherein the exosomes containing selected
genetic material are taken from a patient with a malignant disease;
and are further processed to contain particular genetic constructs;
and then are reintroduced to the patient.
15. A composition of isolated exosomes, wherein the isolated
exosomes contain one or more selected nucleic acid constructs, and
wherein the isolated exosomes do not contain any substantial amount
of nucleic acids other than the selected nucleic acid
constructs.
16. The composition of claim 15, wherein the selected nucleic acid
constructs are selected from the group consisting of mRNA, tRNA,
rRNA, siRNA, microRNA, regulating RNA, non-coding and coding RNA,
DNA fragments, and DNA plasmids.
17. The composition of claim 1, wherein the isolated exosomes
containing one or more selected nucleic acid constructs are
isolated from donor cells, wherein the donor cells express the
nucleic acid constructs or have been transfected with the nucleic
acid constructs.
18. The composition of claim 1, wherein the exosomes were isolated
from a specific donor cell, or from a person with a particular
disease or condition, or from a genetically modified donor
cell.
19. The composition of claim 1, wherein the exosomes were obtained
from (i) tumor tissue, (ii) intestinal epithelial cells,
lymphocytes, mast cells or dendritic cells, (iii) in vitro growing
cells, (iv) cell culture supernatant, (v) body fluid, or (vi) any
combination of (i) to (v).
20. A recipient cell, wherein the recipient cell comprises a
selected nucleic acid construct introduced to the recipient cell
via an isolated exosome of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 16/201,937, filed Nov. 27, 2018 (currently
allowed), which is a continuation application of U.S. application
Ser. No. 15/857,539, filed Dec. 28, 2017 (now abandoned), which is
a continuation application of U.S. application Ser. No. 15/476,844,
filed Mar. 31, 2017 (now U.S. Pat. No. 9,889,210), which is a
continuation application of U.S. application Ser. No. 14/750,457,
filed Jun. 25, 2015 (now U.S. Pat. No. 9,629,929), which is a
continuation of U.S. application Ser. No. 11/799,148, filed Apr.
30, 2007 (now U.S. Pat. No. 9,085,778), which claims benefit to
U.S. Prov. Appl. No. 60/797,149, filed May 3, 2006, each of which
is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention is based on the unexpected findings
that exosomes released to the extracellular milieu can carry
selective RNA from the parental cells. This can, according to this
invention, be used to transfer genetic material to recipient cells
by exosomes. By transferring nucleic acids to recipient cells,
exosomes affect another cell's (recipient cells) protein machinery
and thus protein content, and the invention demonstrates for the
first time that nucleic acids, for example RNA and DNA,
deliberately can be transferred between the cells or organs using
exosomes, and can thus be utilized for gene modulation and therapy
in mammalian cells.
2. Description of the Related Art
[0003] Exosomes are small membrane vesicles of endocytic origin
that are released into the extracellular environment following
fusion of multivesicular bodies with the plasma membrane. The size
of exosomes ranges between 30 and 100 nm in diameter. Their surface
consists of a lipid bilayer from the donor cell's cell membrane,
and they contain cytosol from the cell that produced the exosome,
and exhibit membrane proteins from the parental cell on the
surface.
[0004] Exosomes exhibit different composition and function
depending on the cell type from which they are derived. There are
no "exosome-specific" proteins; however several proteins identified
in these vesicles are associated with endosomes and lysosomes
reflecting their origin. Most exosomes are enriched in MHC I and II
(major histocompatibility complex I and II; important for antigen
presentation to immunocompetent cells such as T-lymphocytes),
tetraspanins, several heat shock proteins, cytoskelatal components
such as actins and tubulins, proteins involved in intracellular
membrane fusion, signal transduction proteins and cytosolic
enzymes.
[0005] Exosomes are produced by many cells including epithelial
cells, B and T lymphocytes, mast cells (MC) as well as dendritic
cells (DC). In humans, exosomes have been found in blood plasma,
urine, bronchoalveolar lavage fluid, intestinal epithelial cells
and tumor tissues.
[0006] All functions of exosomes have not been elucidated, but data
strongly indicates they mediate communication between cells. This
communication could take place in different ways. First, exosomes
could bind to cell surface receptor in a similar way as cell to
cell interaction. Second, exosomes could attach to the cell
membrane and give the cells new receptors and properties. Thus,
exosomes can also fuse with target cells and exchange membrane
proteins and cytosol between two cell types.
[0007] We have put extra effort into understanding the content and
biological function of exosomes specifically released by mast
cells. In proteomic assays we have found that these exosomes
contain a larger number of proteins than previously understood.
However, the unique finding from our research is that we have
discovered a substantial amount of selective RNA in exosomes from
mast cells. Furthermore, use of different recipient cells displays
an uptake of exosomal RNA indicating transfer of genetic material
from exosomes into recipient cells, which in turn will lead to
translation of specific protein in the target cells.
[0008] Considering the exosomal protein content and their capacity
to communicate with different recipient cells, it is particularly
useful to be able to modify the genetic content of exosomes in
order to add or regulate a gene in recipient cells. The method
using the exosomes' capacity of carrying specific genetic material
and transferring it to recipient cells is described in this
application. In this method, the recipient is affected in its
function, as well as in its ability to stay alive, further develop,
proliferate or mature.
[0009] The method is unique and different from any previous
described methods. Several patents and patent applications use
exosomes to influence the immune system through stimulatory or
inhibitory function via exosomal protein interaction with immune
cells, and for treatment of viral disease by influencing the immune
system. It has been suggested that exosomal proteins can be
modified by mutation to affect the immune system. However, no
patent or patent application or any publicly available information
that we have found describes or suggests use of exosomes to
transfer genetic material or nucleic acids to cells.
SUMMARY OF THE INVENTION
[0010] The present invention discloses novel methods of delivering
nucleic acid constructs by exosomes to cells. The invention method
includes RNA and DNA constructs that are transferred into exosomes
by using their parental cells (transformation, transfection, or
modification of the parental cells), or using conventional methods
for instance, transformation and transfection, to introduce nucleic
acids directly into exosomes. The method of the invention is an
excellent tool for gene therapy to introduce genetic material into
recipient cells to compensate for abnormal genes or to introduce
RNA or DNA that produces proteins that affect the function of the
recipient cells or their ability to stay alive or develop or
mature. For gene therapy, usually viruses or liposomes are used as
vectors for transfer of genetic material, because they can deliver
the new gene by infecting the cell. Use of exosomes has several
advantages compared to conventional methods, and most importantly
exosomes are derived from cells, and possibly even the recipient's
own cells, and are thus not a foreign body for the immune system,
avoiding adverse immune reactions. Exosomes are thus easily
produced, isolated and modified for use in gene modulation and
therapy.
[0011] Other objects and features of the inventions will be more
fully apparent from the following disclosure and appended
claims.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
THEREOF
[0012] Using exosome vesicles for transferring genetic material,
nucleic acids, to the cytosol or nucleus of a cell as described in
the invention herein, can treat inherited diseases, cellular or
body dysfunctions, induce or repress cell death (apoptosis), change
cellular ageing, induce tolerance, re-direct existing immune
responses, change intracellular activity or cellular behaviour. It
can also be used in all kinds of gene therapy of genetic disorders,
malignant diseases or diseases involving immune cells or any other
cell type in the body including vasculature, epithelial cells,
interstitial cells, musculature, skeletal system, nervous system,
liver cells, kidney cells, gut cells, lung cells, skin cells or any
other cell in the body.
[0013] Genetic material that can be transferred by exosomes,
described in the invention herein is: microRNA, mRNA, tRNA, rRNA,
siRNA, regulating RNA, non-coding and coding RNA, DNA fragments,
and DNA plasmids, including nucleic acids of any type. The methods
of transferring the genetic material (constructs of DNA or RNA, or
any type of nucleic acids) directly into exosomes are
transformation, transfection and microinjection.
[0014] Genetically dissimilar exosomes can be isolated for further
delivery to recipient cells using either their donor cells or by
introducing specific nucleic acids into them. Different approaches
for production of exosomes containing a set of genetic material are
described below.
[0015] Different procedures for production of genetic dissimilar
exosomes using the exosome producing cells for further delivery to
recipient cells include:
[0016] (a) Exosomes from different donor cells. Exosomes containing
different set of genetic material can be isolated from different
donor cells (e.g. B and T lymphocytes, mast cells, and dendritic
cells) for further delivery to recipient cells. Since the genotype
of the donor cells are dissimilar, which is due to their role and
environment, the isolated exosomes from different cell type give
rise to exosomes with dissimilar genotypes.
[0017] (b) Exosomes from different conditions. Growth of the
exosome producing cells in a certain condition give rise to
cellular changes, and subsequently to genetically modified
exosomes. The exosomes can subsequently be used for transferring
the set of nucleic acids to recipient cells.
[0018] (c) Exosomes from different human being or disease. Exosomes
from different persons or diseases contain diverse set of nucleic
acids. The genotype of the exosomes is different due to their donor
cell's source and conditions. The exosomes with the unique set of
nucleic acids can be isolated for further delivery of the nucleic
acids to recipient cells.
[0019] (d) Exosomes from genetically modified donor cells. Gene
disruption or mutations in exosome producing cells give rise to
genetically modified exosomes that can be used for delivery of
their nucleic acids to recipient cells. Expression of any kind of
nucleic acids present in exosomes can be influenced (erased or
up/down regulated) by gene manipulation of the exosome producing
cells. For instance, disruption of a gene in exosome producing
cells results in a lack of corresponding mRNA in exosomes.
Simultaneously, up or down regulation of a gene in exosome
producing cells affects amount of corresponding mRNA in
exosomes.
[0020] Specific nucleic acid construct(s) (cloned genes, DNA
fragments and plasmids, microRNA, siRNA, coding and none coding DNA
and RNA) can be transferred to recipient cells via genetic modified
exosomes. The nucleic constructs can be manufactured using standard
techniques e.g. cloning, isolation and amplification of RNA or DNA
sequences, for further transformation into exosomes.
[0021] (a) Insertion of specific constructs into exosomes using
their donor cells. New constructs (RNA and DNA) can be introduced
into exosomes using their donor cells. The nucleic acid constructs
can be introduced into the donor cells (the exosome producing
cells) and consequently by the intracellular functions the
transcript or the constructed by itself will be translocated into
exosomes.
[0022] (b) Insertion of specific constructs directly into exosomes.
Exosomes can be isolated from different origin, e.g. in vitro
growing cells, human body, or cells originated from a human and
animal. Genetic constructs of RNA or DNA can be introduced into
these exosomes directly by using conventional molecular biology
techniques such as in vitro transformation, transfection, and
microinjection. The genetically modified exosomes can further be
used for transferring their nucleic acids to recipient cells.
Methods for Production of Genetically Modified Exosomes
[0023] Transformation or transfection of genetic material into
exosomes More generally the term transformation and transfection is
used to describe mechanisms of DNA and RNA transfer in molecular
biology and it was described for the first time 1944 by Oswald
Avery, Colin MacLeod, and Maclyn McCarty (Lederberg J., Genetics.
1994 February; 136(2):423-6).
[0024] (a) Electroporation. By this methods number of holes is made
in cells/exosomes by briefly shocking them with and electric field
of 100-200 V/cm. The DNA/RNA can enter the cells/exosomes through
the holes that made by the electric field.
[0025] (b) Lipofection. The method commonly called transfection and
can be used to transform cells/exosomes with DNA/RNA via vesicles
containing the desired genetic constructs. The vesicles fuses with
the cell membrane (similar to how two oil spots at the top of a
broth will fuse) and the contents of the vesicles and the cells are
combined. There are a number of transfection kits in the market,
ready for use, e.g. DeliverX siRNA Transfection Kit (cat. No.
DX0002) from Panomics, FuGENE.RTM. HD Transfection Reagent (Cat.
no. 04709691001) from Roche and LIPOFECTAMINE.TM. 2000 (Cat. No.
11668-027) from Invitrogen.
[0026] (c) Transformation using heat shock. Chilling cells/exosomes
in the presence of divalent cations such as Ca.sup.2+ (in
CaCl.sub.2)) makes their membranes become permeable to RNA or DNA
plasmids/fragments fragments. Cells/exosomes are incubated with the
DNA and then briefly heat shocked (42.degree. C. for 30-120
seconds), which causes the DNA to enter the cell. This method works
well for circular plasmid DNAs.
[0027] The above methods describe briefly how genetically modified
exosomes can be achieved to transfer RNA and DNA to recipient
cells. Exosomes that contain RNA/DNA or that are modified to
contain the gene of interest will be isolated and shifted to the
recipient cells, to affect their biological function or survival.
Consequently, the exosomes will dispose their content into the
cytoplasm of the target cells, which in turn leads to translation
of mRNA to specific proteins in the target cell through the cells
own protein machinery. Further exosomes are capable to carry and
transfer small coding and none coding RNA such as microRNA and
siRNA that may regulate translation of a specific gene.
[0028] Exosomes being vesicles as carrier of DNA or RNA as
described in the invention herein can be used to treat inherited
diseases in hematopoietic, non-hematopoietic, stem cells, and
organs. Exosome vesicles can also be used as carriers of DNA or RNA
constructs for treatments of microbiological infections or diseases
or dysfunctions in humans or animals, or transfer of genetic
material of any biological membrane.
[0029] Because in humans CD4 T-cells are the target for HIV
infections, infected cells can be treated with RNA or DNA
constructs (siRNA, RNAi, or DNA) carried and transferred by
exosomes to the infected T-cells, specifically designed for
silencing of the translation of the viral RNA. Thus our invention
discloses that exosomes that are capable of transferring their
nucleic acids to CD4 T-cells can be used for treatment of HIV
infected T-cells, as well as T-cell malignancies such as lymphoma
or lymphatic leukemias.
[0030] Changing or modifying the genetic material of exosomes by
altering the condition for the exosome-producing cells, is achieved
by changing pH, temperature, growing conditions, or using
antibodies/chemicals toward exosome-producing cells. This results
in alteration of the nucleic acid content. Also, over-expression or
repression of cytokines, chemokines and other genes in the
exosome-producing cells can be used to change or modify the genetic
content of exosomes
[0031] Transferring sense or anti-sense RNA to specific cells using
exosome vesicles to switch off genes instead of adding new ones
results in down regulation (slow down) or prevention of translation
of the particular gene. The method is called RNA interference
(siRNA).
[0032] The invention herein makes it possible to transfer genetic
materials in vitro to stem-cells acquired from a patient or a donor
prior to administration of this stem cell to a patient or a
recipient human, or animal.
[0033] To administer nucleic acids to recipient cells or tissues,
DNA or RNA-containing exosomes can be administered to cells by
addition of the exosomes to cell cultures in vitro, or injection of
these exosomes intravenously, or by any other route, in vivo as is
known in the art. Exosomes can be targeted to any cell in the body,
including cells in the cardiovascular system, skeletal muscle
cells, joint cells, neural cells, gut cells, lung cells, liver
cells or kidney cells, or cells in the immune system, or to any
type of cell with any function or dysfunction in the body of humans
or animals, including malignant cells.
[0034] As disclosed in the invention herein, exosomes can be used
to deliver genetic material to recipient cells to use the cell's
own protein machinery to produce any drug or precursor of any drug,
or to affect the function or metabolism of any drug, in any cell in
humans or animals.
[0035] To avoid interference with undesirable or irrelevant genetic
material, it is preferable to use exosomes that are lacking genetic
contents. Empty exosomes can be used for direct transfer to
recipient cells or for direct transfection/transformation of a
specific gene (RNA or DNA) into exosomes.
Detection of Mast Cell Derived Exosomes.
[0036] Exosomes released from the murine mast cell line MC/9 (ATCC
Manassas, Va., USA, Number: CRL-8306) were isolated, adsorbed to
carbon-coated grids, and detected using electron microscopy. For
detection of the exosome specific surface protein CD63, exosomes
were also purified from both primary bone marrow mast cells (BMMC)
and the cell line MC/9. The exosomes were adhered onto aldehyde
beads and stained with a CD63 antibody followed by a secondary
PE-coupled antibody. These beads were analysed using flow
cytometric detection, confirming the presence of CD63 on the
surface of both BMMC and MC9 derived exosomes.
Identification of Exosomal Proteins.
[0037] To understand the biological function of the mast cell
derived exosomes, the protein content was analysed using nano-flow
LC-MS/MS. The total protein content was extracted from the isolated
exosomes and collected on a SDS-PAGE gel. The protein band was
trypsinated and analysed by LC-MS/MS. The results of all the tandem
mass spectra were searched by the MASCOT (Matrix Science, London)
program for identification. The results revealed that these
exosomes contain a larger number of proteins than were previously
known. Approximately 150 proteins were identified, many of which
are associated with cellular transcription, translation, and
protein folding. The identified proteins include several ribosomal
proteins as well as heat shock proteins, chaperones, annexines,
cytoskeleton proteins and membrane-bound proteins such as CD63,
CD54, CD43 and WIC class I.
Detection of DNA and RNA from Mast Cell Derived Exosomes.
[0038] Since a certain number of the identified proteins are
associated with RNA and the transcriptional machinery, we
hypothesised that exosomes may also contain DNA and/or RNA. To
examine this, we performed RNA and DNA extraction from both
exosomes and the full exosome producing mast cells. Presence of DNA
and RNA were determined by spectrophotometry and on an agarose gel.
No DNA could be detected in the exosome sample, whereas a
substantial amount of selective RNA was observed from the exosomes.
Specifically exosomal RNA contained very low, or undetectable,
amounts of ribosomal RNA, which indicates the presence of other
types of RNA, i.e. mRNA.
Microarray Analysis of Exosomal RNA.
[0039] In order to characterize the RNA from the mast cell derived
exosomes, Affymetrix mouse DNA microarray (Affymetrix) was applied
using RNA from both mast cells and their exosomes. The results
revealed that exosomes carry mRNA from approximately 2500 genes,
which is approximately 10% of genes that are expressed in the
mother mast cells. Furthermore, the gene profile analysis displayed
essential differences in the mRNA between the exosomes and their
parental cells. The most abundant transcripts in the exosomes
differ from abundant transcripts in the parental cells which
indicate selectivity of the exosomal RNA. Interestingly, the
exosomes carried mRNA from 180 genes whose transcript were absent
in their mother mast cells. The results indicate that mRNA is
transported into exosomes in a highly selective way and it seems
that the parental cells express a certain number of genes
exclusively for exosomal delivery.
Detection of RNA from BMMC Exosomes.
[0040] To identify RNA in exosomes from a non-cell line source,
bone marrow cells were harvested from mice and cultured to become
mast cells (Bone Marrow derived Mast Cells: BMMC) for 4 weeks.
During the last 48 hours, the cells were cultured in the presence
of radioactive uracil (see example 1). The exosomes from the
culture were isolated and the RNA labelled with radioactivity was
detected by scintillator counting. The results showed that the
incorporated radioactive uracil in cellular RNA could be found in
exosomes. The amount of RNA from the bone marrow derived exosomes
is not as abundant as in the mast cell line exosomes, which can be
explained by a lower number of BMMC cells used for the harvest, and
the in vitro growth condition of these cells.
Transfer of RNA by Exosomes between Cells.
[0041] In order to examine whether exosomes can transfer the mRNA
they contain to another cell, mast cell derived exosomes containing
radioactive uracil mRNA were added to cultured dendritic cells
(DC), CD4 T-cells, and MC/9 mast cells. Samples from the cultures
were taken at different intervals and the cells were isolated and
washed by centrifugation. RNA from the recipient cells was isolated
and examined for radioactive uracil using a scintillator. Most
importantly, DC, CD4 and MC/9 cells exposed to the exosomes
contained increased amount of radioactive uracil, thus having been
absorbed from the exosomes. The results show that mast cell derived
exosomes can transfer RNA to other cells, in this case DC, CD4 and
MC/9 cells.
[0042] The data show that mRNA can be transferred between two
mammalian cells through exosomes. Biologically, this suggests that
one cell can affect another cell's protein production by signalling
via exosomes. This has substantial biotechnological applications,
since exosomes may be used as carriers to deliver mRNA, or
DNA-probes, to target cells such as malignant cells or cells in the
immune system. Therefore according to this invention, the exosomes
provide a vehicle for gene modulation and therapy that is likely to
be without the side effects of other gene therapy vehicles, such as
viruses or other types of lipid bodies.
[0043] The features of the present invention will be more clearly
understood by reference to the following examples, which are not to
be construed as limiting the invention.
Example 1
Cell Preparation
[0044] MC/9 cells (ATCC) were cultured according to manufacturer's
recommendations. To eliminate exosomes present in serum, Rat T-Stim
and FBS were ultracentrifuged at 120,000 g for 90 min using a Ti70
rotor (Beckman optima LE-80k Ultracentrifuge). The human mast cell
line HMC-1 (Dr Joseph Butterfield, Mayo Clinic, USA), was cultured
in IMDM containing 10% FBS, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 2 mM L-glutamine and 1.2 mM alph-thioglycerol. For
release of exosome, the HMC-1 cells were cultured in the presence
of 1 .muM calcium ionophore for 30 min. Bone marrow mast cells
(BMMC) were prepared by culturing bone marrow cells from femurs of
7-10 wk old male BALB/c in the presence of IL-3 (R&D systems)
as described previously (Razin, E. et al. Interleukin 3: A
differentiation and growth factor for the mouse mast cell that
contains chondroitin sulfate E proteoglycan. J Immunol. 132,
1479-1486 (1984)). After 4 weeks of culture, the cells were
harvested and consisted of 96% pure MCs as analysed by morphology.
During the last 48 h, BMMC were cultured at 3.times.10.sup.6
cells/ml in complete medium with ultracentrifuged FBS supplemented
with 10 ng/ml IL-4 (R&D-systems), and in some experiments in
the presence of 1 .mu.l/ml.sup.3H-Uracil (Amersham Biosciences).
For culture of CD4.sup.+ T cells, mouse spleens were collected and
passed through a 70 .mu.m followed by 30 .mu.m filter. CD4.sup.+ T
cells were purified by negative selection using the SPINCEP.RTM.
mouse CD4.sup.+ T cells enrichment cocktail (Stemcell Technologies)
according to the manufacturer's instructions. The purity of the
CD4.sup.+ T cells ranged from 89 to 91%, as analysed by flow
cytometry. The cells were cultured in RPMI 1640 containing 10% FBS,
100 U/ml penicillin and 100 .mu.g/ml streptomycin at
1.times.10.sup.6 cells per ml in flat bottom 48 well plates.
Example 2
Exosome Purification
[0045] Exosomes were prepared from the supernatant of MC/9, BMIVIC
and HMC-1 cells by differential centrifugations as previously
described (Raposo, G. et al. B lymphocytes secrete
antigen-presenting vesicles. J. Exp. Med. 183, 1161-1172 (1996)).
Cells were harvested, centrifuged at 500 g for 10 min to eliminate
cells and at 16,500 g for 20 min, followed by filtration through
0.22 .mu.m filter to remove cell debris. Exosomes were pelleted by
ultracentrifugation (Beckman Ti70 rotor) at 120,000 g for 70 min.
For mass spectrometry, the exosome pellet was washed once in PBS.
Exosomes were measured for their protein content using BCA.TM.
Protein Assay Kit (Pierce). For the density gradient experiment the
120,000 g exosome pellet was floated in a sucrose gradient (0.25-2
M sucrose, 20 mM Hepes/NaOH, pH 7.2). The exosomes were dissolved
in 2.5 M sucrose and the gradient was layered on top of the exosome
suspension. The gradient was centrifuged at 100,000 g for 15 h
according to (Rapso et al 1996). Briefly, the gradient fractions
(10.times.3.8 ml) were collected from the bottom of the tube,
diluted with 10 ml PBS and ultracentrifuged for 2 h at 150,000 g
(Beckman Ti70.1 rotor), and the pellets were extracted by
Trizol.RTM. (Invitrogen).
Example 3
Isolation of RNA, DNA and Proteins
[0046] RNA, DNA and proteins were isolated using Trizol.RTM.
(Invitrogen) or RNEASY.RTM. mini kit (Qiagen) according to the
manufacturer's protocol. For co-purification of microRNA and total
RNA, the RNA was extracted using Trizol, followed by the
RNEASY.RTM. mini kit. Cells and exosomes were disrupted and
homogenized in Buffer RLT (Qiagen) and 3.5 volumes of 100% ethanol
were added to the samples prior use of the RNEASY mini spin column.
The rest of the procedure was performed according to the
manufacturer's protocol.
Example 4
[0047] Introducing DNA/RNA Fragments or Constructs into
Exosomes
[0048] Insertion of Specific Constructs Directly into Exosomes.
[0049] Exosomes can be isolated from different origin, e.g. in
vitro growing cells, human body, or cells originated from a human
and animal. Genetic constructs of RNA or DNA can be introduced into
these exosomes directly by using conventional molecular biology
techniques such as in vitro transformation, transfection, and
microinjection.
Example 5
Administration of DNA or RNA-Containing Exosomes to Cells
Transfer Experiments
[0050] To label MC/9 exosome RNA, cells were cultured in complete
medium supplemented with 1 .mu.l/ml.sup.3H-Uracil 72 h before
exosome isolation. Exosomes were isolated according to the
isolation protocol and washed by ultra-filtration (10 kDa,
Millipore) to remove free nucleotides. The exosomes were added to
MC/9, CD4.sup.+, and HMC-1 cells in the ratio of 8:1 between donor
cells and recipients at the starting point of labeling. At 0 h and
24 h, cells were harvested and washed twice. RNA was isolated by
RNEASY.RTM. mini kit and the signal of radioactive RNA was measured
using scintillation. Medium supplemented with 1
.mu.l/ml.sup.3H-Uracil absent from donor cells was treated equally
and used as negative control.
In Vitro Translation
[0051] Total exosomal RNA was purified using RNEASY.RTM. mini kit
and 0.5 .mug was used for the translation. The in vitro rabbit
lysate translation kit (Promega Corporation) was used according to
the manufacturer's recommendation to translate exosomal mRNA to
proteins. A sample without exosomal RNA was treated equally and
used as negative control. After the translation procedure was
accomplished, total proteins were precipitated using acetone and
determined using RC DC protein assay (BioRad). The protein content
of the samples (presence and absence of the exosomal RNA) was
compared using 2D-PAGE, BioRad instruments (Mini-Protean.RTM.3cell)
and recommendation. The 2D-gels were visualized using SyproRuby
(BioRad) and digitalized using phosphoimager. Protein spots of the
samples were compared and a selection of the newly produced
proteins was cut, trypsinated, and identified using LC-MS/MS
followed by MASCOT program search. The newly produced proteins of
mouse origin were compared to the genes identified from the DNA
microarray analysis.
In Vivo Translation
[0052] MC/9 exosomes (1000 .mug) were added to HMC-1 cells
(8.times.10.sup.6) in three different time points (0, 3, 6 h) and
the cells were incubated for approximately 24 h. The HMC-1 cells
were harvested, washed, and the total proteins of the cells were
separated by 2D-PAGE according to Proteomics Core facility. A
sample without exosomes was treated equally and used as negative
control. The newly produced proteins were detected using PDQUEST
and 96 spots were cut and identified using MALDI-tof followed by
MASCOT program search, according to Proteomics Core Facility
(University of Gothenburg).
[0053] The exosomes then deliver the nucleic acids to recipient
cells and consequently affect their biological function or
survival.
Example 6
[0054] Production of Exosomes that are Lacking Genetic Material
[0055] The empty exosomes are used for direct transfer to recipient
cells or for direct transfection/transformation of a specific gene
(RNA or DNA) into exosomes. The methods to produce empty exosomes
(empty of genetic material) are multiple as known by one skilled in
the art; including UV-exposure, mutation of proteins that carry RNA
into exosomes, as well as electroporation and chemical treatments
to open pores in the exosomal membranes. The methods include
mutation/deletion of any protein that can modify loading of any
nucleic acid into exosomes.
Example 7
[0056] Production of Mouse Proteins in Human Mast Cells after
Transfer of Mouse MC/9 Exosomes
[0057] To test whether mouse proteins could be produced in human
mast cells after transfer of mouse MC/9 exosomes, we determined the
presence of mouse proteins in the recipient cell, by 2D-PAGE
followed by MALDI-tof. After incubation of the human cells with
mouse MC/9 exosomes for 24 hours, 96 new or enhanced protein spots
were identified. Interestingly, three distinct mouse proteins were
identified in the human cells that are not present in MC/9
exosomes. These proteins were mouse CDCl.sub.6 (089033), mouse Zinc
finger protein 271 (P15620) and mouse CX7A2 (P48771). The mRNA of
the first two proteins was present in two of the microarray
experiments, and the last one was present in all four microarrays
performed, suggesting that mRNA delivered by exosomes to a
recipient cell can be translated to proteins.
[0058] The proteomic results from transfer of MC/9 exosomes to
HMC-1 cells when human mast cells HMC-1 were incubated with the
mouse MC/9 exosomes and without for 24 hours show that mouse
proteins could be produced in the human mast cells. Proteins
between the two gels were matched and 96 newly produced proteins
were identified by MALDI-tof and mouse proteins were produced from
the exosomal mRNA.
Example 8
Using Exosomes as Gene Therapy of Malignant Disease
[0059] Exosomes are produced by malignant cells, taken from a
patient suffering from a malignant disease. These exosomes are
processed to contain genetic constructs of any type or specificity,
to be reintroduced to the patient. The exosomes from the malignant
cell then preferentially fuse with cells of the same type, which
deliver the DNA and or RNA constructs to the malignant cell
specifically, as gene therapy of malignant disease. The procedure
is performed according to example 1-6, but with exosomes produced
by malignant cells.
[0060] While the invention has been described with reference to
specific embodiments, it will be appreciated that numerous
variations, modifications, and embodiments are possible, and
accordingly, all such variations, modifications, and embodiments
are to be regarded as being within the spirit and scope of the
invention.
* * * * *