# Patent

## Title
Methods and compositions for the packaging of nucleic acids into microglial exosomes for the targeted expression of polypeptides in neural cells

## Abstract

Methods for expressing a polypeptide of interest in an astroglial cell. The methods permit the localization and transport of nucleic acids in microglial exosomes, the reprogramming of astroglial cells to neuronal cells, and the treatment of ischemic stroke or traumatic brain injury patients.

## Background
RELATED APPLICATION
This application claims benefit of 35. U.S.C. 119 based on priority of U.S. Provisional Patent Application No. 62/217,137 filed on Sep. 11, 2015, which is incorporated herein by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
A computer readable form of the Sequence Listing “21806-P49086US01_SequenceListing.txt” (122,880 bytes), submitted via EFS-WEB and created on Sep. 7, 2016 and amended on Nov. 10, 2016, is herein incorporated by reference.
FIELD OF THE DISCLOSURE
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
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.
It is estimated that traumatic brain injury (TBI) 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 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
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.
In one aspect, the present disclosure relates to neural cells, including microglial cells, neuronal cells, and astroglial cells.
In another aspect, the present disclosure relates to methods for the expression of polypeptides of interest in neural cells.
In another aspect, the present disclosure relates to methods for the reprogramming of astroglial cells into neuronal cells.
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;
        
        
        (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.
    
    


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 a human or an animal in need thereof.
In some embodiments, the exosomes are produced in microglia cells in vivo. In such embodiments, the microglia cells may be provided to a human or an animal in need thereof.
In some embodiments, the polypeptide of interest is a protein capable of reprogramming astroglial cells into neuronal cells.
In some embodiments, the polypeptide of interest is NeuroD1.
In another aspect, the present disclosure provides microglia cells capable of producing exosomes, wherein the exosomes comprise:

    
    
        (I) a 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
        
        
        (II) a 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 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
            (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 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
        
        
        (II) a 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 microsomes 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.
        
        
    
    


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:
        
            (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.
    
    


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.
        
        
    
    


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.
    
    


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.
    
    


In yet another aspect, the present disclosure provides a use of exosomes to treat a person in need thereof wherein the exosomes comprise:

    
    
        (I) a 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
        
        
    
    


In yet another aspect the present disclosure provides, a use of exosomes to treat an animal in need thereof wherein the exosomes comprise:

    
    
        (I) a 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
        
        
        (II) a 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.
        
        
    
    


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 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
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.
FIG. 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.
FIG. 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.
FIG. 3 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 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.
FIG. 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 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.
FIG. 5 depicts confocal microscopy images of both control and lipofected human embryonic kidney (HEK-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.
FIG. 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.
FIG. 7 depicts the quantitative real-time polymerase chain reaction (qRT-PCR) results on DNA samples isolated from exosomes from cell cultures lipofected without plasmid (Control), with both pcDNA3.0-RVG-LAMP-2B-NLS-TALE and gfaABC1D-NeuroD1 with the TAL effector binding site (BS), or with both pcDNA3.0-RVG-LAMP-2B-NLS-TALE and gfaABC1D-NeuroD1 without the TAL effector binding site (No BS). U6 small nuclear RNA cDNA was used as an internal control. (A) Relative quantity (delta quantification cycle; ΔCq) of gfaABC1D-NeuroD1 plasmid and U6 cDNA evident in each sample. (B) Relative normalized expression (delta delta quantification cycle; ΔΔCq) of gfaABC1D-NeuroD1 corrected for U6 cDNA in each sample.
FIG. 8 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.



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
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.
All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Definitions
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.
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).
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 & Miller, 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.
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.
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, LIMP-2/SCARB2 polypeptide, Flotillin-1 polypeptide, and any other protein capable of association with or integration within exosomal membranes.
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).
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, 83, 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).
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 base pairs, or at least 50 base pairs in length. The term nucleic acid binding polypeptide recognition sequence includes, for example, the Gal4 Upstream Activator Sequence (specific to the Gal4 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).
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.
“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.
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.
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 coli, 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).
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.
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., O-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., 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



Gln
Asn, Gln, His



Glu
Asp, Gln, His



Gly
Ala



His
Asn, Arg, Gln, Glu



Ile
Leu, Val



Leu
Ile, Val



Lys
Arg, Gln, 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










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 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.
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 (Log 10 [Na+])+0.41(% (G+C)−600/l), 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 5× sodium chloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at Tm (based on the above equation) −5° C., followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC 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.
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 polypeptide 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.
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.
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: 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.
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.
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.
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.
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, 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.
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.
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.
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 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.
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.
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 Gal4 UAS nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions, and capable of binding a Gal4 UAS polypeptide.
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 nucleic acid recognition sequences set forth herein under at least moderately stringent hybridization conditions, and capable of binding a TAL effector polypeptide.
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.
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.
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 human or animal.
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 human or 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.
The term “animal”, as used herein, refers to any non-human animal belonging to the animal kingdom.
The term “transgenic” as used herein refers to an entity, e.g. a cell or an animal or human, having received nucleic acid material wherein such material is integrated into 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.
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.
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.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication.
General Implementation
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 alia in the reprogramming of astroglial cells to replace damaged neuronal cells, such as those lost due to traumatic brain injury or ischemic stroke.
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;
            (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.
    
    


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.
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
         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;
    
    


Referring now to FIG. 1, shown therein is a schematic overview of an embodiment of the present disclosure, namely an embodiment comprising 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.
    
    


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.
    
    


Further shown are regulatory elements included in an expression vector ensuring expression of the first and second chimeric sequence.
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.
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 some embodiments, the exosomal membrane polypeptide is the polypeptide set forth in SEQ. ID NO: 33 or SEQ. ID NO: 34.
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.
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.
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.
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.
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. In some embodiments, the exosomal membrane polypeptide is the polypeptide set forth in SEQ. ID NO: 43 or SEQ. ID NO: 44.
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.
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.
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. ID NO: 45.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 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.
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.
    
    


In accordance herewith the nucleic acid sequence encoding a cleavable polypeptide linker may be any nucleic acid sequence encoding a cleavable polypeptide linker.
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.
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 SEQ. ID NO: 27. In some embodiments, the protease-cleavable linker is the polypeptide set forth in SEQ. ID NO: 57.
In accordance herewith, the nucleic acid sequence encoding a polypeptide providing a signal for nuclear localization in the target cell may be any nucleic acid sequence encoding a polypeptide providing a signal for nuclear localization in the target cell.
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.
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.
    
    


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.
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 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.
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 coli 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.
In some embodiments, the lethal polypeptide is encoded by the nucleic acid sequence set forth in SEQ. ID NO: 32.
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:

    
    
        (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.
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.
        
        
    
    


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 will 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.
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, β-galactosidase, β-glucuronidase (GUS), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and green fluorescent protein (GFP) including derivatives such as Clover and enhanced GFP (eGFP).
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 or human 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 or human 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.
In some embodiments, microglial cells from a human or a specific animal species, e.g. microglial cells originating from mice, are used.
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).
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+, CD45low, 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 detected immunologically using antibodies raised against GFAP, microglial cells will demonstrate no immunoreactivity with this astrocyte-specific marker.
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 20 ng/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 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).
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), Felgner et al. (1987), and Kingston, Chen, and Rose (2003).
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., 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.
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:

    
    
        (I) a 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
        
        
        (II) a 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 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
            (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 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).
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 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
        
        
        (II) a 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.
        
        
    
    


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 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.
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.
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.
        
        
    
    


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.
    
    


A schematic representation of the foregoing embodiment of the present disclosure is further shown in FIG. 3.
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.
    
    


A schematic representation of the foregoing embodiment in accordance with the present disclosure is further shown in FIG. 4.
In one aspect, in some embodiments of the present disclosure, the exosomes are delivered to an astroglial cell. This can be achieved by contacting exosomes with an astroglial cell, for example, by preparing a formulation comprising microglial cells capable of producing exosomes, or by preparing a formulation comprising exosomes, and providing the formulation to an animal or human in need thereof in a manner that allows the exosomes or the microglial cells capable of producing the exosomes, to contact astroglial cells of the human or animal. While microglia innately migrate to sites of neural damage, target cell specificity for exosomes appears to be dictated solely by a combination of antigen and major histocompatibility complex (MHC) class I and II molecules, the expression of which is dependent on the parent cell (McKelvey et al., 2015). Thus, inclusion of a neural cell targeting polypeptide (such as RVG) and/or administration of exosomes derived from microglial cells may improve target cell specificity in some embodiments. Expression of the polypeptide of interest in the target astrocyte cells requires binding of the exosomes to the plasma membrane of the target cell and internalization of the chimeric nucleic acid vector, which is then shuttled to the nucleus of the target cell via the included nuclear localization sequence.
The exosomes may be formulated to prepare a pharmaceutical or veterinary composition comprising exosomes for delivery to an animal or human in need thereof in a manner that permits expression of the protein of interest in the astroglial cells of the animal or human.
In some embodiments, the exosomes are produced in microglia cells in vivo. In such embodiments, microglial cells are prepared for delivery to a human or 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 neural cells, and expression of the protein of interest in the astroglial cells of the human or animal.
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.
    
    


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.
    
    


In yet another aspect, the present disclosure provides a transgenic animal comprising transgenic astroglial cells in which a protein of interest is transgenically expressed in the astroglial cells. In some embodiments the protein of interest is NeuroD1.
In accordance herewith, the astroglial cells in which a protein of interest is expressed includes the astroglial cells of a human or an animal. 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.
In embodiments hereof wherein exosomes are delivered as a pharmaceutical or veterinary compositions to a human or an animal in need thereof, and in embodiments hereof wherein microglial cells are delivered as a pharmaceutical or veterinary composition to an animal or human in need thereof, the animal or human will receive a nucleic acid sequence encoding a protein of interest which is incorporated into astroglial cells of the human or 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 astroglial 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.
Veterinary compositions include compositions for the treatment of any animal including, without limitation, compositions for the treatment of a cow, horse, pig, chicken, or fish and further including, without limitation, compositions for the treatment of companion animals such as a dog or a cat.
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, or as a veterinary composition for use as a veterinary drug, therapeutic 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 for delivery to an astroglial cell. Pharmaceutical or veterinary drug preparations comprising the exosomes and 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 or veterinary acceptable agents that may be administered without undue toxicity. Pharmaceutically and veterinary acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically and veterinary 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 or veterinary 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 or veterinary 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 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.
The pharmaceutical and veterinary 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
        (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.
    
    


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.
The current disclosure further includes a use of exosomes to treat a person in need thereof wherein the exosomes comprise:

    
    
        (I) a 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
        
        
        (II) a 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
        
        
    
    


The person treated in accordance herewith may be treated to ameliorate traumatic brain injury or ischemic stroke, or symptoms associated therewith.
In yet further embodiments, the present disclosure provides methods for treating an animal with a veterinary composition comprising exosomes or microglial cells in accordance with the present disclosure. Accordingly, the present disclosure further provides a method for treating an animal with exosomes or microglial cells of the present disclosure, said method comprising administering to the animal exosomes or microglial cells of the present disclosure, wherein the exosomes or microglial cells are administered in an amount sufficient to ameliorate a health condition in the animal. In some embodiments, the health 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 health condition is traumatic brain injury. In some embodiments, the health condition is ischemic stroke.
The current disclosure further includes a use of exosomes to treat an animal in need thereof wherein the exosomes comprise:

    
    
        (I) a 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
        
        
        (II) a 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.
        
        
    
    


The animal treated in accordance herewith may be treated to ameliorate traumatic brain injury or ischemic stroke, or symptoms associated therewith.
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.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1—System Overview
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 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.
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 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
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.
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 125,000×9 for 70 minutes to remove any pre-existing nanovesicles. During exosome collection, the culture medium was centrifuged at 300×g for 10 minutes to remove non-adherent cells, filtered (200 nm), and centrifuged at 125,000×g for 70 minutes. The resulting pellet was resuspended in either 1×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 1× 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.
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 515 nm (FIG. 6).
Example 3—Efficiency of Exosomal Packaging of Reprogramming Plasmid is Improved by Neural Cell Targeting Protein Construct
An immortalized HEK-293 cell culture was co-lipofected (using Lipofectamine 3000 as per the manufacturer's protocols) with experimental versions of both the first chimeric nucleic acid sequence (encoding the neural cell targeting protein; pcDNA3.0-RVG-LAMP-2B-NLS-TALE) and one of two iterations of the second chimeric nucleic acid sequences (the “reprogramming” plasmid; gfaABC1D-NeuroD1). The first chimeric nucleic acid sequence was comprised by the following operably linked components: a LAMP-2B exosomal membrane polypeptide; a cell targeting RVG polypeptide; a TAL-effector nucleic acid binding polypeptide; and a polypeptide providing a signal for nuclear localization in the target cell. Two versions of the second chimeric nucleic acid sequence were used. The first was comprised by the following operably linked components: a nucleic acid sequence encoding NeuroD1 regulated by a synthetic gfaABC1D promoter; and a TAL-effector nucleic acid binding polypeptide recognition sequence (specific to the TAL-effector polypeptide in the first chimeric nucleic acid sequence). The second version included only the nucleic acid sequence encoding NeuroD1 regulated by a synthetic gfaABC1D promoter (no TAL-effector nucleic acid binding polypeptide recognition sequence was included). Vehicle control cells were lipofected without the addition of either vector.
Co-lipofected cells were incubated in serum-free media for two days. Media was then collected and exosomes were isolated using a Qiagen exoEasy Maxi Kit (as per the manufacturer's protocols). DNA and RNA were extracted from the exosome preparations using a Qiagen QIAamp DNA Mini Kit (as per the manufacturer's protocols). DNA was then PCR amplified with either primers specific for gfaABC1D-NeuroD1 (F: 5′-TCATAAAGCCCTCGCATCCC-3′; SEQ. ID NO: 59; R: 5′-AGACCCCTGAGTTCCTGTCA-3′; SEQ. ID NO: 60) or primers specific for the pcDNA3.0-LAMP-2B-NLS-TALE vector (F: 5′-GGAGGTGGCGGATCAC-3′; SEQ. ID NO: 61; R: 5′-TAGAAGGCACAGTCGAGG-3′; SEQ. ID NO: 62).
RNA (in solution with the DNA) isolated from each of the exosome samples was reverse transcribed using a SuperScript III First-Strand Synthesis System (Invitrogen; as per the manufacturer's protocols) and a U6 small nuclear RNA-specific primer (5′-AAAATATGGAACGCTTCACGAATTTG-3′; SEQ. ID NO: 63). Quantitative real-time PCR (qRT-PCR) experiments were performed using a SsoAdvanced Universal SYBR Green Supermix (Bio-Rad; as per manufacturer's protocols) with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). U6 cDNA was used as the internal control. Primers specific to U6 (F: 5′-CTCGCTTCGGCAGCACATATACT-3′; SEQ. ID NO: 64 R: 5′-ACGCTTCACGAATTTGCGTGTC-3′; SEQ. ID NO: 65) and gfaABC1D-NeuroD1 (F: 5′-TCATAAAGCCCTCGCATCCC-3′; SEQ. ID NO: 59; R: 5′-AGACCCCTGAGTTCCTGTCA-3′; SEQ. ID NO: 60) were used.
Gel electrophoresis of the PCR products confirmed the presence of the gfaABC1D-NeuroD1 plasmid in both samples (with or without inclusion of the TAL effector binding site) based on the presence of an expected 132 base pair band. A band indicating the presence of pcDNA3.0-RVG-LAMP-2B-NLS-TALE was not evident for either DNA preparation.
When normalized to U6 small nuclear RNA expression, qRT-PCR revealed a three-fold increase in gfaABC1D-NeuroD1 plasmid isolated from exosome samples when the lipofected plasmid contained the TAL effector binding site relative to those without the TAL effector binding site, suggesting an increase in exosomal packaging efficiency of the gfaABC1D-NeuroD1 plasmid when anchored to the TAL effector nucleic acid binding protein (FIG. 7).
Example 4—Astrocytic Expression of NeuroD1 Results in a Neuronal Phenotype
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 1×PBS and fixed with 4% PFA for 20 minutes. Fixed cells were rinsed twice with 1×PBS, blocked with 1×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 1×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 1×PBS, coverslipped with Vectashield plus DAPI, and immediately imaged using an Olympus FluoView FV1000 confocal laser scanning microscope.
GFP expression was evident in successfully transfected cells, which do not exhibit co-staining with GFAP (a reactive astrocyte marker; FIG. 8).
Example 5—Functional Recovery from Ischemic Stroke in a Mouse Model by Reprogramming of Reactive Astrocytes In Vivo
Non-adherent bone marrow cells are first obtained from 2-3 month old mice, cultured, and differentiated into microglia according to Hinze and Stolzing (2012). Briefly, femurae and tibiae are isolated, opened and centrifuged to obtain bone marrow, which is cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 10−8M dexamethasone and 100 units/ml Penicillin/Streptomycin. After 24 hours, the non-adherent cells are flushed off and transferred to a new dish. Cells are then differentiated for 6 days in 10 ml DMEM supplemented with 10% FCS, 50% astrocyte-conditioned medium (ACM), and 20 ng/ml granulocyte-monocyte colony stimulating factor (GM-CSF).
Microglial cultures are then lipofected (using Lipofectamine 3000 as per the manufacturer's protocols) with the chimeric pcDNA3.0-RVG-LAMP-2B-NLS-TALE and gfaABC1D-NeuroD1-IRES-GFP plasmids. To assess the in vivo reprogramming capability of these cells, a rodent model is used. Specifically, adult male C57BL/6J mice are given cerebral infarcts using the photothrombotic surgical method (Brown et al., 2007; Labat-gest and Tomasi, 2013). Briefly, animals are sedated using buprenorphine and isoflurane and fitted into a rodent stereotaxic frame. A midline incision on the scalp is made and the skin retracted to expose the skull. A dental drill is then used to thin the skull over motor cortex. Following an intraperitoneal injection (100 mg/kg) of Rose-Bengal (1% w/v), superficial motor cortex is illuminated with a green laser (532 nm, 17 mW) for 15 minutes to induce clotting and an ischemic stroke. Control groups will receive either a Rose-Bengal injection and no laser illumination, or illumination and no injection. One day after surgery, experimental group animals are injected in the tail vein with 500 μl of microglial cells suspended in sterile cerebrospinal fluid (CSF). Control animals receive the CSF injection without microglia cells. Animals are then re-tested on measures of motor skill that they had received training for prior to the infarct surgery. These include but are not limited to: the cylinder test, hanging wire test, pole test, and adhesive removal test (Li et al., 2014). Testing sessions will occur on day 4, 6, 10, and 14 following surgery. Mice that have received treatment with microglia are expected to exhibit gradually improving performance in the behavioral tasks, demonstrating the functional recovery promoted by the reprogramming of reactive astrocytes.
Upon completion of behavioral testing, animals will be deeply anesthetized with an intraperitoneally-administered overdose of sodium pentobarbital (100 mg/kg) and then perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA). Brains will then be post-fixed for 24 hours in 4% PFA and subsequently cryoprotected in 30% sucrose (in PBS) prior to sectioning. 40 μm tissue sections containing motor cortex are then washed with PBS, blocked with 1×PBS plus 0.3% Triton X-100 and 5% goat serum for 2 hours, and incubated overnight with 1:500 rabbit anti-GFAP antibody in 1×PBS plus 0.3% Triton X-100 at room temperature with agitation. Sections are rinsed twice with 1×PBS plus 0.3% Triton X-100 and primary antibodies are then labeled with 1:500 anti-rabbit-Alexa Fluor 594 secondary antibodies in PBS plus 0.3% Triton X-100 overnight at room temperature. Sections are then rinsed twice with 1×PBS, mounted on slides with Vectashield plus DAPI, coverslipped, and immediately imaged using a confocal laser scanning microscope. Similar to what was demonstrated in Example 4, it is expected that successfully reprogrammed reactive astrocytes in the damaged motor cortex will exhibit GFP expression which will not co-localize with GFAP.
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## Claims

1. 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,
(1) 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

(2) 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.

2. The method according to claim 1 wherein the first chimeric nucleic acid sequence additionally comprises:
(iv) a nucleic acid sequence encoding a cleavable polypeptide; or
(v) a nucleic acid sequence encoding a polypeptide providing a signal for nuclear localization in the astroglial cell.

3. The method according to claim 1 wherein the exosomes are produced in microglia cells in vitro.

4. The method according to claim 3 wherein the exosomes are separated from the microglia cells and provided to a human or an animal in need thereof in a manner that permits expression in astroglial cells of the human or the animal.

5. The method according to claim 1 wherein the exosomes are produced in microglia cells in vivo.

6. The method according to claim 5 wherein the microglia cells are provided to a human or an animal in need thereof in a manner that permits expression in astroglial cells of the human or animal.

7. The method according to claim 1 wherein the polypeptide of interest is a polypeptide capable of reprogramming astroglial cells into neurons.

8. The method according to claim 7 wherein the polypeptide of interest is NeuroD1.

9. The method according to claim 7 wherein the polypeptide of interest comprises the polypeptide set forth by SEQ.ID NO: 49; SEQ.ID NO: 50; SEQ.ID NO: 51; SEQ.ID NO: 52; or SEQ.ID NO: 53.

10. The method according to claim 1 wherein the exosomal membrane polypeptide is LAMP-2B.

11. The method according to claim 10 wherein the exosomal membrane polypeptide comprises the polypeptide set forth by SEQ.ID NO: 33; SEQ.ID NO: 34; SEQ.ID NO: 35; SEQ.ID NO: 36; SEQ.ID NO: 37; SEQ.ID NO: 38 SEQ.ID NO: 39; SEQ.ID NO: 40; SEQ.ID NO: 41; SEQ.ID NO: 42; SEQ.ID NO: 43 or SEQ.ID NO: 44.

12. The method according to claim 1 wherein the neural cell targeting polypeptide is RVG.

13. The method according to claim 12 wherein the neural cell targeting polypeptide comprises the polypeptide set forth by SEQ.ID NO: 45 or SEQ.ID NO: 46.

14. The method according to claim 1 wherein the nucleic acid binding polypeptide is Gal4 or TAL effector polypeptide.

15. The method according to claim 14 wherein the Gal4 polypeptide comprises the polypeptide set forth by SEQ.ID NO: 47.

16. The method according to claim 1 wherein the nucleic acid sequence comprising a nucleic acid binding polypeptide recognition sequence is the Gal4 UAS sequence or the TAL effector polypeptide recognition sequence.

17. The method according to claim 16 wherein the nucleic acid sequence comprising a nucleic acid binding polypeptide recognition sequence comprises the nucleic acid sequence set forth by SEQ.ID NO: 17 or SEQ.ID NO: 18.

18. The method according to claim 7 wherein the astroglial cell is reprogrammed into neurons.

19. A composition comprising microglia cells capable of producing exosomes, wherein the exosomes comprise:
(I) a 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

(II) a 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.

20. A composition comprising substantially pure exosomes comprising:
(I) a 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

(II) a 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.

21. The method of claim 14 wherein the TAL effector polypeptide comprises the polypeptide set forth by SEQ.ID NO: 48.