Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
TITLE: METHODS AND COMPOSITIONS FOR THE PACKAGING OF NUCLEIC ACIDS
INTO MICROGLIAL EXOSOMES FOR THE TARGETED EXPRESSION OF
POLYPEPTIDES IN NEURAL CELLS
FIELD OF THE DISCLOSURE
[001] The present disclosure relates to neural cells, notably astroglial
cells,
microglial cells, and neuronal cells. The present disclosure further relates
to methods
for the modification of microglial exosomes to package and deliver nucleic
acid
molecules, expression of polypeptides in neural cells, and to methods of
replenishing
damaged or destroyed neuronal cells.
BACKGROUND OF THE DISCLOSURE
[002] The following paragraphs are provided by way of background to the
present disclosure. They are not however an admission that anything discussed
therein is prior art or part of the knowledge of persons skilled in the art.
1003] It is estimated that traumatic brain injury (TM) and stroke
collectively
cost the Canadian and American medical systems 110 billion dollars annually
(Finkelstein, Corso, and Miller, 2006; Heidenreich et aL, 2011; Public Health
Agency of
Canada, 2009). Both TBI and ischemic stroke are characterized by the death of
neural
tissues, which initiates a molecular signaling cascade that induces the
formation of
astroglial scar tissue that protects surrounding tissue from further damage.
However,
this scar tissue can also inhibit neuronal regrowth or regeneration, and thus
functional recovery. Currently, ischemic stroke therapies are directed at
emergency
care, notably at dissolving or removing blood clots. While these immediate
care
stroke treatments substantially limit the acute neural damage to a stroke
patient by
targeting the direct causal agent, and while these known treatments, if
initiated in a
timely manner, save lives, they are not effective in regenerating injured or
destroyed
neuronal cells. Hence stroke symptoms, such as cognitive, motor, and memory
.. impairments, are commonly experienced by surviving stroke patients, and are
frequently permanent. It is therefore desirable to develop effective medical
therapies
that replace damaged or destroyed neurons, or reduce the inhibitory astroglial
scar to
promote recovery and alleviate these functional deficits. The efficacy of the
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heretofore known therapeutic methodologies to restore neurons and astroglial
scar is
limited. Therefore there exists a need in the art for methods to restore
functional
neurons following stroke or TBI.
SUMMARY OF THE DISCLOSURE
[004] The following paragraphs are intended to introduce the reader to the
more detailed description that follows and not to define or limit the claimed
subject
matter of the present disclosure.
[005] In one aspect, the present disclosure relates to neural cells,
including
microglial cells, neuronal cells, and astroglial cells.
[006] In another aspect the present disclosure relates to methods for the
expression of polypeptides of interest in neural cells.
[007] In another aspect, the present disclosure relates to methods
for the
reprogramming of astroglial cells into neuronal cells.
1008] In another aspect, the present disclosure provides, in at
least one
embodiment, a method of expressing a polypeptide of interest in an astroglial
cell, the
method comprising
a) introducing a first and second chimeric nucleic acid sequence
in a
microglial host cell, the first chimeric nucleic acid sequence comprising as
operably linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest;
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(b) growing the microglial host cell to produce exosomes;
(c) delivering the exosomes to an astroglial cell; and
(d) expressing the polypeptide of interest in the astroglial cell.
In some embodiments, the first chimeric nucleic acid sequence may further
additionally comprise one or more of the following:
(iv) a nucleic acid sequence encoding a cleavable polypeptide; and
(v) a nucleic acid sequence encoding a polypeptide providing a signal for
nuclear localization in the astroglial cell.
[009] In some embodiments, the exosomes are produced in microglia
cells in
vitro. In such embodiments, the exosomes may be separated from the microglia
cells
and provided to an animal in need thereof.
[0010] In some embodiments, the exosomes are produced in microglia
cells in
vivo. In such embodiments, the microglia cells may be provided to an animal in
need
thereof.
[0011] In some embodiments, the polypeptide of interest is a protein
capable
of reprogramming astroglial cells into neuronal cells.
[0012] In some embodiments, the polypeptide of interest is NeuroD1.
[0013] In another aspect, the present disclosure provides microglia
cells
capable of producing exosomes, wherein the exosomes comprise:
(I) a chimeric polypeptide encoded by a first chimeric nucleic acid
sequence; and
(II) a second chimeric nucleic acid sequence,
(a) the first chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
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(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
(b) the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
In another aspect, the present disclosure provides a preparation comprising
substantially pure exosomes comprising:
(I) a chimeric polypeptide encoded by a first chimeric nucleic acid
sequence; and
(II) a second chimeric nucleic acid sequence,
(a) the first chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
(b) the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
[0014] In another aspect, the present disclosure provides a method
for
reprogramming astroglial cells into neuronal cells, the method comprising:
introducing a first and second chimeric nucleic acid sequence in a microglial
host cell, the first chimeric nucleic acid sequence comprising as operably
linked components:
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(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide capable of
reprogramming astroglial cells into neuronal cells;
(b) growing the microglial host cell to produce exosomes;
(c) delivering the exosomes to an astroglial cell; and
(d) expressing the polypeptide to reprogram astroglial cells into neuronal
cells in the astroglial cell.
[0015] In another aspect, the present disclosure provides a
transgenic
microglial cell line, wherein the microglial cells have been obtained by:
introducing a chimeric nucleic acid sequence into the genome of a microglial
host cell, the chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence.
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L00161 In yet another aspect, the present disclosure provides a
transgenic
animal comprising transgenic astroglial cells in which a protein of interest
is
expressed, wherein the transgenic astroglial cells have been obtained by:
introducing a first and second chimeric nucleic acid sequence in a microglial
host cell, the first chimeric nucleic acid sequence comprising as operably
linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest;
(b) growing the microglial host cell to produce exosomes;
(c) delivering the exosomes to an astroglial cell; and
(d) expressing the polypeptide of interest in the astroglial cell.
[0017] In yet another aspect, the present disclosure provides a
transgenic
animal comprising transgenic astroglial cells wherein the transgenic
astroglial cells
comprise a chimeric nucleic acid sequence comprising:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
100181 Other features and advantages of the present disclosure will
become
apparent from the following detailed description. It should be understood,
however,
that the detailed description, while indicating preferred implementations of
the
disclosure, are given by way of illustration only, since various changes and
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modifications within the spirit and scope of the disclosure will become
apparent to
those of skill in the art from the detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The disclosure is in the hereinafter provided paragraphs
described in
relation to its figures. The figures provided herein are provided for
illustration
purposes and are not intended to limit the present disclosure.
[0020] FIGURE 1 depicts an overview of the first and second chimeric
nucleic
acid sequences. The first chimeric nucleic acid sequence comprises as operably
linked
components: (i) a nucleic acid sequence encoding an exosomal membrane
polypeptide; (ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide;
and (iii) a nucleic acid sequence encoding a nucleic acid binding polypeptide
capable
of binding a nucleic acid binding polypeptide recognition sequence. The second
chimeric nucleic acid sequence comprises as operably linked components: (i) a
nucleic acid binding polypeptide recognition sequence; and (ii) a nucleic acid
sequence encoding a polypeptide of interest regulated by a promoter element
specific
to the target cell of interest.
[0021] FIGURE 2 depicts a schematic overview of an exosome produced
by a
microglial cell after introduction therein of a first and second chimeric
nucleic acid
sequence in accordance with the simplest embodiment of the present disclosure.
Shown is a first chimeric polypeptide embedded in the exosomal membrane via an
exosomal membrane polypeptide. A neural cell targeting polypeptide is located
at the
N-terminus of the exosomal membrane polypeptide, which is external to the
exosome
and allows for targeting of the exosome to neural cells. The C-terminus of the
first
chimeric polypeptide is a nucleic acid binding polypeptide that binds the
second
chimeric nucleic acid sequence via a specific nucleic acid binding polypeptide
recognition sequence. The second chimeric nucleic acid sequence (in plasmid
vector
form) contains a regulatory element specific to the target cell operably
linked to a
nucleic acid sequence encoding a polypeptide of interest.
[0022] FIGURE 3 depicts a schematic overview of an exosome produced
by a
microglial cell after introduction therein of a first and second chimeric
nucleic acid
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sequence in accordance with another embodiment of the present disclosure.
Shown is
a first chimeric polypeptide embedded in the exosomal membrane via an exosomal
membrane polypeptide. A neural cell targeting polypeptide is located at the N-
terminus of the exosomal membrane polypeptide, which is external to the
exosome
and allows for targeting of the exosome to neural cells. The C-terminus of the
first
chimeric polypeptide is a nucleic acid binding polypeptide that binds the
second
chimeric nucleic acid sequence via a specific nucleic acid binding polypeptide
recognition sequence. In this embodiment, the exosomal membrane polypeptide is
linked to the nucleic acid binding polypeptide via a cleavable polypeptide
linker and a
nuclear localization polypeptide. When the first chimeric polypeptide is
cleaved, the
second chimeric nucleic acid sequence (bound to the nucleic acid binding
polypeptide
and nuclear localization polypeptide) is released for transport to the nucleus
of the
target neural cell (guided by the nuclear localization polypeptide). The
second
chimeric nucleic acid sequence (in plasmid vector form) contains a regulatory
element specific to the target cell operably linked to a nucleic acid sequence
encoding
a polypeptide of interest. The plasmid also contains a selectable marker for
preparation in bacteria.
[0023] FIGURE 4 depicts a schematic overview of an exosome produced
by a
microglial cell after introduction therein of a first and second chimeric
nucleic acid
sequence in accordance with another embodiment of the present disclosure.
Shown is
a first chimeric polypeptide embedded in the exosomal membrane via a LAMP-2B
polypeptide transmembrane domain. An RVG is located near the N-terminus of
LAMP-2B, which is external to the exosome and allows for targeting of the
exosome to
neural cells. The C-terminus of the first chimeric polypeptide is a synthetic
transcription activator-like (TAL) effector nucleic acid binding polypeptide
that binds
the second chimeric nucleic acid sequence via the nucleic acid recognition
sequence
specific to the TAL effector. LAMP-2B is linked to the TAL effector
polypeptide via a
cleavable polypeptide and a nuclear localization polypeptide. When the first
chimeric
polypeptide is cleaved, the second chimeric nucleic acid sequence (bound to
the TAL
effector and nuclear localization polypeptide) is released for transport to
the nucleus
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of the target neural cell (guided by the nuclear localization polypeptide).
The second
chimeric nucleic acid sequence contains an astrocyte-specific promoter
operably
linked to NeuroD1, a gene encoding a transcription factor necessary for
neuronal
differentiation. The plasmid also contains a small selectable nucleic acid
sequence
encoding an RNA polynucleotide for preparation in bacteria, namely an RNA-OUT
sequence.
[0024] FIGURE 5 depicts confocal microscopy images of both control
and
lipofected human embryonic kidney (lEK-293) cells and lipofected mouse
microglia
(EOC 13.31) counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue;
counterstained nuclei indicated with arrows). Cell cultures were lipofected
with a
variant of the first chimeric polypeptide where the cleavable polypeptide and
the
nucleic acid binding polypeptide were replaced with Clover, a green
fluorescent
protein (GFP) variant. (A) Both non-lipofected and vehicle control (not shown)
HEK-
293 cell cultures do not exhibit green cytoplasmic fluorescence. (B) HEK-293
cells
lipofected with pcDNA3.0-Clover exhibit green fluorescence in the cytoplasm
which
can be attributed to Clover expression (green fluorescence indicated with
arrowheads). (C) HEK-293 cells lipofected with pcDNA3.0-RVG-LAMP-2B-Clover
exhibit green fluorescence in the cytoplasm which can be attributed to Clover
expression and indicates that a C-terminal modification of LAMP-2B is viable.
(D)
Murine microglia cells transfected with pcDNA3.0-RVG-LAMP2B-Clover similarly
exhibit green fluorescence in the cytoplasm attributable to Clover expression,
indicating that this chimeric construct is also viable in mouse models.
[0025] FIGURE 6 depicts the fluorescence exhibited by exosomes
isolated
from unlipofected cell cultures and those lipofected with pcDNA3.0-RVG-LAMP-2B-
Clover. Exosomes were excited at 489 nm and monitored for fluorescence with a
spectrofluorometer from 505 nm - 620 nm. Lysis (with sodium deoxycholate) of
exosomes from cells transfected with pcDNA3.0-RVG-LAMP-2B-Clover results in
elevated fluorescence peaking at 520 nm which is consistent with the
fluorescence
spectrum of Clover and indicates that the chimeric polypeptide is
appropriately
localized to exosomal lumens.
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[00261 FIGURE 7 depicts confocal microscopy images of an immortal
astrocyte
(C8-D30) cell culture lipofected with an experimental version of the second
chimeric
nucleic acid sequence comprising NeuroD1 operably linked to a constitutive
cytomegalovirus (CMV) promoter and an internal ribosome entry site (IRES)
linked
to a green fluorescent protein (GFP). This experimental embodiment provides a
method of visually confirming cellular expression of NeuroD1 by associating it
with
GFP expression. Adherent astrocytes were fixed with 4% paraformaldehyde,
immunolabeled with 1:100 anti-GFAP and 1:200 anti-rabbit-Alexa Fluor 594
(red),
and counterstained with DAPI (blue). GFP expression (green) is evident in the
cytoplasm of successfully transfected cells, which do not exhibit co-staining
with
GFAP (a reactive astrocyte marker). The loss of GFAP expression suggests
reprogramming of the reactive astrocytes.
[0027] The figures together with the following detailed description
make
apparent to those skilled in the art how the disclosure may be implemented in
practice.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0028] Various compositions and methods will be described below to
provide
an example of an embodiment of each claimed subject matter. No embodiment
described below limits any claimed subject matter and any claimed subject
matter
may cover methods, processes, compositions or systems that differ from those
described below. The claimed subject matter is not limited to compositions or
methods having all of the features of any one composition, method, system or
process
described below or to features common to multiple or all of the compositions,
systems or methods described below. It is possible that a composition, system,
method or process described below is not an embodiment of any claimed subject
matter. Any subject matter disclosed in a composition, system, method or
process
described below that is not claimed in this document may be the subject matter
of
another protective instrument, for example, a continuing patent application,
and the
applicants, inventors or owners do not intend to abandon, disclaim or dedicate
to the
public any such subject matter by its disclosure in this document.
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Definitions
[0029] The term "glial cell", as used herein refers to connective
tissue cells of
the central nervous system providing structural and functional support to the
neuronal cells of the central nervous system, including, for example, in the
form of
providing nutrition and homeostasis and/or by participation in signal
transmission in
the nervous system. Glial cells include, but are not limited to, astrocytes
(also referred
to herein as astroglial cells), microglia, and oligodendrocytes.
[0030] The term "microglial cell" or "microglia", as used herein,
refers to a
class of glial cells involved in the mediation of an immune response within
the central
nervous system by acting as macrophages. Microglial cells are capable of
producing
exosomes, and further include different forms of microglial cells, including
amoeboid
microglial cells, ramified microglial cells and reactive microglial cells.
Microglial cells
include reactive microglia, which are defined as quiescent ramified microglia
that
transform into a reactive, macrophage-like state and accumulate at sites of
brain
injury and inflammation to assist in tissue repair and neural regeneration
(Kreutzberg, 1996).
[0031] The term "astroglial cell" or "astrocyte", as used herein,
refers to a class
of glial cells involved in the structural and nutritional support of central
nervous
system cell populations and in the repair and scarring process following
neural injury.
Astroglial cells further include different forms including reactive astroglial
cells,
fibrous astroglial cells, protoplasmic astroglial cells, and radial astroglial
cells. The
term astroglial cell includes reactive astrocytes which, in response to brain
injury,
proliferate and become the primary cellular component of the resulting glial
scar
(Stitchel & Muller, 1988). Reactive astrocytes undergo morphological changes,
increase synthesis of glial fibrillary acidic protein (GFAP), and secrete
molecules to
modulate neuronal outgrowth thereby restricting neuronal regeneration and
functional recovery following a TBI or stroke.
[0032] The term "exosome" as used herein refers to nanometer sized
(having a
diameter in the range of approximately 30 nm - 150 nm) membrane-derived
vesicles,
secreted by a mammalian cell, including, for example, a microglial cell.
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[0033] The term "exosomal membrane polypeptide", as used herein
refers to
any protein associated with or integrated within exosomal membranes. The term
exosomal membrane polypeptide includes, without limitation, LAMP-1
polypeptide,
LAMP-2A polypeptide, LAMP-2B polypeptide, LAMP-2C polypeptide, LIM P-2/SCARB2
polypeptide, Flotillin-1 polypeptide, and any other protein capable of
association with
or integration within exosomal membranes.
[0034] The term "neural cell targeting polypeptide", as used herein
in refers to
any polypeptide capable of associating with or binding to neural cells. The
term
neural cell targeting polypeptide includes, without limitation, the entirety,
or any
functional portion of viral envelope proteins known to associate with or bind
to
neural cells, including rabies virus glycoprotein (RVG). The term neural cell
targeting
polypeptide further also includes cell-surface expression of antibodies
including, but
not limited to, anti-GLAST IgG, which confers in vivo and in vitro selective
targeting to
astrocytes (Fassler et al., 2013; Balyasnikova et al., 2010). Neural cell
targeting
polypeptide further also includes a T7/transferrin receptor-binding
polypeptide
operably linked to a cell membrane permeant peptide (such as penetratin or
transportan) as described by Youn, Chen and Furgeson (2014) or Muratovska and
Eccles (2004).
[0035] The term "nucleic acid binding polypeptide" or "nucleic acid
binding
domain", as may be used interchangeably herein, refers to a polypeptide
capable of
specifically binding to a specific nucleic acid recognition sequence. The term
nucleic
acid binding polypeptide includes, without limitation, any polypeptide
comprising a
helix-turn-helix, zinc finger, leucine zipper, winged helix, winged helix-turn-
helix,
helix-loop-helix, HMG-box, Wor3, immunoglobulin fold, B3, TAL effector, or RNA-
guided DNA-binding domain. Further included are the Gal4 polypeptide and any
TAL
effector, including a synthetically engineered TAL effector (Sanjana et al.,
2013).
[0036] The term "nucleic acid binding polypeptide recognition
sequence" as
used herein, refers to a nucleic acid sequence capable of specifically
associating with a
polypeptide capable of binding to the sequence. The nucleic acid sequence may
vary
in length and may for example be a DNA sequence of at least 10 base pairs, at
least 20
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base pairs, or at least 50 base pairs in length. The term nucleic acid binding
polypeptide recognition sequence includes, for example, the Ga14 Upstream
Activator
Sequence (specific to the Ga14 polypeptide) or any TAL effector recognition
sequence,
including any synthetically engineered TAL effector recognition sequence
(specific to
its corresponding TAL effector polypeptide; Sanjana et al., 2013).
[0037] The term "nucleic acid sequence", as used herein, refers to a
sequence
of nucleoside or nucleotide monomers consisting of naturally occurring bases,
sugars
and intersugar (backbone) linkages. The term also includes modified or
substituted
sequences comprising non-naturally occurring monomers or portions thereof. The
nucleic acid sequences of the present disclosure may be deoxyribonucleic acid
sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally
occurring bases including adenine, guanine, cytosine, thymidine and uracil.
The
sequences may also contain modified bases. Examples of such modified bases
include
aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine
and
hypoxanthine.
[0038] "Operably linked" refers to a configuration of nucleic acids
in which a
first nucleic acid sequence is placed in a functional relationship with a
second nucleic
acid sequence. For instance, a promoter is operably linked to a coding
sequence if the
promoter affects the transcription or expression of the coding sequence.
Operably
linked DNA sequences may be in close proximity or contiguous and, where
necessary
to join two protein coding regions, in the same reading frame. Furthermore,
the
polynucleotide sequences contemplated herein may be present in expression
vectors.
[0039] The term "vector" or "expression vector" refers to a means by
which
nucleic acid (e.g., DNA) can be introduced into a host organism or host
tissue. There
are various types of vectors including plasmid vector, bacteriophage vectors,
cosmid
vectors, bacterial vectors, and viral vectors. The term vector, as used
herein, may
refer to a recombinant nucleic acid that has been engineered to express a
heterologous polypeptide (e.g., the fusion proteins disclosed herein), or a
heterologous promoter (e.g., a eukaryotic or prokaryotic promoter) operably
linked
to a polynucleotide that encodes a protein.
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[0040] A "heterologous promoter" refers to a promoter that is not the
native
or endogenous promoter for the protein that is being expressed. For example,
as
contemplated herein, heterologous promoters for NeuroD1 include a synthetic
gfaABC1D promoter, a eukaryotic GFAP promoter, or a prokaryotic CMV promoter,
none of which are the native, endogenous promoter for NeuroD1. The vectors
contemplated herein may be introduced and propagated in a prokaryote, such as
Escherichia coil, which may be used to amplify copies of a vector to be
introduced into
a eukaryotic cell or as an intermediate vector in the production of a vector
to be
introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a
viral vector
packaging system).
[0041] The term "expression", as used herein, refers to the process
by which a
polynucleotide is transcribed from a DNA template (such as into and mRNA or
other
RNA transcript) and/or the process by which a transcribed mRNA is subsequently
translated into peptides, polypeptides, or proteins. If the polynucleotide is
derived
from genomic DNA, expression may include splicing of the mRNA in a eukaryotic
cell.
[0042] The terms "peptide", "protein", or "polypeptide", as used
herein,
typically comprises a polymer of naturally occurring amino acids (e.g.,
alanine,
arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine,
threonine, tryptophan, tyrosine, and valine). Typically, a "polypeptide" or
"protein" is
defined as a longer polymer of amino acids, of a length typically of greater
than 50, 60,
70, 80, 90, or 100 amino acids. A "peptide" is defined as a short polymer of
amino
acids, of a length typically of 50, 40, 30, 20 or less amino acids. The
polypeptides
contemplated herein may be further modified in vitro or in vivo to include non-
amino
acid moieties. These modifications may include but are not limited to
acylation (e.g.,
0-acylation (esters), N-acylation (amides), S-acylation (thioesters)),
acetylation (e.g.,
the addition of an acetyl group, either at the N-terminus of the protein or at
lysine
residues), formylation, lipoylation (e.g., attachment of a lipoate, a C8
functional
group), myrtstoylation (e.g., attachment of myristate, a C14 saturated acid),
palmitoylation (e.g., attachment of palmitate, a C16 saturated acid),
alkylation (e.g.,
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the addition of an alkyl group, such as an methyl at a lysine or arginine
residue),
isoprenylation or prenylation (e.g., the addition of an isoprenoid group such
as
farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g.,
the addition
of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine,
resulting
in a glycoprotein). Distinct from glycation, which is regarded as a
nonenzymatic
attachment of sugars, polysialylation (e.g., the addition of polysialic acid),
glypiation
(e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation,
iodination
(e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a
phosphate
group, usually to serine, tyrosine threonine or histidine). The amino acid
sequences of
polypeptide variants, mutants, or derivatives as contemplated herein may also
include conservative amino acid substitutions relative to a reference amino
acid
sequence. For example, a variant, mutant, or derivative protein may include
conservative amino acid substitutions relative to a reference molecule.
Conservative
amino acid substitutions are those substitutions that are a substitution of an
amino
acid for a different amino acid where the substitution is predicted to
interfere least
with the properties of the reference polypeptide. In other words, conservative
amino
acid substitutions substantially conserve the structure and the function of
the
reference polypeptide. Conservative amino acid substitutions generally
maintain (a)
the structure of the polypeptide backbone in the area of the substitution, for
example,
as a beta sheet or alpha helical conformation. (b) the charge or
hydrophobicity of the
molecule at the site of the substitution, and/or (c) the bulk of the side
chain. The
following table provides a list of exemplary conservative amino acid
substitutions
which are contemplated herein:
Original residue Conservative substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
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Gin Asn, Gin, His
Glu Asp, Gin, His
Gly Ala
His Asn, Arg, Gin, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gin, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
[0043] By the term "substantially identical" it is meant that two
polypeptide
sequences preferably are at least 70% identical, and more preferably are at
least 85%
identical and most preferably at least 95% identical, for example 96%, 97%,
98% or
99% identical. In order to determine the percentage of identity between two
polypeptide sequences the amino acid sequences of such two sequences are
aligned,
using for example the alignment method of Needleman and Wunsch, 1970, as
revised
by Smith and Waterman, 1981, so that the highest order match is obtained
between
the two sequences and the number of identical amino acids is determined
between
the two sequences. Methods to calculate the percentage identity between two
amino
acid sequences are generally art recognized and include, for example, those
described
by Carillo and Lipman, 1988 and those described in: Lesk, 1988. Generally,
computer
programs will be employed for such calculations. Computer programs that may be
used in this regard include, but are not limited to, GCG (Devereux et al.
1984) BLASTP,
BLASTN and FASTA (Altschul et al. 1990). A particularly preferred method for
determining the percentage identity between two polypeptides involves the
Clustal
16
CA 2903933 2018-01-10
W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994) together with
the
BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992) using a gap
opening
penalty of 10 and a gap extension penalty of 0.1, so that the highest order
match
obtained between two sequences wherein at least 50% of the total length of one
of
the two sequences is involved in the alignment.
[0044] By "at least moderately stringent hybridization conditions" it
is meant
that conditions are selected which promote selective hybridization between two
complementary nucleic acid molecules in solution. Hybridization may occur to
all or a
portion of a nucleic acid sequence molecule. The hybridizing portion is
typically at
least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in
the art will
recognize that the stability of a nucleic acid duplex, or hybrids, is
determined by the
Tm, which in sodium containing buffers is a function of the sodium ion
concentration
and temperature (Tm=81.5 C.-16.6 (Log10 [Na+])+0.41(% (G+C)-600/1), or
similar
equation). Accordingly, the parameters in the wash conditions that determine
hybrid
stability are sodium ion concentration and temperature. In order to identify
molecules that are similar, but not identical, to a known nucleic acid
molecule a 1%
mismatch may be assumed to result in about a 1 C. decrease in Tm, for example
if
nucleic acid molecules are sought that have a >95% identity, the final wash
temperature will be reduced by about 5 C. Based on these considerations those
skilled in the art will be able to readily select appropriate hybridization
conditions. In
preferred embodiments, stringent hybridization conditions are selected. By way
of
example the following conditions may be employed to achieve stringent
hybridization: hybridization at 5x sodium chloride/sodium citrate
(SSC)/5xDenhardt's solution/1.0% SDS at Tm (based on the above equation) -5
C.,
followed by a wash of 0.2xSSC/0.1% SDS at 60 C. Moderately stringent
hybridization
conditions include a washing step in 3xSSC at 42 C. It is understood however
that
equivalent stringencies may be achieved using alternative buffers, salts and
temperatures. Additional guidance regarding hybridization conditions may be
found
in: Ausubel, 1989 and in: Sambrook et al., 1989.
17
CA 2903933 2018-01-10
[0045] The term "chimeric", as used herein in the context of nucleic
acid
sequences or proteins, refers to at least two linked nucleic acid sequences or
polypeptide sequences, which are not naturally linked. Chimeric nucleic acid
sequences and chimeric polypeptide sequences include linked nucleic acid
sequences
or polypeptide sequences of different natural origins. For example a nucleic
acid
sequence constituting an astrocyte specific promoter linked to a nucleic acid
sequence encoding a NeuroD1 polypeptide is considered a chimeric nucleic acid
sequence. For example a polypeptide sequence constituting an exosomal membrane
polypeptide linked to a neural targeting polpypeptide is considered a chimeric
protein Chimeric nucleic acid sequences and protein sequences also may
comprise
nucleic acid sequences or polypeptide sequences of the same natural origin,
provided
they are not naturally linked. For example a nucleic acid sequence
constituting a
promoter obtained from a particular cell-type may be linked to a nucleic acid
sequence encoding a polypeptide obtained from that same cell-type, but not
normally
linked to the nucleic acid sequence constituting the promoter. Chimeric
nucleic acid
sequences and protein sequences also include nucleic acid sequences comprising
any
naturally occurring nucleic acid sequence linked to any non-naturally
occurring
nucleic acid sequence or polypeptide sequence.
[0046] The terms "LAMP-2B protein", "LAMP-2B polypeptide" and "LAMP-
2B",
as may be used interchangeably herein, refer to any and all proteins
comprising a
sequence of amino acid residues which is (i) substantially identical to the
amino acid
sequences constituting any LAMP-2B polypeptide set forth herein, including,
for
example, SEQ.ID. NO: 33, or (ii) encoded by a nucleic acid sequence capable of
hybridizing under at least moderately stringent conditions to any nucleic acid
sequence encoding any LAMP-2B polypeptide set forth herein, but for the use of
synonymous codons.
[0047] The terms "RVG protein", "RVG polypeptide" and "RVG", as may
be used
interchangeably herein, refer to any and all proteins comprising a sequence of
amino
acid residues which is (i) substantially identical to the amino acid sequences
constituting any RVG polypeptide set forth herein, including, for example,
SEQ.ID. NO:
18
CA 2903933 2018-01-10
45, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at
least
moderately stringent conditions to any nucleic acid sequence encoding any RVG
polypeptide set forth herein, but for the use of synonymous codons.
[0048] The terms "Gal4 protein", "Gal4 polypeptide" and "Gal4", as
may be
.. used interchangeably herein, refer to any and all proteins comprising a
sequence of
amino acid residues which is (i) substantially identical to the amino acid
sequences
constituting any Gal4 polypeptide set forth herein, including, for example,
SEQ.ID. NO:
47, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at
least
moderately stringent conditions to any nucleic acid sequence encoding any Gal4
polypeptide set forth herein, but for the use of synonymous codons.
[0049] The terms "TAL effector protein", "TAL effector polypeptide"
and "TAL
effector", as may be used interchangeably herein, refer to any and all
proteins
comprising a sequence of amino acid residues which is (i) substantially
identical to
the amino acid sequences constituting any TAL effector polypeptide set forth
herein,
.. including, for example, SEQ.ID. NO: 48, or (ii) encoded by a nucleic acid
sequence
capable of hybridizing under at least moderately stringent conditions to any
nucleic
acid sequence encoding any TAL effector polypeptide set forth herein, but for
the use
of synonymous codons.
[0050] The terms "NeuroD1 protein", "NeuroD1 polypeptide" and
"NeuroD1",
as may be used interchangeably herein, refer to any and all proteins
comprising a
sequence of amino acid residues which is (i) substantially identical to the
amino acid
sequences constituting any NeuroD1 polypeptide set forth herein, including,
for
example, SEQ.ID. NO: 49, or (ii) encoded by a nucleic acid sequence capable of
hybridizing under at least moderately stringent conditions to any nucleic acid
sequence encoding any NeuroD1 polypeptide set forth herein, but for the use of
synonymous codons.
[0051] The terms "LAMP-2B nucleic acid sequence", "nucleic acid
sequence
encoding LAMP-2B protein", "nucleic acid sequence encoding LAMP-2B
polypeptide"
and "nucleic acid sequence encoding LAMP-2B", as may be used interchangeably
.. herein, refer to any and all nucleic acid sequences encoding a LAMP-2B
polypeptide,
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CA 2903933 2018-01-10
including, for example, SEQ.ID. NO: 1. Nucleic acid sequences encoding a LAMP-
2B
polypeptide further include any and all nucleic acid sequences which (i)
encode
polypeptides that are substantially identical to the LAMP-2B polypeptide
sequences
set forth herein; or (ii) hybridize to any LAMP-2B nucleic acid sequences set
forth
herein under at least moderately stringent hybridization conditions or which
would
hybridize thereto under at least moderately stringent conditions but for the
use of
synonymous codons.
[0052] The terms "RVG nucleic acid sequence", "nucleic acid sequence
encoding RVG protein", "nucleic acid sequence encoding RVG polypeptide" and
"nucleic acid sequence encoding RVG", as may be used interchangeably herein,
refer
to any and all nucleic acid sequences encoding a RVG polypeptide, including,
for
example, SEQ.ID. NO: 13. Nucleic acid sequences encoding a RVG polypeptide
further
include any and all nucleic acid sequences which (i) encode polypeptides that
are
substantially identical to the RVG polypeptide sequences set forth herein; or
(ii)
hybridize to any RVG nucleic acid sequences set forth herein under at least
moderately stringent hybridization conditions or which would hybridize thereto
under at least moderately stringent conditions but for the use of synonymous
codons.
[0053] The terms "Gal4 nucleic acid sequence", "nucleic acid sequence
encoding Gal4 protein", "nucleic acid sequence encoding Gal4 polypeptide" and
"nucleic acid sequence encoding Gal4", as may be used interchangeably herein,
refer
to any and all nucleic acid sequences encoding a Gal4 polypeptide, including,
for
example, SEQ.ID. NO: 15. Nucleic acid sequences encoding a Gal4 polypeptide
further
include any and all nucleic acid sequences which (i) encode polypeptides that
are
substantially identical to the Gal4 polypeptide sequences set forth herein; or
(ii)
hybridize to any Gal4 nucleic acid sequences set forth herein under at least
moderately stringent hybridization conditions or which would hybridize thereto
under at least moderately stringent conditions but for the use of synonymous
codons.
[0054] The terms "TAL effector nucleic acid sequence", "nucleic acid
sequence
encoding TAL effector protein", "nucleic acid sequence encoding TAL effector
polypeptide" and "nucleic acid sequence encoding TAL effector", as may be used
CA 2903933 2018-01-10
interchangeably herein, refer to any and all nucleic acid sequences encoding a
TAL
effector polypeptide, including, for example, SEQ.ID. NO: 16. Nucleic acid
sequences
encoding a TAL effector polypeptide further include any and all nucleic acid
sequences which (i) encode polypeptides that are substantially identical to
the TAL
effector polypeptide sequences set forth herein; or (ii) hybridize to any TAL
effector
nucleic acid sequences set forth herein under at least moderately stringent
hybridization conditions or which would hybridize thereto under at least
moderately
stringent conditions but for the use of synonymous codons.
[0055] The terms "NeuroD1 nucleic acid sequence", "nucleic acid
sequence
encoding NeuroD1 protein", "nucleic acid sequence encoding NeuroD1
polypeptide"
and "nucleic acid sequence encoding NeuroD1", as may be used interchangeably
herein, any and all nucleic acid sequences encoding a NeuroD1 polypeptide,
including,
for example, SEQ.ID. NO: 19. Nucleic acid sequences encoding a NeuroD1
polypeptide
further include any and all nucleic acid sequences which (i) encode
polypeptides that
are substantially identical to the NeuroD1 polypeptide sequences set forth
herein; or
(ii) hybridize to any NeuroD1 nucleic acid sequences set forth herein under at
least
moderately stringent hybridization conditions or which would hybridize thereto
under at least moderately stringent conditions but for the use of synonymous
codons.
[0056] The terms "Gal4 Upstream Activator Sequence", "Gal4 UAS", and
"Gal4
UAS nucleic acid sequence", as may be used interchangeably herein, refer to
any and
all nucleic acid sequences capable of binding Gal4 polypeptide, including, for
example,
SEQ.ID. NO: 17. Gal4 UAS further includes any and all nucleic acid sequences
which
hybridize to any Ga14 UAS nucleic acid sequences set forth herein under at
least
moderately stringent hybridization conditions, and capable of binding a Gal4
UAS
polypeptide.
[0057] The terms "TAL effector recognition sequence", and "TAL
effector
nucleic acid recognition sequence", as may be used interchangeably herein,
refer to
any and all nucleic acid sequences capable of binding a TAL effector
polypeptide,
including, for example, SEQ.ID. NO: 18. TAL effector recognition sequence
further
includes any and all nucleic acid sequences which hybridize to any TAL
effector
21
CA 2903933 2018-01-10
nucleic acid recognition sequences set forth herein under at least moderately
stringent hybridization conditions, and capable of binding a TAL effector
polypeptide.
[0058] The terms "substantially pure" and "isolated", as may be used
interchangeably herein describe a compound, cell, subcellular structure,
chemical
compound or pharmaceutical compound, for example, a glial cell, an exosome or
a
polypeptide, which has been separated from other components that naturally
accompany it. Typically, a compound is substantially pure when at least 60%,
more
preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%,
and
most preferably at least 99% of the total material (by volume, by wet or dry
weight,
or by mole percent or mole fraction) in a sample is the compound of interest.
Purity
can be measured by any appropriate method, e.g., in the case of polypeptides,
by
chromatography, gel electrophoresis, or HPLC analysis.
[0059] The terms "reprogramming" and "reprogram", as used herein,
refer to a
process involving the re-differentiation or de-differentiation of a cell
having the
morphological and functional characteristics representative of a certain class
of cells,
into a cell having known morphological and functional characteristics of a
different
class of cells. Thus for example astroglial cells may be reprogrammed to
neuronal
cells.
[0060] The term "in vivo", as used herein to describe methods of
producing
exosomes, refers to methods involving the production of exosomes by a cell,
including,
for example, a microglial cell, while such cell is present within a living
animal.
[0061] The term "in vitro" as used herein to describe methods of
making
exosomes refers to methods involving the production of exosomes by a cell,
including
for example a microglial cell, while such cell is not present within a living
animal,
including, without limitation, for example, in a microwell plate, a tube, a
flask, a
beaker, a tank, a reactor and the like, to form the exosomes.
[0062] The term "animal", as used herein, refers to any animal
belonging to the
animal kingdom, including humans.
[0063] The term "transgenic" as used herein refers to an entity, e.g.
a cell or an
animal, having received nucleic acid material wherein such material is
integrated into
22
CA 2903933 2018-01-10
the genome of the entity, and wherein the material is received through other
than
naturally occurring processes or events, e.g. breeding, non-recombinant
bacterial or
viral infection, spontaneous mutation and the like.
[0064] It should be noted that terms of degree such as
"substantially",
"essentially" "about" and "approximately" as used herein mean a reasonable
amount
of deviation of the modified term such that the end result is not
significantly changed.
These terms of degree should be construed as including a deviation of the
modified
term if this deviation would not negate the meaning of the term it modifies.
[0065] As used herein, the wording "and/or" is intended to represent
an
inclusive-or. That is, "X and/or Y" is intended to mean X or Y or both, for
example. As
a further example, "X, Y, and/or Z" is intended to mean X or Y or Z or any
combination
thereof.
General implementation
[0066] As hereinbefore mentioned, the present disclosure relates to
neural
cells. Neural cells, including astrocytes, microglia and neurons, are located
in the
central and peripheral nervous system, and the heretofore known techniques for
the
selective delivery of therapeutics specific to neural cell types are
suboptimal. The
herein provided novel methods permit the specific delivery of protein or
nucleic acid
therapeutics to neural cells, by introducing in vivo nucleic acid sequences
encoding a
polypeptide of interest into microglial cells. The methods of the present
disclosure
are useful inter alio in the reprogramming of astroglial cells to replace
damaged
neuronal cells, such as those lost due to traumatic brain injury or ischemic
stroke.
[0067] Accordingly, the present disclosure provides, in at least one
aspect, in at
least one embodiment, a method of expressing a polypeptide of interest in an
astroglial cell, the method comprising
a) introducing a first and second chimeric nucleic acid sequence
in a
microglial host cell, the first chimeric nucleic acid sequence comprising as
operably linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
23
CA 2903933 2018-01-10
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest;
(b) growing the microglial host cell to produce exosomes;
(c) delivering the exosomes to an astroglial cell; and
(d) expressing the polypeptide of interest in the astroglial cell.
[00681 The present disclosure provides, in at least one aspect,
methods for the
expression of polypeptides in a glial cell, the methods involving introducing
a first
.. and second chimeric nucleic acid sequence into a microglial cell. In
accordance
herewith the first and second chimeric nucleic acid sequence are separate,
unlinked
nucleic acid sequences. In accordance with some embodiments, the first and
second
chimeric nucleic acid sequences are introduced into microglial cells by means
of a
first and second expression vector. The first and second chimeric nucleic acid
sequence may be introduced into the microglial cell together or separately.
[0069] In accordance with the present disclosure, the first chimeric
nucleic
acid sequence comprises as operably linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
24
CA 2903933 2018-01-10
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest;
[0070] Referring now to FIG. 1, shown therein is a schematic overview of an
embodiment of the present disclosure, namely a first and second chimeric
nucleic
acid sequence. Shown at the top panel is the a first chimeric nucleic acid
sequence
comprising as operably linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence.
[0071] Shown at the bottom panel is a second chimeric nucleic acid
sequence
comprising as operably linked components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
[0072] Further shown are regulatory elements included in an expression
vector ensuring expression of the first and second chimeric sequence.
[0073] In accordance herewith, the nucleic acid sequence encoding an
exosomal membrane polypeptide may be any nucleic acid sequence encoding an
exosomal membrane polypeptide, including any polypeptide capable of
integrating
into, or associating with, an exosomal membrane.
[0074] In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is a nucleic acid sequence encoding a LAMP-2B
polypeptide.
In some embodiments, the nucleic acid sequence encoding an exosomal membrane
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 1 or SEQ.ID
NO:2. In
CA 2903933 2018-01-10
some embodiments, the exosomal membrane polypeptide is the polypeptide set
forth
in SEQ.ID NO: 33 or SEQ.ID NO: 34.
[0075] In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is a nucleic acid sequence encoding the LAMP-2A
polypeptide.
In some embodiments, the nucleic acid sequence encoding an exosomal membrane
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 3 or SEQ.ID
NO: 4. In
some embodiments, the exosomal membrane polypeptide is the polypeptide set
forth
in SEQ.ID NO: 35 or SEQ.ID NO: 36.
[0076] In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is a nucleic acid sequence encoding the LAMP-2C
polypeptide.
In some embodiments, the nucleic acid sequence encoding an exosomal membrane
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 5 or SEQ.ID
NO: 6. In
some embodiments, the exosomal membrane polypeptide is the polypeptide set
forth
in SEQ.ID NO: 37 or SEQ.ID NO: 38.
[0077] In some embodiments, the nucleic acid sequence encoding an exosomal
membrane polypeptide is a nucleic acid sequence encoding the LAMP-1
polypeptide.
In some embodiments, the nucleic acid sequence encoding an exosomal membrane
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 7 or SEQ.ID
NO: 8. In
some embodiments, the exosomal membrane polypeptide is the polypeptide set
forth
in SEQ.ID NO: 39 or SEQ.ID NO: 40.
[0078] In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is a nucleic acid sequence encoding the Limp-2/SCARB2
polypeptide. In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 9 or
SEQ.ID NO: 10. In some embodiments, the exosomal membrane polypeptide is the
polypeptide set forth in SEQ.ID NO: 41 or SEQ.ID NO: 42.
[0079] In some embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide is a nucleic acid sequence encoding the Flotillin-1
polypeptide.
In some embodiments, the nucleic acid sequence encoding an exosomal membrane
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 11 or SEQ.ID
NO: 12.
26
CA 2903933 2018-01-10
In some embodiments, the exosomal membrane polypeptide is the polypeptide set
forth in SEQ.ID NO: 43 or SEQ.ID NO: 44.
[0080] In other embodiments, the nucleic acid sequence encoding an
exosomal
membrane polypeptide may be a nucleic acid sequence encoding a suitable
substitute
for the LAMP-2B polypeptide, LAMP-2A polypeptide, LAMP-2C polypeptide, LAMP-
1polypeptide, Limp-2/SCARB2 polypeptide or Flotillin-1 polypeptide.
[0081] In accordance herewith the nucleic acid sequence encoding a
neural
cell targeting polypeptide may be any nucleic acid sequence encoding a neural
cell
targeting polypeptide, including any polypeptide capable of being directed to
or
targeted to a neural cell. The neural cell targeting polypeptide may be
targeted to any
neural cell.
[0082] In some embodiments, the nucleic acid sequence encoding a
neural cell
targeting polypeptide is a nucleic acid sequence encoding a Rabies Virus
Glycoprotein
(RVG). In some embodiments, the nucleic acid sequence encoding a neural cell
targeting polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 13.
In some
embodiments, the neural cell targeting polypeptide is the polypeptide set
forth in
SEQ.1D NO: 45.
[0083] In some embodiments, the nucleic acid sequence encoding a
neural cell
targeting polypeptide is a nucleic acid sequence encoding a T7/transferrin
receptor
binding and cell permeating polypeptide. In some embodiments, the nucleic acid
sequence encoding a neural cell targeting polypeptide is the nucleic acid
sequence set
forth in SEQ.ID NO: 14. In some embodiments, the neural cell targeting
polypeptide is
the polypeptide set forth in SEQ.ID NO: 46.
[0084] In other embodiments, the nucleic acid sequence encoding a
neural cell
targeting polypeptide may be a nucleic acid sequence encoding a suitable
substitute
for the RVG polypeptide or the T7/transferrin receptor binding and cell
permeating
polypeptide.
[0085] In accordance herewith the nucleic acid sequence encoding a
nucleic
acid binding polypeptide may be any nucleic acid sequence encoding a
polypeptide
capable of binding a specific nucleic acid binding polypeptide recognition
sequence.
27
CA 2903933 2018-01-10
[0086] In some embodiments, the nucleic acid sequence encoding a
nucleic
acid binding polypeptide is a nucleic acid sequence encoding a Gal4
polypeptide. In
some embodiments, the nucleic acid sequence encoding a nucleic acid binding
polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 15. In some
embodiments, the nucleic acid binding polypeptide is the polypeptide set forth
in
SEQ.ID NO: 47.
[0087] In some embodiments, the nucleic acid sequence encoding a
nucleic
acid binding polypeptide is a nucleic acid sequence encoding a synthetic TAL
effector
polypeptide. In some embodiments, the nucleic acid sequence encoding a nucleic
acid
binding polypeptide is the nucleic acid sequence set forth in SEQ.ID NO: 16.
In some
embodiments, the nucleic acid binding polypeptide is the polypeptide set forth
in
SEQ.ID NO: 48.
[0088] In other embodiments, the nucleic acid sequence encoding a
nucleic
acid binding polypeptide may be a nucleic acid sequence encoding a suitable
substitute for the Gal4 polypeptide or a nucleic acid sequence encoding a
synthetic
TAL effector polypeptide.
[0089] In accordance herewith, the nucleic acid sequence comprising a
nucleic
acid binding polypeptide recognition sequence may be any nucleic acid sequence
comprising a nucleic acid binding polypeptide recognition sequence.
[0090] In some embodiments, the nucleic acid sequence comprising a nucleic
acid binding polypeptide recognition sequence is a nucleic acid sequence
comprising
the Gal4 Upstream Activator Sequence. In some embodiments, the nucleic acid
sequence comprising a nucleic acid binding polypeptide recognition sequence is
the
nucleic acid sequence set forth in SEQ.ID NO: 17.
[0091] In some embodiments, the nucleic acid sequence comprising a nucleic
acid binding polypeptide recognition sequence is a nucleic acid sequence
comprising
the synthetic TAL effector polypeptide recognition sequence. In some
embodiments,
the nucleic acid sequence comprising a nucleic acid binding polypeptide
recognition
sequence is the nucleic acid sequence set forth in SEQ.ID NO: 18.
28
CA 2903933 2018-01-10
[0092] In other
embodiments, the nucleic acid sequence encoding a nucleic
acid binding polypeptide recognition sequence may be a nucleic acid sequence
encoding a suitable substitute for the Gal4 Upstream Activator Sequence, or
the TAL
effector polypeptide recognition sequence.
[0093] In accordance
herewith the nucleic acid sequence encoding a
polypeptide of interest may be any nucleic acid sequence encoding a
polypeptide of
interest. In some embodiments the polypeptide of interest is a polypeptide
capable of
reprogramming an astroglial cell.
[0094] In some
embodiments, the nucleic acid sequence encoding a
polypeptide of interest is a nucleic acid sequence encoding a NeuroD1
polypeptide. In
some embodiments, the nucleic acid sequence encoding a polypeptide of interest
is
the nucleic acid sequence set forth in SEQ.ID NO: 19 or SEQ.ID NO: 20. In some
embodiments, the polypeptide of interest is the polypeptide set forth in
SEQ.ID NO:
49 or SEQ.ID NO: 50.
[0095] In some
embodiments, the nucleic acid sequence encoding a
polypeptide of interest is a nucleic acid sequence encoding a Sox2
polypeptide. In
some embodiments, the nucleic acid sequence encoding a polypeptide of interest
is
the nucleic acid sequence set forth in SEQ.ID NO: 21 or SEQ.ID NO: 22. In some
embodiments, the polypeptide of interest is the polypeptide set forth in
SEQ.ID NO:
51 or SEQ.ID NO: 52.
[0096] In other
embodiments, the nucleic acid sequence encoding a
polypeptide of interest may be a nucleic acid sequence encoding a suitable
substitute
for NeuroD1 polypeptide, or Sox2 polypeptide.
[0097] In other
embodiments, the nucleic acid sequence encoding a
polypeptide of interest is a nucleic acid sequence encoding a fluorescent
polypeptide.
Such polypeptide may be used for research purposes. In some embodiments, the
nucleic acid sequence of interest is a nucleic acid sequence encoding a green
fluorescent polypeptide or a variant thereof. In some embodiments, the nucleic
acid
sequence encoding a polypeptide of interest is a nucleic acid sequence
encoding
enhanced green fluorescent protein (eGFP). In some embodiments, the nucleic
acid
29
CA 2903933 2018-01-10
sequence encoding a polypeptide of interest is the nucleic acid sequence set
forth in
SEQ.ID NO: 23. In some embodiments, the polypeptide of interest is the
polypeptide
set forth in SEQ.ID NO: 53.
[0098] The first chimeric nucleic acid sequence may, in accordance
with some
embodiments hereof, further additionally comprise one or more of the following
nucleic acid sequences:
(iv) a nucleic acid sequence encoding a cleavable polypeptide; and
(v) a nucleic acid sequence encoding a polypeptide providing a signal for
nuclear localization in the target cell.
[0099] In accordance herewith the nucleic acid sequence encoding a
cleavable
polypeptide linker may be any nucleic acid sequence encoding a cleavable
polypeptide linker.
[00100] In some embodiments, the nucleic acid sequence encoding a
cleavable
polypeptide linker is capable of undergoing autocatalytic cleavage. In some
embodiments, the autocatalytic polypeptide linker is an intein that is cleaved
in
reducing environments including, but not limited to, the Ssp DnaE, Npu DnaE,
or Prp8
inteins. In some embodiments, these inteins have been modified to include a
disulfide
bond between the N-terminal extein and the intein. In some embodiments, the
nucleic
acid sequence encoding an autocatalytic intein-based polypeptide linker is the
nucleic
acid sequence set forth in SEQ.ID NO: 24 or SEQ.ID NO: 25 or SEQ.ID NO: 26. In
some
embodiments, the autocatalytic intein-based polypeptide linker is the
polypeptide set
forth in SEQ.ID NO: 54 or SEQ.ID NO: 55 or SEQ.ID NO: 56.
[00101] In other embodiments, the nucleic acid sequence encoding a
cleavable
polypeptide linker is protease-cleavable. In some embodiments, this protease-
.. cleavable linker is cleavable specifically by Furin. In some embodiments,
the nucleic
acid sequence encoding a protease-cleavable linker is the nucleic acid
sequence set
forth in SEQID NO: 27. In some embodiments, the protease-cleavable linker is
the
polypeptide set forth in SEQ.ID NO: 57.
[00102] In accordance herewith, the nucleic acid sequence encoding a
polypeptide providing a signal for nuclear localization in the target cell may
be any
CA 2903933 2018-01-10
nucleic acid sequence encoding a polypeptide providing a signal for nuclear
localization in the target cell.
[00103] In some embodiments, the nucleic acid sequence encoding a
polypeptide providing a signal for nuclear localization in the target cell is
the nucleic
acid sequence encoding the simian virus 40 (SV40) nuclear localization signal.
In
some embodiments, the nucleic acid sequence encoding a polypeptide providing a
signal for nuclear localization in the target cell is the nucleic acid
sequence set forth in -
SEQ.ID NO: 28. In some embodiments, the polypeptide providing a signal for
nuclear
localization in the target cell is the polypeptide set forth in SEQ.ID NO: 58.
[00104] The second chimeric nucleic acid sequence may, in accordance with
some embodiments hereof, further additionally comprise one or more of the
following nucleic acid sequences:
(iii) a nucleic acid sequence encoding a regulatory element, such as a
promoter, capable of expressing a polypeptide of interest in a astroglial
cell;
and
(iv) a nucleic acid sequence providing a selectable or screenable marker.
[00105] In some embodiments, the nucleic acid sequence encoding a
regulatory
element capable of expressing a polypeptide of interest in an astroglial cell
is an
astrocyte-specific promoter, such as a glial fibrillary acidic protein (GFAP)
promoter
or a derivative thereof. In some embodiments, the nucleic acid sequence
encoding a
regulatory element capable of expressing a polypeptide of interest in an
astroglial cell
is the nucleic acid sequence set forth in SEQ.ID NO: 29 or SEQ.ID NO: 30. In
some
embodiments, the nucleic acid sequence encoding a regulatory element capable
of
expressing a polypeptide of interest in an astroglial cell is the synthetic
nucleic acid
sequence set forth in SEQ.ID NO: 31.
[00106] In accordance herewith the nucleic acid sequence providing a
selectable or screenable marker may be any nucleic acid sequence providing a
selectable or screenable marker, permitting screening and selection of
prokaryotic or
eukaryotic cells comprising the marker. This marker enables for the selection
of
prokaryotic cells which contain DNA vectors useful in the generation of the
above
31
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described chimeric nucleotide sequences. In some embodiments, the nucleic acid
providing a selectable or screenable marker encodes a polypeptide. In other
embodiments, the selectable or screenable marker provides for a
polynucleotide, for
example an RNA polynucleotide.
[00107] In some embodiments, the nucleic acid sequence providing a
selectable
or screenable marker encodes an antibiotic resistance marker. In some
embodiments,
the nucleic acid sequence providing a selectable or screenable marker encodes
an
RNA polynucleotide capable of preventing expression of an otherwise lethal
polypeptide. Such RNA polynucleotides may prevent the expression of lethal
.. polypeptides via hybridization with the ribosome binding site of the lethal
peptide
coding sequence. One example of such RNA polynucleotides is the RNA-OUT
polynucleotide from the Escherichia coil insertion sequence IS10 and
engineered
variants thereof (Mutalik et al., 2012). Such a lethal polypeptide may be
encoded by a
nucleic acid sequence separately introduced into host prokaryotic cells, and
its
expression may be controlled by an inducible promoter. In some embodiments,
the
nucleic acid sequence encoding a lethal polypeptide encodes the cytotoxic
protein
known as "control of cell death B" or CcdB. RNA polynucleotides capable of
preventing expression of an otherwise lethal polypeptide, in accordance
herewith are
deemed preferred as a selectable marker, since they avoid the use of
antibiotic
resistance markers and are typically smaller in size. The latter is deemed
beneficial in
view of the limited size and space available for exosomes to accept nucleic
acid
sequences.
[00108] In some embodiments the lethal polypeptide is encoded by the
nucleic
acid sequence set forth in SEQ. ID NO: 32.
[00109] As hereinbefore mentioned, the first and second chimeric nucleic
acid
sequence may be introduced into microglial cells by means of an expression
vector.
Accordingly, the present disclosure further comprises:
a first recombinant expression vector comprising as operably linked
components:
32
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(a) one or more nucleic acid sequences capable of controlling expression in
a host glial cell; and
(b) (i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence,
wherein the expression vector is suitable for expression in a host glial cell.
[00110] The present disclosure further comprises:
a second recombinant expression vector comprising as operably linked
components:
(a) one or more nucleic acid sequences capable of controlling expression in
a host glial cell; and
(b) (i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest,
wherein the expression vector is suitable for expression in a host glial cell.
[00111] The term "suitable for expression in a host glial cell" means
that the
recombinant expression vector comprises the first or second chimeric nucleic
acid
sequences of the present disclosure linked to genetic elements required to
achieve
expression in a host glial cell. Genetic elements that may be included in the
expression vector in this regard include a transcriptional termination region,
one or
more nucleic acid sequences encoding marker genes, one or more origins of
replication, enhancer sequences, and the like. The genetic elements are
operably
linked, typically as well be known to those of skill in the art, by linking
e.g. a promoter
in the 5' to 3' direction of transcription to a coding sequence. In preferred
embodiments, the expression vector further comprises genetic elements required
for
the integration of the vector or a portion thereof in the glial host cell's
genome.
33
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[00112] Pursuant to the present disclosure, the expression vector may
further
contain a marker gene. Marker genes that may be used in accordance with the
present disclosure include all genes that allow the distinction of transformed
cells
from non-transformed cells, including all selectable and screenable marker
genes. A
marker gene may be a resistance marker such as an antibiotic resistance marker
against, for example, kanamycin or ampicillin. Screenable markers that may be
employed to identify transformants through visual inspection include, but are
not
limited to, 8-galactosidase, 13-glucuronidase (GUS), red fluorescent protein
(RFP),
cyan fluorescent protein (CFP), and green fluorescent protein (GFP) including
derivatives such as Clover and enhanced GFP (eGFP).
[00113] In accordance herewith, a first and second chimeric nucleic
acid
sequence are introduced in a microglial cell. The introduction of a first and
second
chimeric nucleic acid sequence in a microglial cell may be achieved by
transforming
or transfecting cultured microglial cells with expression vectors comprising
the first
and second chimeric nucleic acid sequences. In accordance herewith, a wide
variety
of suitable microglial cell lines may be selected and used, including, for
example, any
commercially available immortalized microglial cell lines (e.g., the mouse
microglial
cell lines EOC 13.31 (CRL-2468), EOC 2 (CRL-2467), or EOC 20 (CRL-2469) from
the
American Type Culture Collection (ATCC)). In some embodiments, microglial
cells are
isolated directly from samples of animal tissue obtained via biopsy, autopsy,
donation,
or other surgical or medical procedure. In some embodiments, microglial cells
are
derived from other cell types taken from samples of animal tissue obtained via
biopsy,
autopsy, donation, or other surgical or medical procedure. Suitable cell types
for
direct isolation or derivation of microglia include, but are not limited to,
stem cells
(e.g., mesenchymal stem cells), cortical cells, or bone marrow cells. Further
suitable
microglial cells include amoeboid microglial cells, ramified microglial cells
and
reactive microglial cells.
[00114] In some embodiments, microglial cells from a specific animal
species,
e.g. microglial cells originating from mice or humans are used.
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[00115] Microglial cells may be obtained using a variety of techniques
and
methodologies including, but not limited to, subculture from an immortalized
cell line,
density separation, derivation from other cell types, and cell culture
selection. For
example, mouse microglia may be obtained from mixed cortical cell populations
using
.. the "shake off' cell culture selection method described by Schildge et al.
(2013).
Further guidance describing the isolation of microglial cells may be found in
Lee and
Tansey (2013), and Moussaud and Draheim (2010), among others. In some
embodiments, microglial cells from a specific individual, for example an
individual
selected to receive therapeutic treatment using microglial cells in accordance
with
the present disclosure, may be used, for example by deriving microglial cells
from
cultured patient bone marrow cells. Microglial cells may be derived from bone
marrow cells through cell culture selection and media supplementation as
described,
for example, by Hinze and Stolzing (2012).
[00116] Microglial cells may be distinguished from other cell types
based on
adherence, morphology, silver carbonate staining, lectin staining, flow
cytometry,
membrane ion channel expression, protein profiling, and immunoreactivity,
among
other methods. For example, microglial identification may readily be
accomplished
using flow cytometry as it enables differences in antigen expression levels to
be
reliably quantified. Ramified parenchymal microglia have been demonstrated to
possess the phenotype CD11b+, CD4510w, while reactive microglia and peripheral
macrophages exhibit the phenotype CD11b+,CD45high (Ford et al., 1995; Becher &
Antel 1996). CD11b refers to "cluster of differentiation 11b" and belongs to
the
integrin alpha chain family that is used as a marker to distinguish
macrophages. CD45
refers to "cluster of differentiation 45" and is a membrane tyrosine
phosphatase that
is used as a marker to distinguish cells of the hematopoietic lineage from the
endothelial lineage. As another example, microglia may also be detected
immunologically using antibodies raised against a number of macrophage-
specific
antigens (e.g. OX-42, CD68, and CD11b) although they may not readily
distinguish
microglia from other macrophages. Microglial cells may further be
distinguished from
.. other cell types by lack of immunoreactivity. For example, whereas
astrocytes may be
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detected immunologically using antibodies raised against GFAP, microglial
cells will
demonstrate no immunoreactivity with this astrocyte-specific marker.
[00117] Microglial cells may be grown under controlled in vitro
conditions
allowing multiplication of the microglial cells. The exemplary conditions
described
.. herein below demonstrate at least one functional set of culture conditions
useful for
cultivation of microglial cells. It is to be understood, however, that the
optimal plating
and culture conditions can be determined by one of ordinary skill in the art
using only
routine experimentation. Cells can be plated onto the surface of culture
vessels
without attachment factors (e.g. in a microwell plate, a tube, a flask, a
beaker, a tank, a
reactor, and the like). Alternatively, the vessels can be precoated with
natural,
recombinant or synthetic attachment factors or peptide fragments (e.g.,
collagen or
fibronectin, or natural or synthetic fragments thereof). The cell seeding
densities for
each experimental condition can be optimized for the specific culture
conditions
being used. When cell cultures reach at least 90% confluence, they may be
subcultivated at an optimal ratio between 1:2 and 1:4 of confluent cells to
fresh media.
Microglial cells may be cultivated in a humidified cell incubator at about 37
C and the
incubator should contain about 3-10% carbon dioxide in air. Appropriate
culture
media are known in the art and may comprise, for example, a combination of any
number of the following: an N2-medium (i.e. Dulbecco's Modified Eagle's Medium
(DMEM) or DMEM: Nutrient Mixture F-12), L-glutamine, fetal bovine serum (FBS),
sodium bicarbonate, glucose, sodium pyruvate, penicillin/streptomycin,
dexamethasone, ascorbic acid, granulocyte-monocyte colony stimulating factor,
astrocyte-conditioned media, and/or LADMAC-conditioned media. For example, a
preferred media for differentiating a cell population comprising bone marrow
cells
into microglial cells is: 40% DMEM, 10% FBS, 50% astrocyte-conditioned media,
and
20ng/mL granulocyte-monocyte colony stimulating factor. As another example, a
preferred media for culturing immortalized mouse microglia (ATCC EOC 13.31)
is:
70% DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate
and 4.5 g/L glucose, 10% FBS, and 20% LADMAC-conditioned media. Culture medium
pH should be in the range of about 7.0-7.6. Cells in closed or batch culture
should
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CA 2903933 2018-01-10
undergo complete medium exchange (i.e., replacing spent media with fresh
media)
about every 2-3 days, or more or less frequently as required by the specific
cell type.
Further guidance describing growth and cultivation of microglial cells may be
found,
as examples, in Bronstein et al. (2013), Hinze and Stolzing (2012), and
Witting and
Moller (2011).
[00118] Transformation or transfection describes a process by which
exogenous nucleic acids (for example, DNA or RNA) is introduced into a
recipient cell.
Transformation or transfection may occur under natural or artificial
conditions
according to various methods well known in the art, and may rely on any known
method for the insertion of foreign nucleic acid sequences into a prokaryotic
or
eukaryotic host cell. The method for transformation or transfection is
selected based
on the type of host cell being transformed and may include, but is not limited
to,
bacteriophage or viral infection or non-viral delivery. Methods of non-viral
delivery of
nucleic acids include lipofection, nucleofection, microinjection,
electroporation, heat
shock, particle bombardment, biolistics, virosomes, liposomes,
immunoliposomes,
polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions,
and agent-
enhanced uptake of DNA. Lipofection agents are sold commercially (e.g.,
Lipofectamine 3000). Delivery can be to cells (e.g. in vitro or ex vivo
administration)
or target tissues (e.g. in vivo administration). The term "transformed cells"
or
"transfected cells" includes stably transformed or transfected cells in which
the
inserted DNA is capable of replication either as an autonomously replicating
plasmid
or as part of the host chromosome, as well as transiently transformed or
transfected
cells which express the inserted DNA or RNA for limited periods of time.
Further
guidance describing the transformation or transfection of microglial cells may
be
found, as examples, in Kim and Eberwine (2010), Feigner et al. (1987), and
Kingston,
Chen, and Rose (2003).
[00119] In the methods contemplated herein, a host cell may be
transiently or
non-transiently stably transfected with one or more vectors described herein.
In
some embodiments, a cell is transfected as it naturally occurs in a subject
(i.e., in situ).
In some embodiments, a cell that is transfected is taken from a subject (i.e.,
37
CA 2903933 2018-01-10
explanted). In some embodiments, the cell is derived from cells taken from a
subject,
such as a cell line. A cell transfected with one or more vectors described
herein may
be used to establish a new cell line comprising one or more vector-derived
sequences.
In the methods contemplated herein, a cell may be transiently transfected with
the
components of a system as described herein (such as by transient transfection
of one
or more vectors) in order to establish a new cell line comprising cells
containing the
modification but lacking any other exogenous sequence.
[00120] In accordance herewith, microglial cells are grown to produce
exosomes. In accordance with one embodiment, the microglial cells are grown to
produce exosomes in vitro. In accordance with another embodiment the
microglial
cells are grown to produce exosomes in vivo. Thus the present disclosure
further
provides, in another aspect, microglia cells capable of producing exosomes,
wherein
the exosomes comprise:
a chimeric polypeptide encoded by a first chimeric nucleic acid
sequence; and
(II) a second chimeric nucleic acid sequence:
(a) the first chimeric nucleic acid sequence comprising as
operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
(b) the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
38
CA 2903933 2018-01-10
[00121] In
some embodiments, the exosomes are produced in microglia cells in
vitro. In such embodiments, the exosomes may be separated from the microglia
cells
and an isolated exosome preparation may be obtained. Isolation of exosomes
from
microglial cells may be achieved using a variety of methodologies and
techniques
including, but not limited to, ultracentrifugation, precipitation, or affinity
chromatography. Further guidance describing the isolation of exosomes may be
found in El-Andaloussi et al. (2012).
[00122] Upon
separation of the exosomes from the microglial cells, a
substantially pure exosome preparation may be obtained. Accordingly, in
another
aspect the present disclosure further provides:
a preparation comprising substantially pure exosomes comprising:
(I) a chimeric polypeptide encoded by a first chimeric nucleic acid sequence;
and
(II) a second chimeric nucleic acid sequence:
(a) the first
chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
(b) the
second chimeric nucleic acid sequence comprising as operably
linked components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
[00123] In
accordance herewith, the exosomes comprise a chimeric polypeptide
comprising an exosomal membrane polypeptide embedded in the exosomal
membrane linked to a neural targeting polypeptide and a nucleic acid binding
39
CA 2903933 2018-01-10
polypeptide capable of binding a specific nucleic acid binding polypeptide
recognition
sequence. In some embodiments, the neural targeting polypeptide is located N-
terminally relative to the exosomal binding polypeptide. In some embodiments,
the
nucleic acid binding polypeptide capable of binding a specific nucleic acid
binding
polypeptide recognition sequence is located C-terminally from the exosomal
membrane polypeptide.
[00124] In accordance herewith, the second chimeric nucleic acid
sequence is
located within the lumen of the exosome. The second chimeric nucleic acid
sequence
is connected to the polypeptide encoded by the first chimeric nucleic acid
sequence
via the nucleic acid binding polypeptide recognition sequence bound to the
nucleic
acid binding polypeptide capable of binding the recognition sequence.
[00125] Referring now to FIG. 2, shown therein is a schematic
representation of
an embodiment of present disclosure, namely an exosome in an exosome
preparation
comprising a chimeric polypeptide and chimeric nucleic acid sequence,
the chimeric polypeptide comprising as operably linked components:
(i) an exosomal membrane polypeptide;
(ii) a neural cell targeting polypeptide; and
(iii) a nucleic acid binding polypeptide capable of binding a nucleic acid
binding polypeptide recognition sequence; and
the chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
[00126] In some embodiments of the present disclosure, the chimeric
polypeptide additionally comprises:
(iv) a cleavable polypeptide; and
(v) a nucleic acid sequence encoding a polypeptide providing a
signal for
nuclear localization in the target cell.
[00127] A schematic representation of the foregoing embodiment of the
present
disclosure is further shown in FIG. 3.
CA 2903933 2018-01-10
[00128] In some embodiments of the present disclosure, the chimeric
polypeptide comprises:
(i) exosomal membrane polypeptide wherein the exosomal polypeptide
is
a LAMP-2B polypeptide;
(ii) a neural cell targeting polypeptide wherein the neural targeting
polypeptide is an RVG polypeptide;
(iii) a nucleic acid binding polypeptide capable of binding a nucleic acid
binding polypeptide recognition sequence, wherein nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide recognition
sequence is a TAL-effector polypeptide;
(iv) a cleavable polypeptide; and
(v) a polypeptide providing a signal for nuclear localization in the target
cell; and
and the chimeric nucleic acid sequence comprises:
(i) a nucleic acid binding polypeptide recognition sequence, wherein the
nucleic acid binding polypeptide recognition sequence is a TAL-effector
sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest
wherein the
polypeptide of interest is NeuroD1.
[00129] A schematic representation of the foregoing embodiment in
accordance
with the present disclosure is further shown in FIG. 4.
[00130] The exosomes may be formulated to prepare a pharmaceutical
composition comprising exosomes for delivery to an animal in need thereof in a
manner that permits expression of the protein of interest in the astroglial
cells of the
animal.
[00131] In some embodiments, the exosomes are produced in microglia
cells in
vivo. In such embodiments, microglial cells are prepared for delivery to an
animal in
need thereof. Delivery of the prepared microglial cells stimulates the
production in
vivo of exosomes by microglial cells, in vivo contacting of the produced
exosomes with
41
CA 2903933 2018-01-10
neural cells, and expression of the protein of interest in the astroglial
cells of the
animal.
[00132] Thus in another aspect, the present disclosure provides a
transgenic
animal comprising transgenic astroglial cells in which a protein of interest
is
expressed, wherein the transgenic astroglial cells have been obtained by:
introducing a first and second chimeric nucleic acid sequence in a microglial
host cell, the first chimeric nucleic acid sequence comprising as operably
linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest;
(b) growing the microglial host cell to produce exosomes;
(c) delivering the exosomes to an astroglial cell; and
(d) expressing the polypeptide of interest in the astroglial cell.
[00133] In yet another aspect, the present disclosure provides a
transgenic
animal comprising transgenic astroglial cells wherein the transgenic
astroglial cells
comprise a chimeric nucleic acid sequence comprising:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest..
[00134] In yet another aspect, the present disclosure provides a
transgenic
animal comprising transgenic astroglial cells in which a protein of interest
is
42
CA 2903933 2018-01-10
transgenically expressed in the astroglial cells. In some embodiments the
protein of
interest is NeuroDl.
[00135] In
accordance herewith, the astroglial cells in which a protein of
interest is expressed includes the astroglial cells of an animal including
astroglial cells
of a human. The astroglial cells may be any astroglial cells of the central
nervous
system or peripheral nervous system. The astroglial cells in which the protein
of
interest is expressed include reactive astrocyte cells, fibrous astroglial
cells,
protoplasmic astroglial cells, and radial astroglial cells. The astroglial
cells, as a result
of the expression of the protein of interest, may reprogram. Thus, for
example, certain
astroglial cells, e.g. reactive astrocyte cells, may as a result of the
expression of a
protein of interest, such as NeuroD1, re-differentiate to form neurons. The
astroglial
cells further include astroglial cells that have formed scar tissue as a
result of a brain
injury. In further embodiments, the astroglial cells are astroglial cells that
have
formed scar tissue as a result of an ischemic stroke or traumatic brain
injury.
[00136] In
embodiments hereof wherein exosomes are delivered as a
pharmaceutical compositions to an animal in need thereof, and in embodiments
hereof wherein microglial cells are delivered as a pharmaceutical composition
to an
animal in need thereof, the animal will receive a nucleic acid sequence
encoding a
protein of interest which is incorporated into astroglial cells of the animal,
and
expressed therein. Accordingly, the genetic constitution of the astroglial
cell is
modulated in such a manner that at least one protein that is not naturally
produced
by the glial cell, or not normally produced at certain levels by the cell, is
produced by
the astroglial cell, or produced at altered levels by the cell.
[00137] As
hereinbefore described, the exosomes and the microglial cells of the
present disclosure obtained in accordance with the present disclosure may be
used to
prepare a pharmaceutical composition for use as a pharmaceutical drug,
therapeutic
agent or medicinal agent. Thus the present disclosure further includes
pharmaceutical and veterinary compositions comprising the exosomes or the
microglial cells prepared in accordance with the methods of the present
disclosure.
Pharmaceutical or veterinary drug preparations comprising the exosomes and
43
CA 2903933 2018-01-10
microglial cells in accordance with the present disclosure in some embodiments
further comprise vehicles, excipients, diluents, and auxiliary substances,
such as
wetting or emulsifying agents, pH buffering substances and the like. These
vehicles,
excipients and auxiliary substances are generally pharmaceutically acceptable
agents
.. that may be administered without undue toxicity. Pharmaceutically
acceptable
excipients include, but are not limited to, liquids such as water, saline,
polyethylene
glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable
salts can
also be included therein, for example, mineral acid salts such as
hydrochlorides,
phosphates, sulfates, and the like; and the salts of organic acids such as
acetates,
propionates, benzoates, and the like. It is also preferred, although not
required, that
the preparation will contain a pharmaceutically acceptable excipient that
serves as a
stabilizer. Examples of suitable carriers that also act as stabilizers for
peptides
include, without limitation, pharmaceutical grades of dextrose, sucrose,
lactose,
sorbitol, inositol, dextran, and the like. Other suitable carriers include,
again without
limitation, starch, cellulose, sodium or calcium phosphates, citric acid,
glycine,
polyethylene glycols (PEGs), and combinations thereof. The pharmaceutical
composition may be formulated for intravenous administration and other routes
of
local or systemic administration including, but not limited to, inhalation as
a nasal
spray, rectal compositions such as enemas or suppositories, and direct
injection into
the cerebrospinal fluid, spinal cord, or brain as desired. Dosing may vary and
may be
optimized using routine experimentation. Powder formulations for exosome
delivery
can be prepared by conventional methods for inhalation into the lungs of the
subject
to be treated or for intranasal administration into the nose and sinus
cavities of a
subject to be treated. For example, the compositions can be delivered in the
form of
an aerosol spray presentation from pressurized packs or a nebulizer, with the
use of a
suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Capsules and
cartridges of, for example, gelatin for use in an inhaler or insufflator may
be
formulated containing a powder mix of the desired compound and a suitable
powder
base such as lactose or starch. Exosomes can also be formulated as rectal
44
CA 2903933 2018-01-10
compositions, such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other glycerides.
Further, the
exosomal compositions can also be formulated as a depot preparation by
combining
the compositions with suitable polymeric or hydrophobic materials (for example
as
an emulsion in an acceptable oil) or ion exchange resins, or as sparingly
soluble
derivatives, for example, as a sparingly soluble salt. For intravenous
injections, water
soluble versions of the microglial cell or exosome compositions can be
administered
by the drip method or direct injection, whereby a formulation including a
pharmaceutical composition of the present invention and a physiologically-
acceptable excipient is infused. Physiologically-acceptable excipients can
include, for
example, 5% dextrose, 0.9% saline, Ringer's solution, human serum albumin, or
other
suitable excipients.
[00138] The
pharmaceutical compositions of the present disclosure may be
used as a neuro-regenerative therapeutic agent, including as an agent for
reprogramming astroglial cells as a method of replenishing lost or damaged
neuronal
populations. Thus, in yet another aspect, the present disclosure provides a
method for
regenerating neurons, the method comprising:
(a)
introducing a first and second chimeric nucleic acid sequence in
a microglial host cell, the first chimeric nucleic acid sequence
comprising as operably linked components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
CA 2903933 2018-01-10
(ii) a nucleic acid sequence encoding a polypeptide capable of
reprogramming an astroglial cell;
(b) growing the microglial host cell to produce exosomes;
(c) delivering the microglia and/or exosomes to sites of neural damage
including, but not limited to, reactive astroglial cell populations; and
(d) expressing the polypeptide to reprogram an astroglial cell into a
neuronal cell.
[00139] In yet further embodiments, the present disclosure provides
methods
for treating a patient with a pharmaceutical composition comprising exosomes
or
microglial cells in accordance with the present disclosure. Accordingly, the
present
disclosure further provides a method for treating a patient with exosomes or
microglial cells of the present disclosure, said method comprising
administering to
the patient exosomes or microglial cells of the present disclosure, wherein
the
exosomes or microglial cells are administered in an amount sufficient to
ameliorate a
.. medical condition in the patient. In some embodiments, the medical
condition is a
neurodegenerative condition that may be ameliorated by administration of the
exosomes or microglial cells of the present disclosure. In some embodiments,
the
medical condition is traumatic brain injury. In some embodiments, the medical
condition is ischemic stroke.
[00140] The current disclosure further includes a use of exosomes to treat
a
person in need thereof wherein the exosomes comprise:
(I) a chimeric polypeptide encoded by a first chimeric nucleic acid
sequence; and
(II) a second chimeric nucleic acid sequence:
(a) the first chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid sequence encoding an exosomal membrane
polypeptide;
(ii) a nucleic acid sequence encoding a neural cell targeting
polypeptide; and
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CA 2903933 2018-01-10
(iii) a nucleic acid sequence encoding a nucleic acid binding
polypeptide capable of binding a nucleic acid binding polypeptide
recognition sequence; and
(b) the second chimeric nucleic acid sequence comprising as operably linked
components:
(i) a nucleic acid binding polypeptide recognition sequence; and
(ii) a nucleic acid sequence encoding a polypeptide of interest.
[00141] The person treated in accordance herewith may be treated to
ameliorate traumatic brain injury or ischemic stroke, or symptoms associated
therewith.
[00142] The above disclosure generally describes various aspects of
methods
and compositions of the present disclosure. A more complete understanding can
be
obtained by reference to the following specific examples. These examples are
described solely for the purpose of illustration and are not intended to limit
the scope
of the disclosure. Changes in form and substitution of equivalents are
contemplated
as circumstances might suggest or render expedient. Although specific terms
have
been employed herein, such terms are intended in a descriptive sense and not
for
purposes of limitation.
[00143] The following non-limiting examples are illustrative of the
present
invention:
Examples
Example 1 ¨ System overview
100144] In accordance with one aspect, the mode of action of the
herein
disclosed system is that microglia, even when intravenously administered,
innately
migrate to regions of neural damage in the brain. In accordance with the
methodology
of the present disclosure, microglia are modified to include a chimeric
polypeptide
which is capable both of localizing to exosomes through its transmembrane LAMP-
2B
domain and binding a nucleic acid recognition sequence through its nucleic
acid
binding polypeptide domain. During the process of exosome biogenesis, the
nucleic
acid binding domain is initially localized in the cytoplasm, where it has
access to
47
CA 2903933 2018-01-10
cytoplasmic DNA species, including a second transfected chimeric nucleic acid
plasmid vector. The inward budding of the multivesicular body (MVB) membrane
to
form intraluminal vesicles (ILVs), results in the nucleic acid binding
polypeptide
domain localizing in the lumen of ILVs. Bound nucleic acid vectors move in
concert
with the nucleic acid binding polypeptide, also localizing to the ILV lumen.
As ILVs are
released from the exosome-producing cell, as exosomes, the nucleic acid
binding
polypeptide and bound nucleic acid vector remain in the vesicle lumen,
ultimately
resulting in their presence in the lumen of exosomes (FIG. 2- FIG. 4). After
being
released from an exosome-producing cell, such as a microglial cell, the
exosomes may
be delivered to a target cell (i.e., recipient cell) where the exosomes are
taken up and
the cargo is delivered to the cytoplasm of the target cell. To enhance
delivery of the
nucleic acid vector to the target cell cytoplasm and, more specifically, its
nucleus, the
nucleic acid binding domain may be operably linked to a NLS and fused to LAMP-
2B
via a cleavable linker. In some embodiments, the cleavable linker is intein-
based and
autocatalytic in the reducing environment of the exosome, allowing release of
the
nucleic acid vector from the exosomal membrane. The bound NLS and nucleic acid
binding polypeptide then targets the nucleic acid vector for expression in the
nucleus
of the target cell. In some embodiments, as a therapy for ischemic stroke or
traumatic
brain injury, expression of the nucleic acid vector encoding NeuroD1 (under
the
regulation of an astrocyte-specific promoter such as gfaABC1D) by recipient
reactive
astrocytes may provide a method of replenishing damaged or dead neuronal
cells.
[00145] This system may be implemented as follows: (a) a chimeric
polypeptide
comprising a neural cell targeting polypeptide (e.g., RVG) on the external
terminus
and a nucleic acid binding protein (e.g., a synthetic TAL effector) on the
internal
terminus of an exosomal membrane protein (e.g., LAMP-2B) is designed (FIG. 1);
(b)
a chimeric nucleic acid encoding the polypeptide of interest is designed (for
example,
NeuroD1 operably linked to a gfaABC1D promoter; FIG. 1); (c) DNA sequences are
generated (by traditional recombinant DNA assembly techniques such as
restriction
cloning and/or DNA synthesis) and inserted into a suitable expression vector
containing the nucleic acid binding protein recognition sequence (e.g., a
plasmid
48
CA 2903933 2018-01-10
vector containing a TAL effector recognition sequence); (e) the chimeric
vectors are
transfected into a suitable cell line for producing exosomes (e.g.,
microglia); (f) the
microglia or their exosomes are harvested; and (g) the microglia or their
exosomes
are administered (e.g., by intravenous injection).
Example 2 - Exosomal expression and localization of neural cell targeting
protein construct
[00146] To specifically target exosomes to neural cells, the N-
terminus of the
mouse exosomal membrane protein, LAMP-2B, was modified to include a Rabies
Virus Glycoprotein (RVG) as previously described (Alvarez-Erviti et al.,
2011). To
verify appropriate expression and exosomal membrane localization of the
modified
LAMP-2B protein and to validate the viability of C-terminal modifications,
Clover (a
green fluorescent protein variant) was operably linked to the C-terminus.
[00147] Immortalized mouse microglia (EOC 13.31) and human embryonic
kidney (HEK-293) cell cultures were lipofected (using Lipofectamine 3000 as
per the
manufacturer's protocols) with the chimeric pcDNA3.0-RVG-LAMP-2B-NLS-Clover
plasmid. Unlipofected cultures were used as controls. One day prior to exosome
isolation, the microglia culture medium was replaced with fresh medium
centrifuged
at 125,000 x g for 70 minutes to remove any pre-existing nanovesicles. During
exosome collection, the culture medium was centrifuged at 300 x g for 10
minutes to
remove non-adherent cells, filtered (200nm), and centrifuged at 125,000 x g
for 70
minutes. The resulting pellet was resuspended in either 1X PBS (for
fluorescence
detection) or 2% PFA (for transmission electron microscopy; TEM) and stored at
4 C.
The presence of exosomes was verified with TEM and samples were quantified and
standardized with a Nanodrop 2000 (A280). Half of each of the exosome samples
were lysed with sodium deoxycholate and fluorescence was measured using a
QuantaMaster 60 fluorescence spectrofluorometer. The adherent microglia and
HEK-
293 cell cultures were rinsed with 1X phosphate-buffered saline (PBS), fixed
with 4%
paraformaldehyde (PFA) for 20 minutes, coverslipped with Vectashield plus
DAPI,
and immediately imaged using an Olympus FluoView FV1000 confocal laser
scanning microscope.
49
CA 2903933 2018-05-17
[00148] Unlike
unlipofected controls, cultured microglia and HEK-293 cells
lipofected with the chimeric RVG-LAMP-2B-Clover plasmid demonstrated
cytoplasmic green fluorescence (FIG. 5). Upon lysis, exosomes isolated from
transfected microglia and HEK-293 culture media exhibited elevated
fluorescence
(approximately three times that of both control and unlysed exosome samples)
with
an emission peak at approximately 515nm (FIG. 6).
Example 3 - Astrocytic expression of NeuroD1 results in a neuronal phenotype
, [00149] An immortalized
astrocyte (C8-D30) cell culture was lipofected (using
Lipofectamine 3000 as per the manufacturer's protocols) with an experimental
version of the second chimeric nucleic acid sequence comprising NeuroD1
operably
linked to a CMV promoter and an IRES-GFP element. Briefly, adherent astrocytes
were rinsed with 1X PBS and fixed with 4% PFA for 20 minutes. Fixed cells were
rinsed twice with lx PBS, blocked with 1X PBS plus 0.3% Triton-X and 3% goat
serum for 45 minutes, and incubated with 1:100 anti-GFAP for 2 hours at room
temperature. Cells were rinsed twice with 1X PBS and primary antibodies were
labeled with 1:200 anti-rabbit-Alexa Fluor 594 in blocking buffer for 1 hour
at room
temperature. Slides were rinsed twice with 1X PBS, coverslipped with
Vectashield
plus DAPI, and immediately imaged using an Olympus FluoView FV1000 confocal
laser scanning microscope.
[00150] GFP expression
was evident in successfully transfected cells, which do
not exhibit co-staining with GFAP (a reactive astrocyte marker; FIG. 7).
[00151] While the
present disclosure has been described with reference to what
are presently considered to be the preferred examples, it is to be understood
that the
disclosure is not limited to the disclosed examples. To the contrary, the
disclosure is
intended to cover various modifications and equivalent arrangements included
within the spirit and scope of the appended claims.
CA 2903933 2018-05-17
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