U.S. patent application number 16/190058 was filed with the patent office on 2019-06-13 for loading of extracellular vesicles through imparting of mechanical shear.
The applicant listed for this patent is Codiak BioSciences, Inc.. Invention is credited to Raymond W. Bourdeau, Michael F. Doherty, Kathryn E. Golden, Konstantin Konstantinov, Michael P. Mercaldi, Aaron R. Noyes, Douglas E. Williams.
Application Number | 20190175506 16/190058 |
Document ID | / |
Family ID | 66734871 |
Filed Date | 2019-06-13 |
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United States Patent
Application |
20190175506 |
Kind Code |
A1 |
Noyes; Aaron R. ; et
al. |
June 13, 2019 |
Loading of Extracellular Vesicles through Imparting of Mechanical
Shear
Abstract
Methods of loading extracellular vesicles with payload molecules
via homogenization are disclosed herein.
Inventors: |
Noyes; Aaron R.; (Melrose,
MA) ; Mercaldi; Michael P.; (Wilmington, MA) ;
Golden; Kathryn E.; (Braintree, MA) ; Bourdeau;
Raymond W.; (Watertown, MA) ; Doherty; Michael
F.; (Somerville, MA) ; Konstantinov; Konstantin;
(Waban, MA) ; Williams; Douglas E.; (Boston,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Codiak BioSciences, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
66734871 |
Appl. No.: |
16/190058 |
Filed: |
November 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62588143 |
Nov 17, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1275 20130101;
A61K 9/1278 20130101; C12N 15/111 20130101; C12N 2310/3515
20130101; C12N 15/113 20130101; C12N 2310/14 20130101; C12N 2320/32
20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C12N 15/113 20060101 C12N015/113 |
Claims
1. A method for producing an isolated extracellular vesicle for
delivery of a payload molecule, the method comprising: modifying an
extracellular vesicle with a payload molecule via homogenization,
isolating the modified extracellular vesicle containing the payload
molecule, and optionally formulating the isolated modified
extracellular vesicle into a pharmaceutical composition.
2. The method of claim 1, wherein the vesicle is an exosome, a
nanovesicle, an apoptotic body, a microvesicle, a lysosome, an
endosome, an enveloped virus, a viral vector, a liposome, a lipid
nanoparticle, a micelle, a multilamellar structure, a revesiculated
vesicle, or an extruded cell.
3. The method of claim 1, wherein the payload molecule is a
therapeutic molecule.
4. The method of claim 1, wherein the homogenization is
microfluidization.
5. The method of claim 4, wherein the microfluidization is a single
pass.
6. The method of claim 4, wherein the microfluidization is
performed between 10,000 to 30,000 psi.
7. (canceled)
8. (canceled)
9. (canceled)
10. The method of claim 1, wherein the microfluidization is
multiple passes, wherein the pressure is different in the multiple
passes.
11. (canceled)
12. The method of claim 10, wherein the pressure is different in
the multiple passes.
13. The method of claim 1, wherein the vesicle is in a buffered
solution.
14. (canceled)
15. (canceled)
16. The method of claim 13, wherein the buffer is between pH 7 and
8.
17. (canceled)
18. (canceled)
19. (canceled)
20. The method of claim 1, wherein homogenization occurs in a
volume of at least 1 ml.
21. The method of claim 1, wherein the temperature is 15.degree. C.
to 80.degree. C.
22. (canceled)
23. (canceled)
24. The method of claim 1, wherein the payload is an siRNA, an
miRNA, an antisense RNA, a DNA, a plasmid, an mRNA, a tRNA, a
protein, a carbohydrate, a lipid, a small molecule drug, a STING
agonist, a toxin, an antibody, a recombinant protein, a viral
vector, or a vaccine.
25. The method of claim 1, wherein the payload is siRNA.
26. The method of claim 1, wherein the vesicle and the payload are
first mixed in a solution and the solution is homogenized.
27. The method of claim 1, wherein a solution comprising the
vesicle is homogenized first and the payload is added to the
homogenized vesicle solution.
28. The method of claim 1, wherein the vesicle is modified with a
plurality of payload molecules.
29. (canceled)
30. (canceled)
31. The method of claim 1, wherein the vesicle in solution is both
homogenized and treated with another technology for loading the
payload molecules.
32. The method of claim 31, wherein the vesicle is pre-treated
before homogenization or post-treated after homogenization.
33. (canceled)
34. The method of claim 31, wherein the additional treatment is a
chemical treatment.
35. (canceled)
36. The method of claim 25, wherein the siRNA is conjugated to a
cholesterol moiety.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/588,143, filed Nov. 17, 2017, the
disclosure of which is incorporated by reference in its
entirety.
BACKGROUND
[0002] There is a need in the field for improved methods and
compositions for delivering therapeutic agents to cells. The
therapeutic properties of extracellular vesicles can be enhanced by
the complexation of a molecule or molecules in a process referred
to as loading. Loading represents the encapsulation, partial
engulfment, and/or external association of a cargo molecule with
the vesicle or vesicles. Methods that facilitate the loading of
molecules into or onto vesicles can improve efficiency and enable
the loading of diverse classes of molecules. Homogenization, the
application of shear and cavitation, can be applied simultaneously
to both vesicles and the desired payload to promote loading of the
payload into and/or on to the vesicle.
[0003] Homogenization, including microfluidization (a branded from
of homogenization) has been previously employed for
nanoencapsulation and loading of liposomes but has not been used
for the loading of vesicles. Provided herein are methods for
loading exosomes with therapeutic agents, and the exosomes created
by those methods.
SUMMARY OF THE INVENTION
[0004] Disclosed herein are methods for producing an isolated
extracellular vesicle for delivery of a payload molecule comprising
modifying an extracellular vesicle with a payload molecule via
homogenization, isolating the modified extracellular vesicle
containing the payload molecule, and optionally formulating the
isolated modified extracellular vesicle into a pharmaceutical
composition. In one embodiment, the vesicle is an exosome, a
nanovesicle, an apoptotic body, a microvesicle, a lysosome, an
endosome, an enveloped virus, a viral vector, a liposome, a lipid
nanoparticle, a micelle, a multilamellar structure, a revesiculated
vesicle, or an extruded cell. In another embodiment the payload
molecule is a therapeutic molecule. In another embodiment, the
vesicle is modified with a plurality of payload molecules.
[0005] In some embodiments, the homogenization is
microfluidization. In some embodiments, the microfluidization is a
single pass. In other embodiments, the microfluidization is
multiple passes. In some embodiments, the pressure of the
microfluidization is performed between 10,000 to 30,000 pounds per
square inch (psi). In one embodiment, the microfluidization is
10,000 psi. In one embodiment, the microfluidization is 20,000 psi.
In one embodiment, the microfluidization is 30,000 psi. In some
embodiments, the pressure is the same in multiple passes. In other
embodiments, the pressure is different in multiple passes.
[0006] In some embodiments, the vesicle is in buffered solution. In
one embodiment, the solution is between a pH 3 and 13. In another
embodiment, the pH of the solution is between 7 and 8. In a further
embodiment, the pH of the solution is 7.4. In some embodiments, the
buffered solution comprises phosphate buffered saline and 0.5-5%
sucrose.
[0007] In some embodiments, the volume of the microfluidization is
less than a liter. In other embodiments, the volume of the
microfluidization is more than a liter. In another embodiment, the
volume of the microfluidization is at least 1 ml.
[0008] In some embodiments, the microfluidization is performed at
15.degree. C. to 80.degree. C. In another embodiment, the
microfluidization is performed at room temperature. In some
embodiments, the microfluidization is performed at 70-80.degree.
C.
[0009] In one embodiment the payload is siRNA, miRNA, antisense
RNA, DNA, a plasmid, mRNA, tRNA, a protein, a carbohydrate, a
lipid, a small molecule drug, a toxin, an antibody, a recombinant
protein, a viral vector, or a vaccine. In another embodiment, the
payload is siRNA. In certain embodiments, the payload is a modified
nucleic acid. In some embodiments, said modified nucleic acid is an
siRNA or miRNA that is modified to additionally comprise one or
more cholesterol molecules. In some embodiments, said cholesterol
is chemically conjugated to the 5' end of the sense, or passenger
strand of the siRNA or miRNA. In some embodiments, said cholesterol
is chemically conjugated to the 3' end of the sense, or passenger
strand of the siRNA or miRNA. In some embodiments, said cholesterol
is chemically conjugated to the 5' end of the antisense, or guide
strand of the siRNA or miRNA. In some embodiments, said cholesterol
is chemically conjugated to the 3' end of the antisense, or guide
strand of the siRNA or miRNA.
[0010] In some embodiments, a solution comprising the vesicle and
the payload are first mixed and the mixture is homogenized. In some
embodiments, a solution comprising the vesicle is homogenized first
and the payload is added to the homogenized vesicle solution.
[0011] In some embodiments, the vesicle is modified with a
plurality of payload molecules.
[0012] In one embodiment, the payload molecules are predominantly
on the surface of the modified vesicle. In another embodiment, the
payload molecules are predominantly inside the modified
vesicle.
[0013] In some embodiments, the vesicle in solution is both
homogenized and treated with another technology for loading the
payload molecules. In one embodiment, the additional treatment is a
chemical treatment. In one embodiment, vesicle is post-treated
after homogenization. In another embodiment, the vesicle is
pre-treated before homogenization.
[0014] The vesicles may be homogenized in a batch, a semi-batch, or
a continuous process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the qPCR results from kras gene knockdown cell
based assay in Panc-1 cells as a function of shear pressure. The
left bar shows samples homogenized at 10,000 psi, the middle bar
shows the samples homogenized at 20,000 psi, and the right bar
shows the samples homogenized at 30,000 psi.
[0016] FIG. 2 shows the qPCR results from kras gene knockdown cell
based assay in Panc-1 cells as a function of siRNA
concentration.
[0017] FIG. 3 shows the qPCR results from kras gene knockdown cell
based assay in Panc-1 cells using unmodified siRNA and
cholesterol-tagged siRNA loaded in exosomes by microfluidic
homogenization.
[0018] FIGS. 4A-B show the anion exchange chromatogram profile of
unmodified siRNA (4A) and cholesterol-tagged siRNA (4B) after
repeated rounds of microfluidic homogenization.
[0019] FIGS. 5A-B show the results from measuring the diameter of
purified exosomes after repeated rounds of microfluidic
homogenization as determined by size exclusion chromatography (5A)
and nanoparticle tracking assay (5B).
DETAILED DESCRIPTION
Definitions
[0020] Terms used in the claims and specification are defined as
set forth below unless otherwise specified.
[0021] As used herein, the term "extracellular vesicle" refers to a
cell-derived vesicle comprising a membrane that encloses an
internal space. Extracellular vesicles comprise all membrane-bound
vesicles that have a smaller diameter than the cell from which they
are derived. Generally extracellular vesicles range in diameter
from 20 nm to 1000 nm, and may comprise various macromolecular
cargo either within the internal space, displayed on the external
surface of the extracellular vesicle, and/or spanning the membrane.
Said cargo may comprise nucleic acids, proteins, carbohydrates,
lipids, small molecules, and/or combinations thereof. By way of
example and without limitation, extracellular vesicles include
apoptotic bodies, fragments of cells, vesicles derived from cells
by direct or indirect manipulation (e.g., by serial extrusion or
treatment with alkaline solutions), vesiculated organelles, and
vesicles produced by living cells (e.g., by direct plasma membrane
budding or fusion of the late endosome with the plasma membrane).
Extracellular vesicles may be derived from a living or dead
organism, explanted tissues or organs, and/or cultured cells.
[0022] As used herein, the term "nanovesicle" refers to a
cell-derived small (between 20-250 nm in diameter, more preferably
30-150 nm in diameter) vesicle comprising a membrane that encloses
an internal space, and which is generated from said cell by direct
or indirect manipulation such that said nanovesicle would not be
produced by said producer cell without said manipulation.
Appropriate manipulations of said producer cell include but are not
limited to serial extrusion, treatment with alkaline solutions,
sonication, or combinations thereof. The production of nanovesicles
may, in some instances, result in the destruction of said producer
cell. Preferably, populations of nanovesicles are substantially
free of vesicles that are derived from producer cells by way of
direct budding from the plasma membrane or fusion of the late
endosome with the plasma membrane. The nanovesicle comprises lipid
or fatty acid and polypeptide, and optionally comprises a payload
(e.g., a therapeutic agent), a receiver (e.g., a targeting moiety),
a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar
(e.g., a simple sugar, polysaccharide, or glycan) or other
molecules. The nanovesicle, once it is derived from a producer cell
according to said manipulation, may be isolated from the producer
cell based on its size, density, biochemical parameters, or a
combination thereof.
[0023] As used herein, the term "exosome" refers to a cell-derived
small (between 20-300 nm in diameter, more preferably 40-200 nm in
diameter) vesicle comprising a membrane that encloses an internal
space, and which is generated from said cell by direct plasma
membrane budding or by fusion of the late endosome with the plasma
membrane. Generally, production of exosomes does not result in the
destruction of the producer cell. The exosome comprises lipid or
fatty acid and polypeptide, and optionally comprises a payload
(e.g., a therapeutic agent), a receiver (e.g., a targeting moiety),
a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar
(e.g., a simple sugar, polysaccharide, or glycan) or other
molecules. The exosome can be derived from a producer cell, and
isolated from the producer cell based on its size, density,
biochemical parameters, or a combination thereof.
[0024] As used herein, the terms "parent cell" or "producer cell"
include any cell from which an extracellular vesicle may be
isolated. The terms also encompasses a cell that shares a protein,
lipid, sugar, or nucleic acid component of the extracellular
vesicle. For example, a "parent cell" or "producer cell" may
include a cell which serves as a source for the extracellular
vesicle membrane.
[0025] As used herein, the terms "purify," "purified," and
"purifying" or "isolate," "isolated," or "isolating" or "enrich,"
"enriched" or "enriching" are used interchangeably and refer to the
state of a population (e.g., a plurality of known or unknown amount
and/or concentration) of desired extracellular vesicles, that have
undergone one or more processes of purification, e.g., a selection
or an enrichment of the desired extracellular vesicles composition,
or alternatively a removal or reduction of residual biological
products as described herein. In some embodiments, a purified
extracellular vesicles composition has no detectable undesired
activity or, alternatively, the level or amount of the undesired
activity is at or below an acceptable level or amount. In other
embodiments, a purified extracellular vesicle composition has an
amount and/or concentration of desired extracellular vesicles at or
above an acceptable amount and/or concentration. In other
embodiments, the purified extracellular vesicle composition is
enriched as compared to the starting material (e.g., biological
material collected from tissue, bodily fluid, or cell preparations)
from which the composition is obtained. This enrichment may be by
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as
compared to the starting material.
[0026] A "payload" as used herein is a therapeutic agent that acts
on a target (e.g. a target cell) that is contacted with the
extracellular vesicles. Payloads that may be introduced into a
extracellular vesicles and/or a producer cell include therapeutic
agents such as, nucleotides (e.g. nucleotides comprising a
detectable moiety or a toxin or that disrupt transcription),
nucleic acids (e.g. DNA or mRNA molecules that encode a
polypepetide such as an enzyme, or RNA molecules that have
regulatory function such as miRNA, dsDNA, lncRNA, siRNA), amino
acids (e.g. amino acids comprising a detectable moiety or a toxin
or that disrupt translation), polypeptides (e.g. enzymes), lipids,
carbohydrates, and small molecules (e.g. small molecule drugs and
toxins). The payload may comprise nucleotides, e.g. nucleotides
that are labeled with a detectable or cytotoxic moiety (e.g. a
radiolabel).
[0027] "Transgene" or "exogenous nucleic acid" refers to a foreign
or native nucleotide sequence that is introduced into an
extracellular vesicle. Transgene and exogenous nucleic acid are
used interchangeably herein and encompass recombinant nucleic
acids.
[0028] A "therapeutic agent" or "therapeutic molecule" includes a
compound or molecule that, when present in an effective amount,
produces a desired therapeutic effect, pharmacologic and/or
physiologic effect on a subject in need thereof. It includes any
compound, e.g., a small molecule drug, or a biologic (e.g., a
polypeptide drug or a nucleic acid drug) that when administered to
a subject has a measurable or conveyable effect on the subject,
e.g., it alleviates or decreases a symptom of a disease, disorder
or condition.
[0029] The term "pharmaceutically-acceptable" and grammatical
variations thereof, refers to compositions, carriers, diluents and
reagents capable of administration to or upon a subject without the
production of undesirable physiological effects to a degree that
would prohibit administration of the composition.
[0030] As used herein, the term "pharmaceutical composition" refers
to one or more of the compounds described herein, such as, e.g., an
extracellular vesicles mixed or intermingled with, or suspended in
one or more other chemical components, such as pharmaceutically
acceptable carriers and excipients. One purpose of a pharmaceutical
composition is to facilitate administration of preparations of
extracellular vesicles to a subject.
[0031] The term "about" indicates and encompasses an indicated
value and a range above and below that value. In certain
embodiments, the term "about" indicates the designated value
.+-.10%, .+-.5%, or .+-.1%. In certain embodiments, where
applicable, the term "about" indicates the designated value(s)
.+-.one standard deviation of that value(s).
[0032] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
[0033] Ranges recited herein are understood to be shorthand for all
of the values within the range, inclusive of the recited endpoints.
For example, a range of 1 to 50 is understood to include any
number, combination of numbers, or sub-range from the group
consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
and 50.
[0034] Methods of the Invention
[0035] Methods of Loading Extracellular Vesicles
[0036] Described herein are methods for producing modified
extracellular vesicles via mechanical shear such as homogenization
and pharmaceutical preparations thereof. Extracellular vesicles can
include, but are not limited to, an exosome, a nanovesicle, an
apoptotic body, a microvesicle, a lysosome, an endosome, an
enveloped virus, a viral vector, a liposome, a lipid nanoparticle,
a micelle, a multilamellar structure, a revesiculated vesicle, or
an extruded cell.
[0037] Homogenization, the application of shear and cavitation, can
stress the vesicular structure resulting in many effects, including
distortion to the phospholipid membrane and associated cytoskeleton
leading to the creation of transient pores; and increase
permeability, exposure of binding sites, and/or invagination of the
surface. Homogenization may also lead to disaggregation of lipid
rafts leading to increased permeability and membrane fluidity;
formation of multilamellar bodies, both through invagination and
disaggregation; conformational shifts in peripheral and
transmembrane proteins that expose binding sites, alter surface
properties, and enable the transmembrane passage of molecules; heat
the vesicular membrane and alter permeability, fluidity, and
diffusion; and/or denature proteins that permanently affect the
structure of the membrane. Homogenization may also alter the
surface properties of the molecule to be loaded or cause shifts in
the chemical equilibria and local concentrations of molecules,
leading to steeper concentration gradients. These effects may
facilitate the loading of molecules into or onto vesicles, and
improve the efficiency of loading diverse classes of molecules.
[0038] Generally, any homogenization method that induces controlled
injury may be used to load an agent, e.g. a payload molecule,
receiver or surface marker into or onto an extracellular vesicle.
The homogenization of the membrane of the producer cell or
extracellular vesicles can be caused by, for example, pressure
induced by mechanical strain or shear forces, subjecting the cell
to deformation, constriction, rapid stretching, rapid compression,
or pulse of high shear rate. The controlled injury leads to uptake
of material, e.g., a payload, receiver or surface marker into the
interior of the extracellular vesicles or the cytoplasm of the
producer cell from the surrounding cell medium.
[0039] Many different homogenization instruments are available,
including microfluidizers, French Press, high pressure
homogenizers, bead mills, rotary blenders and rotor/stator devices.
In some embodiments described herein, vesicles may be homogenized
using a microfluidizer. Microfluidizers may be purchased from
commercial sources, such as the LV1 Microfluidizer made by Idex
Corporation (Newton, Mass.) or the M110P homogenization module made
by Microfluidics Corp (Westwood, Mass.)
[0040] A microfluidizer consists of a fixed-geometry interaction
chamber in which flow is driven by a pump. The incoming flow is
split into two or more streams and the recombined at high velocity
to create steep velocity and pressure gradients, shear, cavitation,
and heating. The intensity of homogenization can be altered by
changing the geometry of the interaction chamber, altering the
temperature, changing the pressure, or processing the same material
through the instrument multiple times. There is also a complex
interaction with the concentration of the incoming material, buffer
composition, and the physiochemical properties of the solution,
emulsion, or suspension.
[0041] Several parameters may affect loading of the payload
molecule, including pressure, temperature, number of homogenization
passes, and buffer conditions. The pressure of the
microfluidization can be from about 5,000 to 50,000 psi. The
pressure of the microfluidization can be at least about 5,000 psi,
10,000 psi, 15,000 psi, 20,000 psi, 25,000 psi, 20,000 psi, 25,000
psi, 40,000 psi, 45,000 psi, or 50,000 psi. The pressure of the
microfluidization can be between about 5,000 to 50,000 psi, 5,000
to 10,000 psi, 7,500 to 12,000 psi, 10,000 to 15,000 psi, 15,000 to
20,000 psi, 15,000-22,000, psi, 18,000-25,000 psi, 18,000-22,000
psi, 20,000 to 25,000 psi, 25,000 to 30,000 psi, 27,500 to 30,000
psi, 27,500 to 32,000 psi, 30,000 to 32,000 psi, 30,000 to 35,000
psi, 35,000 to 40,000 psi, 40,000 to 45,000 psi, or 45,000 to
50,000 psi. In some embodiments disclosed herein, the pressure of
the homogenization or microfluidization is about 10,000 psi, 20,000
psi, or 30,000 psi. In one embodiment, the pressure is between
10,000 and 30,000 psi.
[0042] The homogenization or microfluidization may be a single pass
or run, or comprise multiple passes or runs. In one embodiment, the
pressure of one or more of the multiple passes or runs may be that
same for each pass. For example, the pressure of the first pass can
be about 10,000 psi and the pressure of the second pass can be
about 10,000 psi; the pressure of the first pass can be about
20,000 psi and the pressure of the second pass can be about 20,000
psi; or the pressure of the first pass can be about 30,000 psi and
the pressure of the second pass can be about 30,000 psi; or any of
the microfluidization pressures disclosed herein. Multiple passes
can be two or more passes, such as three passes, four passes, five
passes, six passes, seven passes, eight passes, and so on. In
another embodiment, the pressure of one or more of the multiple
passes or runs may be different. For example, the pressure of the
first pass can be about 10,000 psi and the pressure of the second
pass can be about 20,000 psi; the pressure of the first pass can be
about 20,000 psi and the pressure of the second pass can be about
30,000 psi; the pressure of the first pass can be about 30,000 psi
and the pressure of the second pass can be about 20,000 psi; or the
pressure of the first pass can be about 10,000 psi and the pressure
of the second pass can be about 30,000 psi, or any combination of
the microfluidization pressures disclosed herein. Multiple passes
can be two or more passes, such as three passes, four passes, five
passes, six passes, seven passes, eight passes, and so on.
[0043] The buffer conditions of the solution of extracellular
vesicles may also be altered to optimize homogenization and payload
molecule loading. In one embodiment, the buffer may be a phosphate
buffered saline (PBS) with sucrose. PBS is a well-known buffer to
those skilled in the art. In one embodiment, the PBS buffer also
comprises 0.5-5% sucrose. The buffer can comprise about 0.5-5%,
0.5-1%, 1-2%, 2-3%, 3-4%, 4-5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,
4.5% or 5% sucrose. Additional buffer modifications may also be
used, such as shear protectants, viscosity modifiers, and/or
solutes that affect vesicle structural properties. Excipients may
also be added to improve the efficiency of the homogenization or
microfluidization such as membrane softening materials and
molecular crowding agents. Other modifications to the buffer may
include specific pH ranges and/or concentrations of salts, organic
solvents, small molecules, detergents, zwitterions, amino acids,
polymers, and/or any combination of the above including multiple
concentrations.
[0044] The pH of the buffer may also be altered. The pH can be
between 3-13, 3-5, 5-10, 7-8, 8-10, 10-13. The pH can be about 3,
3.5, 4, 4.5, 5, 5.5, 6, 6.5, 6.8, 7, 7.2, 7.4, 7.5, 7.6, 7.8, 8,
8.2, 8.4, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or 13. In one
embodiment, the pH is between 3 and 13. In one embodiment, the pH
is between 7 and 8. In a further embodiment, the pH is 7.4.
[0045] The temperature of the homogenization or microfluidization
may be changed for optimization of loading the payload molecules.
The temperature can be between about 15-90.degree. C.,
15-20.degree. C., 20-25.degree. C., 25-30.degree. C., 30-35.degree.
C., 35-40.degree. C., 45-50.degree. C., 50-55.degree. C.,
55-60.degree. C., 60-65.degree. C., 65-70.degree. C., 70-75.degree.
C., 75-80.degree. C., 80-85.degree. C., or 85-90.degree. C. The
temperature can be about 15.degree. C., 20.degree. C., 25.degree.
C., 27.degree. C., 30.degree. C., 32.degree. C., 35.degree. C.,
40.degree. C., 45.degree. C., 50.degree. C., 55.degree. C.,
60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C.,
80.degree. C., 85.degree. C., or 90.degree. C. In one embodiment,
the temperature may be room temperature. In another embodiment, the
temperature is 15.degree. C. to 80.degree. C. In another
embodiment, the temperature is 70-80.degree. C.
[0046] The volume of the solution comprising extracellular vesicles
to be homogenized or microfluidized may also be altered. The volume
can be from about 1 ml to about 5000 ml, or more. The volume can be
at least about 1-10 ml, 10-50 ml, 50-100 ml, 100-150 ml, 150-200
ml, 200-250 ml, 250-300 ml, 300-350 ml, 350-400 ml, 400-450 ml,
450-500 ml, 500-550 ml, 550-600 ml, 600-650 ml, 650-700 ml, 750-800
ml, 800-850 ml, 850-900 ml, 950-1000 ml, 1000-1500 ml, 1500-2000
ml, 2000-2500 ml, 2500-3000 ml, 3000-3500 ml, 3500-4000 ml,
4000-4500 ml, 45000-5000 ml, or more. The volume can be at least
about 1 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml,
90 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450
ml, 500 ml, 550 ml, 600 ml, 650 ml, 700 ml, 750 ml, 800 ml, 850 ml,
900 ml, 950 ml, 1000 ml, 1500 ml, 2000 ml, 2500 ml, 3000 ml, 3500
ml, 4000 ml, 4500, ml or 5000 ml. In one embodiment, the volume is
less than a liter. In another embodiment, the volume is more than a
liter. In another embodiment, the volume of the solution may be at
least 1 ml.
[0047] The extracellular vesicles may also be treated with
additional technologies before or after homogenization. In one
embodiment, the vesicles are pre-treated before homogenization. In
one embodiment, the vesicles are post-treated after homogenization.
The treatment may include a chemical treatment, such as acidic or
basic buffers, or other chemical treatments not described
herein.
[0048] The extracellular vesicle may be modified with a single
payload molecule or with a plurality of payload molecules. A
detailed description of such payload molecules is disclosed below.
In one embodiment, the extracellular vesicles may be modified with
a plurality of payload molecules. The payload molecules may be the
same type or class of molecule, or a different type or class of
molecule. Multiple types of payload molecules may be combined to
produce vesicles modified with multivalent payload molecule
cargoes. For example, the vesicle can have two types, three types,
four types, five types, six types, and so on. In another
embodiment, the payload molecules may be predominantly on the
surface of the modified vesicle. In another embodiment, the payload
molecules may be predominantly inside the modified vesicle. The
extracellular vesicles to be modified may be homogenized first and
the payload molecules added to the homogenized vesicle solution. In
a further embodiment, the extracellular vesicles and the payload
molecules are mixed together and the mixture is homogenized.
[0049] The homogenization may be applied in a batch mode, a
semi-batch mode, or a continuous process. Either batch or
continuous process may rely on feedback to control the
homogenization or microfluidization parameters and/or terminate the
process.
[0050] Extracellular Vesicles
[0051] Extracellular vesicles can be extracted from the supernatant
of parent cells and demonstrate membrane and internal protein,
lipid, and nucleic acid compositions that enable their efficient
delivery to and interaction with recipient cells. Extracellular
vesicles can be derived from parent cells that may include, but are
not limited to, reticulocytes, erythrocytes, megakaryocytes,
platelets, neutrophils, tumor cells, connective tissue cells,
neural cells and stem cells. Suitable sources of extracellular
vesicles include but are not limited to, cells isolated from
subjects from patient-derived hematopoietic or erythroid progenitor
cells, immortalized cell lines, or cells derived from induced
pluripotent stem cells, optionally cultured and differentiated.
Cell culture protocols can vary according to compositions of
nutrients, growth factors, starting cell lines, culture period, and
morphological traits by which the resulting cells are
characterized. In some embodiments, the samples comprising
extracellular vesicles are derived from a plurality of donor cell
types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000,
or 10000 donor cell types) and are combined or pooled. Pooling may
occur by mixing cell populations prior to extracellular vesicles
extraction or by mixing isolated extracellular vesicles
compositions from subsets of donor cell types. Parent cells may be
irradiated or otherwise treated to affect the production rate
and/or composition pattern of secreted extracellular vesicles prior
to isolation.
[0052] In some embodiments, the extracellular vesicle comprises a
membrane that forms a particle that has a diameter of between about
10-100 nm, 50-150 nm, 30-100 nm, 40-100 nm, 20-150 nm, 20-200 nm,
80-125 nm, 40-250 nm, 20-500 nm, or between about 10-1000 nm. The
vesicle particle can have diameter of about 10, 150, 100, 120, 125,
130, 135, 140, 145, 150, 155, 160, 175, 200, 225, 250, 275, 300,
325, 250, 275, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950,
975, or 1000 nm. In some embodiments, the membrane comprises lipids
and fatty acids. In some embodiments, the membrane comprises one or
more of phospholipids, glycolipids, fatty acids, sphingolipids,
phosphoglycerides, sterols, cholesterols, and phosphatidylserine.
In addition, the membrane may comprise one or more polypeptides and
one or more polysaccharides, such as glycans.
[0053] In some embodiments, the extracellular vesicle is generated
by a producer cell (or parental cell), such as, e.g., a mammalian
cell. In some embodiments, the membrane of the extracellular
vesicle comprises one or more molecules derived from the producer
cell. The extracellular vesicle may be generated in a cell culture
system and isolated, e.g. by separating the extracellular vesicle
from the producer cell. Separation may be achieved by
sedimentation. For example, the extracellular vesicle can have a
specific density between 0.5-2.0, 0.5-0.75, 0.5-1, 0.75-1.5,
0.75-2, 1-2, 1.5-2, and 0.9-1.1 kg/m.sup.3. The extracellular
vesicle can have a specific density about 0.5, 0.75, 1, 1.25, 1.5,
1.75, or 2 kg/m.sup.3.
[0054] In some embodiments, the extracellular vesicle delivers the
payload molecule to a target. The payload molecule is a therapeutic
agent that acts on a target (e.g. a target cell) that is contacted
with the extracellular vesicle. Contacting may occur, e.g. in vitro
or in a subject. Payloads that may be introduced into an
extracellular vesicle and/or a producer cell include therapeutic
agents such as, nucleotides (e.g. nucleotides comprising a
detectable moiety or a toxin or that disrupt transcription),
nucleic acids (e.g. DNA or mRNA molecules that encode a
polypepetide such as an enzyme, or RNA molecules that have
regulatory function such as miRNA, dsDNA, lncRNA, siRNA), amino
acids (e.g. amino acids comprising a detectable moiety or a toxin
or that disrupt translation), polypeptides (e.g. enzymes), lipids,
carbohydrates, and small molecules (e.g. small molecule drugs and
toxins). The payload may comprise nucleotides, e.g. nucleotides
that are labeled with a detectable or cytotoxic moiety (e.g. a
radiolabel).
[0055] In some embodiments, the extracellular vesicle comprises
nucleotides and/or polynucleotides (e.g. nucleic acids). For
example, the extracellular vesicle may comprise RNA, DNA, mRNA,
miRNA, dsDNA, lncRNA, siRNA, or singular nucleotides. In some
embodiments, the nucleotides and polynucleotides are synthetic. For
example, an exogenous nucleic acid may be introduced into the
extracellular vesicle and/or the producer cell. In some
embodiments, the nucleic acid is DNA that can be transcribed into
an RNA (e.g. an siRNA or mRNA) and in the case of an mRNA may be
translated into a desired polypeptide. In some embodiments, the
nucleic acid is an RNA (e.g. an siRNA or mRNA) and in the case of
an mRNA may be translated into a desired polypeptide.
[0056] In some embodiments, the extracellular vesicle comprises a
nucleic acid, such as a RNA or DNA. The nucleic acid is delivered
to a target cell as a payload. The target cell may transcribe a DNA
payload into an RNA such as a siRNA. In case an mRNA is transcribed
by the target cell form the DNA payload, the cell may translate the
mRNA into a polypeptide (e.g. therapeutic polypeptide). The target
cell may also translate a delivered mRNA payload into a
polypeptide.
[0057] In some embodiments, the producer cell comprises a nucleic
acid that may be transcribed (e.g. a DNA may be transcribed into a
siRNA or mRNA) and in case a mRNA is made the mRNA may be
translated by the producer cell into a polypeptide. The producer
cell may also be modified with a non-translatable RNA (e.g. siRNA
or miRNA). In case a mRNA is transferred the producer cell may
translate the mRNA into a polypeptide. Extracellular vesicles
derived from the producer cell may then carry the non-translatable
RNA, the transcribed RNA or the translated polypeptide as a
payload. In certain embodiments, the nucleic acid payload is a
modified nucleic acid. In some embodiments, said modified nucleic
acid is an siRNA or miRNA that is modified to additionally comprise
one or more cholesterol molecules. The cholesterol modifications on
the nucleic acid payloads may increase the association between the
nucleic acid and the inner and/or outer membrane of the
extracellular vesicles, therefore increasing the maximal loading
capacity of the extracellular vesicles. In some embodiments, said
cholesterol is chemically conjugated to the 5' end of the sense, or
passenger strand of the siRNA or miRNA. In some embodiments, said
cholesterol is chemically conjugated to the 3' end of the sense, or
passenger strand of the siRNA or miRNA. In some embodiments, said
cholesterol is chemically conjugated to the 5' end of the
antisense, or guide strand of the siRNA or miRNA. In some
embodiments, said cholesterol is chemically conjugated to the 3'
end of the antisense, or guide strand of the siRNA or miRNA.
[0058] The extracellular vesicles may interact with the target cell
via membrane fusion and deliver payloads (e.g., therapeutic agents)
in an extracellular vesicle composition to the surface or cytoplasm
of a target cell. In some embodiments, membrane fusion occurs
between the extracellular vesicles and the plasma membrane of a
target cell. In other embodiments, membrane fusion occurs between
the extracellular vesicles and an endosomal membrane of a target
cell.
[0059] Methods of Isolating Extracellular Vesicles
[0060] The extracellular vesicles may be isolated from the producer
cells. It is contemplated that all known manners of isolation of
extracellular vesicles are deemed suitable for use herein. For
example, physical properties of extracellular vesicles may be
employed to separate them from a medium or other source material,
including separation on the basis of electrical charge (e.g.,
electrophoretic separation), size (e.g., filtration, molecular
sieving, etc), density (e.g., regular or gradient centrifugation),
Svedberg constant (e.g., sedimentation with or without external
force, etc). Alternatively, or additionally, isolation may be based
on one or more biological properties, and include methods that may
employ surface markers (e.g., for precipitation, reversible binding
to solid phase, FACS separation, specific ligand binding,
non-specific ligand binding, etc.). In yet further contemplated
methods, the extracellular vesicles may also be fused using
chemical and/or physical methods, including PEG-induced fusion
and/or ultrasonic fusion.
[0061] Isolation (and enrichment) can be done in a general and
non-selective manner (typically including serial centrifugation).
Alternatively, isolation and enrichment can be done in a more
specific and selective manner (e.g., using producer cell-specific
surface markers). For example, specific surface markers may be used
in immunoprecipitation, FACS sorting, affinity purification,
bead-bound ligands for magnetic separation etc.
[0062] In certain preferred embodiments, isolation can be done by
affinity purification. For example, the extracellular vesicle can
be purified by binding a population comprising extracellular
vesicles to a resin, said resin comprising a plurality of ligands
that have specific affinity for one or more target proteins on the
surface of the extracellular vesicle. The one or more target
protein may be a tetraspanin (e.g., CD63, CD81 and/or CD9), an EWI
protein/immunoglobulin superfamily member (e.g., PTGFRN, IGSF8
and/or IGSF3), an integrin (e.g., ITGB1 and/or ITGA4), an ATP
transporter protein (e.g., ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B3,
ATP2B1, ATP2B2, ATP2B3 and/or ATP2B4), SLC3A2, BSG, or CD98hc. The
target protein may additionally be the immunomodulating component
that is displayed on the surface of the exosomes.
[0063] In some embodiments, size exclusion chromatography can be
utilized to isolate the extracellular vesicles. Size exclusion
chromatography techniques are known in the art. Exemplary,
non-limiting techniques are provided herein. In some embodiments, a
void volume fraction is isolated and comprises the extracellular
vesicles of interest. Further, in some embodiments, the
extracellular vesicles can be further isolated after
chromatographic separation by centrifugation techniques (of one or
more chromatography fractions), as is generally known in the art.
In some embodiments, for example, density gradient centrifugation
can be utilized to further isolate the extracellular vesicles.
Still further, in some embodiments, it can be desirable to further
separate the producer cell-derived extracellular vesicles from
extracellular vesicles of other origin. For example, the producer
cell-derived extracellular vesicles can be separated from
non-producer cell-derived extracellular vesicles by immunosorbent
capture using an antigen antibody specific for the producer
cell.
[0064] In some embodiments, the isolation of extracellular vesicles
may involve combinations of methods that include, but are not
limited to, differential centrifugation, size-based membrane
filtration, concentration and/or rate zonal centrifugation.
[0065] Particle size may be quantified after the extracellular
vesicles are isolated using any appropriate technique, including
dynamic light scattering or nanoparticle tracking analysis. The
diameter of the extracellular vesicles in a population can also be
described using particle size distribution (D values). D values
reflect the mass of the extracellular vesicles in a population as a
percentage when the particles are arranged on an ascending mass
basis. For instance, the D10 value is the diameter at which 10% of
the extracellular vesicles population mass is comprised of
extracellular vesicles less than the indicated diameter value. In
such a case, the population of extracellular vesicles is comprised
mainly of vesicles larger than the indicated diameter value. The
D50 value is the diameter at which 50% of the extracellular
vesicles population mass is comprised of vesicles less than the
indicated diameter value and 50% of the extracellular vesicles
population mass is comprised of vesicles larger than the indicated
value. In such a case, the population of extracellular vesicles is
comprised equally of vesicles larger than the indicated diameter
value and smaller than the indicated diameter. The D90 value is the
diameter at which 90% of the extracellular vesicles population mass
is comprised of extracellular vesicles less than the indicated
diameter value. In this case, the population of extracellular
vesicles is comprised mainly of vesicles smaller than the indicated
diameter value.
[0066] Payload Molecules
[0067] Extracellular vesicles may comprise payloads such as
peptides, proteins, DNA, RNA, siRNA, and other macromolecules and
small therapeutic molecules. In some embodiments, the payload is
transferred to a producer cell by applying controlled injury to the
cell for a predetermined amount of time in order to cause
perturbations in the cell membrane such that the payload can be
delivered to the inside of the cell (e.g., cytoplasm). In some
embodiments the payload is transferred to a extracellular vesicles
isolated from a producer cell by applying controlled injury to the
extracellular vesicles for a predetermined amount of time in order
to cause perturbations in the complex membrane such that the
payload can be delivered to the inside of the extracellular
vesicles. In some embodiments the payload of the extracellular
vesicles may be loaded within the membrane or interior portion of
the extracellular vesicles.
[0068] The payload may be a therapeutic agent selected from a
variety of known small molecule pharmaceuticals. Alternatively, the
payload may be a therapeutic agent selected from a variety of
macromolecules, such as, e.g., an inactivating peptide nucleic acid
(PNA), an RNA or DNA oligonucleotide aptamer, an interfering RNA
(iRNA), a peptide, or a protein.
[0069] In some embodiments, the payload that may be delivered to a
target by a extracellular vesicles includes, but is not limited to,
RNA, DNA, siRNA, mRNA, lncRNA, iRNA, polypeptides, enzymes,
cyotkines, antibodies, antibody fragments, small molecules,
chemotherapeutics, metals, viral particles, imaging agents, and
plasmids.
[0070] In some embodiments the payload of the extracellular
vesicles is a nucleic acid molecule, e.g. mRNA or DNA, and the
extracellular vesicles targets the payload to the cytoplasm of the
recipient or target cell, such that the nucleic acid molecule can
be translated (if mRNA) or transcribed and translated (if DNA) and
thus produce the polypeptide encoded by the payload nucleic acid
molecule within the target cell. In one embodiment the polypeptide
encoded by the payload nucleic acid molecule is secreted by the
target cell, thus modulating the systemic concentration or amount
of the polypeptide encoded by the payload nucleic acid molecule in
the subject. In one embodiment the polypeptide encoded by the
payload nucleic acid molecule is not secreted by the target cell,
thus modulating the intracellular concentration or amount of the
polypeptide encoded by the payload nucleic acid molecule in the
subject. In one embodiment the polypeptide encoded by the payload
nucleic acid molecule is toxic to the target cell or to other cell
or tissue in the subject, e.g. toxic to a cancer cell. In one
embodiment, the polypeptide encoded by the payload nucleic acid
molecule is not toxic to the target cell or other cell or tissue in
the subject, e.g. is therapeutically beneficial or corrects a
disease phenotype.
[0071] In some embodiments the payload of the extracellular
vesicles may be a membrane protein delivered to the plasma membrane
or endosomal membrane of the recipient cell.
[0072] Extracellular vesicles may comprise two or more payloads,
including mixtures, fusions, combinations and conjugates, of atoms,
molecules, etc. as disclosed herein, for example including but not
limited to, a nucleic acid combined with a polypeptide; two or more
polypeptides conjugated to each other; a protein conjugated to a
biologically active molecule (which may be a small molecule such as
a prodrug); and the like.
[0073] Suitable payloads include, without limitation,
pharmacologically active drugs and genetically active molecules,
including antineoplastic agents, anti-inflammatory agents, hormones
or hormone antagonists, ion channel modifiers, and neuroactive
agents. Examples of suitable payloads of therapeutic agents include
those described in, "The Pharmacological Basis of Therapeutics,"
Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth
edition, under the sections: Drugs Acting at Synaptic and
Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous
System; Autacoids: Drug Therapy of Inflammation; Water, Salts and
Ions; Drugs Affecting Renal Function and Electrolyte Metabolism;
Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function;
Drugs Affecting Uterine Motility; Chemotherapy of Parasitic
Infections; Chemotherapy of Microbial Diseases; Chemotherapy of
Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting
on Blood-Forming organs; Hormones and Hormone Antagonists;
Vitamins, Dermatology; and Toxicology, all incorporated herein by
reference. Suitable payloads further include toxins, and biological
and chemical warfare agents, for example see Somani, S. M. (ed.),
Chemical Warfare Agents, Academic Press, New York (1992)).
[0074] In some embodiments, the payload is a therapeutic agent,
such as a small molecule drug or a large molecule biologic. Large
molecule biologics include, but are not limited to, a protein,
polypeptide, or peptide, including, but not limited to, a
structural protein, an enzyme, a cytokine (such as an interferon
and/or an interleukin), a polyclonal or monoclonal antibody, or an
effective part thereof, such as an Fv fragment, which antibody or
part thereof, may be natural, synthetic or humanized, a peptide
hormone, a receptor, or a signaling molecule. In certain
embodiments, the protein payload is a Cas9 protein, a TALEN, a zinc
finger nuclease, or other component of a genome-editing
complex.
[0075] Large molecule biologics are immunoglobulins, antibodies, Fv
fragments, etc., that are capable of binding to antigens in an
intracellular environment. These types of molecules are known as
"intrabodies" or "intracellular antibodies." An "intracellular
antibody" or an "intrabody" includes an antibody that is capable of
binding to its target or cognate antigen within the environment of
a cell, or in an environment that mimics an environment within the
cell. Selection methods for directly identifying such "intrabodies"
include the use of an in vivo two-hybrid system for selecting
antibodies with the ability to bind to antigens inside mammalian
cells. Such methods are described in PCT/GB00/00876, incorporated
herein by reference. Techniques for producing intracellular
antibodies, such as anti-.beta.-galactosidase scFvs, have also been
described in Martineau et al., J Mol Biol 280:117-127 (1998) and
Visintin et al., Proc. Natl. Acad. Sci. USA 96:11723-1728
(1999).
[0076] Large molecule biologics include but is not limited to, at
least one of a protein, a polypeptide, a peptide, a nucleic acid, a
virus, a virus-like particle, an amino acid, an amino acid
analogue, a modified amino acid, a modified amino acid analogue, a
steroid, a proteoglycan, a lipid and a carbohydrate or a
combination thereof (e.g., chromosomal material comprising both
protein and DNA components or a pair or set of effectors, wherein
one or more convert another to active form, for example
catalytically).
[0077] A large molecule biologic may include a nucleic acid,
including, but not limited to, an oligonucleotide or modified
oligonucleotide, an antisense oligonucleotide or modified antisense
oligonucleotide, an aptamer, a cDNA, genomic DNA, an artificial or
natural chromosome (e.g., a yeast artificial chromosome) or a part
thereof, RNA, including an siRNA, a shRNA, mRNA, tRNA, rRNA or a
ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like
particles; a nucleotide or ribonucleotide or synthetic analogue
thereof, which may be modified or unmodified. Said modification may
be a lipophilic modification including addition of a cholesterol
molecule, myristoylation, PEGylation, and/or palmitoylation.
[0078] The large molecule biologic can also be an amino acid or
analogue thereof, which may be modified or unmodified or a
non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a
carbohydrate. If the large molecule biologic is a polypeptide, it
can be loaded directly into a producer cell according to the
methods described herein. Alternatively, an exogenous nucleic acid
encoding a polypeptide, which sequence is operatively linked to
transcriptional and translational regulatory elements active in a
producer cell at a target site, may be loaded.
[0079] Small molecules, including inorganic and organic chemicals,
may also be used as payloads of the extracellular vesicles
described herein.
[0080] In some embodiments, the small molecule is a
pharmaceutically active agent. Useful classes of pharmaceutically
active agents include, but are not limited to, antibiotics,
anti-inflammatory drugs, angiogenic or vasoactive agents, growth
factors and chemotherapeutic (anti-neoplastic) agents (e.g., tumour
suppressers).
[0081] In some embodiments, the payload molecule comprises an
agonist or activator of stimulator of interferon genes (STING). The
STING agonist may be a cyclic dinucleotide (CDN). Exemplary CDNs,
including but not limited to cyclic diguanylate monophosphate
(c-di-GMP), are described in Nature 2011, DOI: 10.1038/nature10429
and Nat. Chem. Biol. 2014, DOI: 10.1038/nchembio.1661. The STING
agonist may be a small molecule agonist.
[0082] A payload may be expressed by a target cell from a transgene
or mRNA introduced into a extracellular vesicles by
electroporation, chemical or polymeric transfection, viral
transduction, mechanical membrane disruption, or other method when
the target cell is contacted with the extracellular vesicles.
[0083] In some instances, the exogenous nucleic acid is an RNA
molecule, or a DNA molecule that encodes for an RNA molecule, that
silences or represses the expression of a target gene. For example,
the molecule can be a small interfering RNA (siRNA), an antisense
RNA molecule, or a short hairpin RNA (shRNA) molecule.
[0084] Pharmaceutical Compositions and Dosage Forms
[0085] Provided herein are pharmaceutical compositions comprising
extracellular vesicles that are suitable for administration to a
subject. The pharmaceutical compositions generally comprise a
plurality of extracellular vesicles and a
pharmaceutically-acceptable excipient or carrier in a form suitable
for administration to a subject. Pharmaceutically-acceptable
excipients or carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions
comprising a plurality of extracellular vesicles. (See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. 18th ed. (1990)). The pharmaceutical compositions are generally
formulated sterile and in full compliance with all Good
Manufacturing Practice (GMP) regulations of the U.S. Food and Drug
Administration.
[0086] In some embodiments, the pharmaceutical composition
comprises one or more therapeutic agents and the extracellular
vesicles described herein. In some embodiments, the extracellular
vesicles are co-administered with of one or more separate
therapeutic agents, wherein co-administration includes
administration of the separate therapeutic agent before, after or
concurrent with administration of the extracellular vesicles.
[0087] Pharmaceutically-acceptable excipients include excipients
that are generally safe (GRAS), non-toxic, and desirable, including
excipients that are acceptable for veterinary use as well as for
human pharmaceutical use.
[0088] Examples of carriers or diluents include, but are not
limited to, water, saline, Ringer's solutions, dextrose solution,
and 5% human serum albumin. The use of such media and compounds for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or compound is incompatible with
the extracellular vesicles described herein, use thereof in the
compositions is contemplated. Supplementary therapeutic agents may
also be incorporated into the compositions. Typically, a
pharmaceutical composition is formulated to be compatible with its
intended route of administration. The extracellular vesicles can be
administered by parenteral, topical, intravenous, oral,
subcutaneous, intraarterial, intradermal, transdermal, rectal,
intracranial, intraperitoneal, intranasal; intramuscular route or
as inhalants. In one embodiment, the pharmaceutical composition
comprising extracellular vesicles is administered intravenously,
e.g. by injection. The extracellular vesicles can optionally be
administered in combination with other therapeutic agents that are
at least partly effective in treating the disease, disorder or
condition for which the extracellular vesicles are intended.
[0089] Solutions or suspensions can include the following
components: a sterile diluent such as water, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial compounds such as benzyl alcohol
or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating compounds such as ethylenediaminetetraacetic
acid (EDTA); buffers such as acetates, citrates or phosphates, and
compounds for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. The preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0090] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (if water soluble) or dispersions
and sterile powders. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water,
Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered
saline (PBS). The composition is generally sterile and fluid to the
extent that easy syringeability exists. The carrier can be a
solvent or dispersion medium containing, e.g., water, ethanol,
polyol (e.g., glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, e.g., by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal compounds, e.g., parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. If desired, isotonic
compounds, e.g., sugars, polyalcohols such as manitol, sorbitol,
sodium chloride can be added to the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition a compound which delays absorption,
e.g., aluminum monostearate and gelatin.
[0091] Sterile injectable solutions can be prepared by
incorporating the extracellular vesicles in an effective amount and
in an appropriate solvent with one or a combination of ingredients
enumerated herein, as desired. Generally, dispersions are prepared
by incorporating the extracellular vesicles into a sterile vehicle
that contains a basic dispersion medium and any desired other
ingredients. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The extracellular vesicles can
be administered in the form of a depot injection or implant
preparation which can be formulated in such a manner to permit a
sustained or pulsatile release of the extracellular vesicles.
[0092] Systemic administration of compositions comprising
extracellular vesicles can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, e.g., for transmucosal administration, detergents, bile
salts, and fusidic acid derivatives. Transmucosal administration
can be accomplished through the use of nasal sprays or
suppositories. For transdermal administration, the modified
extracellular vesicles are formulated into ointments, salves, gels,
or creams as generally known in the art.
Examples
[0093] The invention is further illustrated by the following
examples. The examples are provided for illustrative purposes only,
and are not to be construed as limiting the scope or content of the
invention in any way. The practice of the present invention will
employ, unless otherwise indicated, conventional methods of protein
chemistry, biochemistry, recombinant DNA techniques and
pharmacology, within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., T. E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and
Company, 1993); Green & Sambrook et al., Molecular Cloning: A
Laboratory Manual, 4th Edition (Cold Spring Harbor Laboratory
Press, 2012); Colowick & Kaplan, Methods In Enzymology
(Academic Press); Remington: The Science and Practice of Pharmacy,
22nd Edition (Pharmaceutical Press, 2012); Sundberg & Carey,
Advanced Organic Chemistry: Parts A and B, 5th Edition (Springer,
2007).
Example 1: Microfluidization for Loading Exosomes with siRNA
[0094] Exosomes were purified chromatographically from a single-use
bioreactor (SUB) and formulated in PBS-Sucrose buffer (1.8 mM
KH.sub.2PO.sub.4, 10 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl,
5% w/v sucrose, pH 7.4). Exosomes were mixed with an siRNA
targeting kras G12D at a final concentration of 2 .mu.M siRNA and
1.times.10.sup.11 particles per mL in a 1.times.PBS, .about.1%
sucrose buffer. Loading reactions were performed at a 1.0 mL scale,
and processed in duplicate over a Microfluidics M110P
homogenization module equipped with a F12Y (75 .mu.m) interaction
chamber (Microfludics, Westwood, Mass.). Reactions were held at
20-25.degree. C. by using a heat-exchanger coil configured to the
outlet of the interaction chamber submerged in water. Upon
processing, samples were filtered using a 0.45 .mu.m PVDF filter
and stored at 4.degree. C. Process conditions were screened in a
single-pass manner at 1.times.10.sup.4, 2.times.10.sup.4, and
3.times.10.sup.4 psi. Process controls were run in addition to the
loading reactions. The entire experimental matrix is shown in Table
1.
TABLE-US-00001 TABLE 1 EV only siRNA only Process no Processing
(ctrl) (ctrl) Sample (mixing ctrl) 10,000 psi, single pass SUB-6 UF
Pool 274 uL 0 uL 274 uL 274 uL (3.7E11 p/mL) siRNA 0 uL 20 uL 20 uL
20 uL (100 .mu.M) 1X PBS, pH 7.4 726 uL 980 uL 706 uL 706 uL Total
# Samples 2 2 2 2 20,000 psi, single pass SUB-6 UF Pool 274 uL 0 uL
274 uL 274 uL siRNA 0 uL 20 uL 20 uL 20 uL 1X PBS, pH 7.4 726 uL
980 uL 706 uL 706 uL Total # Samples 2 2 2 2 30,000 psi, single
pass SUB-6 UF Pool 274 uL 0 uL 274 uL 274 uL siRNA 0 uL 20 uL 20 uL
20 uL 1X PBS, pH 7.4 726 uL 980 uL 706 uL 706 uL Total # Samples 2
2 2 2
[0095] Upon completion of processing, one set of process samples
and mixing controls were assayed using nanoparticle tracking
analysis (NTA) (Malvern Technologies). The results are shown in
Table 2.
TABLE-US-00002 TABLE 2 Mean D10 D50 D90 Sample p/mL (nm) (nm) (nm)
(nm) Mixing Control 1.11E+11 143.8 104.8 134.4 186.0 Process Sample
4.39E+10 139.9 94.3 134.7 179.0 10,000 psi Process Sample 2.57E+10
132.4 95.1 123.1 184.7 20,000 psi Process Sample 3.36E+10 134.9 93
125.9 177.5 30,000 psi
[0096] Through processing, substantial particle loss was observed
as a result of sample dilution through the microfluidizer, or as a
result of shear-induced homogenization. At this scale of
processing, the sample volume was approximately equal to the
volumetric holdup of the Microfluidics LV-1 and substantial
dilution of the processed sample was expected. Additionally,
relative to the mixing control, an approximately 10% reduction in
the particle D10 was observed
[0097] After processing through the microfluidizer, samples were
filtered using a 0.45 .mu.m polypropylene filter plate, and added
to Panc-1 cells to assay kras knockdown using an mRNA knockdown
qPCR assay. The relative kras expression of treated and control
cells are shown in FIG. 1. Positive (non-transfected Panc-1 cells)
and negative controls (Lipofectamine-transfected Panc-1 with a kras
G12D siRNA) were performed in parallel.
[0098] The homogenized vesicle samples showed greater knockdown of
kras expression as compared to extracellular vesicles (EV), siRNA
only, or Mixing control samples. Mixing controls show similar gene
expression to the negative control, and reagent only process
controls (siRNA only) show normal gene expression. The
3.times.10.sup.4 reagent controls showed knockdown when processed
independently through the LV-1. When vesicles and siRNA were
processed through the LV-1 Microfludizer, a pressure-dependent
gene-knockdown effect is observed--with the greatest knockdown seen
at a single-pass at the highest concentration of 3.times.10.sup.4
psi. These results demonstrate that purified exosomes can be loaded
with bioactive payloads and successfully deliver the payload to the
cytoplasm of recipient cells.
Example 2: Optimization of Microfluidic Loading Conditions
[0099] To determine conditions that maximized siRNA loading of
exosomes, microfluidic homogenization was carried out as in Example
1 at varying concentrations of siRNA. Exosome samples were
processed with kras G12D siRNAs at 0.5 .mu.M, 2 .mu.M, or 8 .mu.M
final concentrations. The products from each run were added to
cultured Panc-1 cells at concentrations of 100 EV/cell, 500
EV/cell, 1.times.10.sup.3 EV/cell or 1.times.10.sup.4 EV/cell. As a
positive control, Panc-1 cells were transfected with the kras G12D
siRNA using Lipofectamine. As shown in FIG. 2, there was a
concentration-dependent and dosage-dependent increase in KRAS mRNA
knockdown with 8 .mu.M siRNA and 1.times.10.sup.4 exosomes
resulting in the greatest knockdown.
[0100] Another method of loading exosomes with siRNA molecules is
to use siRNA duplexes that are covalently fused to a cholesterol
moiety. Cholesterol-conjugated siRNAs can associate with the
exosome membrane through lipid-lipid interactions between the
cholesterol molecule and the phospholipids of the exosome membrane.
This loading strategy can increase effective concentration of
loaded exosomes and allow for delivery of more siRNA molecules to a
recipient cell.
[0101] To determine whether microfluidic homogenization can enhance
the loading of cholesterol-modified siRNAs, exosomes were loaded as
in Example 1 with unmodified kras G12D siRNA or a
cholesterol-tagged version of the same miRNA. For controls, these
siRNAs were mixed with exosomes without microfluidic
homogenization. All samples were processed at either 10,000 psi or
20,000 psi and added to Panc-1 cells at a concentration of
1.times.10.sup.4 exosomes per cell. As shown in FIG. 3, at 30,000
psi both the unmodified and cholesterol-tagged siRNAs resulted in
significant knockdown of the kras G12D transcript, while none of
the mixing controls altered the levels of the transcript.
Interestingly, the cholesterol-tagged siRNA could efficiently knock
down the transcript at 20,000 psi, while there was very modest
knockdown of the transcript at 20,000 psi with the unmodified
siRNA. These results demonstrate that microfluidic homogenization
can be used for loading lipid-modified siRNAs, and that efficient
loading of the siRNAs can be accomplished at relatively low
processing pressure.
Example 3: Characterization of Exosome and siRNA Integrity after
Microfluidization
[0102] To understand the effects of the forces exerted on siRNAs
during microfluidic homogenization, RNA profiles were measured for
siRNAs exposed to repeated rounds of homogenization. siRNA (FIG.
4A) or cholesterol-tagged siRNA (FIG. 4B) were processed at 30,000
psi once, three times or five times and analyzed by anion-exchange
chromatography with detection at 254 nm. High concentration ("Mix")
and low concentration ("110 nM ctrl) control samples that were not
processed were used to determine the chromatographic trace of
unmodified siRNAs. As shown in FIGS. 4A and 4B, both RNA species
were diluted by serial rounds of homogenization, but the shape of
the chromatographic trace was not changed, indicating that the
structure of the siRNA was resistant to multiple rounds of
high-pressure microfluidic processing.
[0103] To determine whether the structure of exosomes was altered
during repeated rounds of microfluidic homogenization, purified
exosome samples were processed once, three times or five times and
were analyzed by size exclusion chromatography (SEC) to determine
the diameter of the exosome population. As shown in FIGS. 5A and
5B, repeated rounds of homogenization resulted in an increase in
retention time (t.sub.r), indicating the elution of smaller
products (FIG. 5A). This alteration in elution time was validated
using NTA, corresponding to a change from .about.160 nm average
diameter to .about.90 nm average diameter (FIG. 5B). These results
demonstrate that repeated microfluidic homogenization of exosomes
results in a modest but significant decrease in diameter, perhaps
due to shear-mediated fragmentation of the exosomes.
INCORPORATION BY REFERENCE
[0104] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
EQUIVALENTS
[0105] The present disclosure provides, inter alia, methods of
producing exosomes for the delivery of payload molecules. While
various specific embodiments have been illustrated and described,
the above specification is not restrictive. It will be appreciated
that various changes can be made without departing from the spirit
and scope of the invention(s). Many variations will become apparent
to those skilled in the art upon review of this specification.
* * * * *