Compositions and methods for treatment of ischemia and cardiomyopathy

Abstract

To provide compositions and methods for the treatment of ischemia and cardiomyopathy.SOLUTION: Provided is a method to promote tissue healing, to treat tissue injury or to treat a condition related to tissue injury, comprising: administering to a subject in need thereof an effective amount of one or both of: a CAMKK1 polypeptide or an equivalent thereof; or an SDF-l polypeptide or an equivalent thereof, in the presence of CXCR4 polypeptide expression, the effective amount being an amount that induces exosome production, secretion and / or function in the subject. Recombinant stem cells are also provided that overexpress one or both of a CAMKK1 protein or polypeptide and / or an SDF-l protein or polypeptides. The stem cells and products produced from them are useful to treat ischemic or inflammatory conditions as well as to promote tissue healing.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 659,667, filed April 18, 2018, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] background Ischemia is a condition in which blood flow to a part of the body is completely occluded or severely reduced, resulting in anoxia, reduced substrate supply, and accumulation of metabolic products. Prolonged ischemia leads to atrophy, degeneration, apoptosis, and necrosis in the affected tissue.

[0003] Inflammation is a protective response intended to remove the initial cause of injury and necrotic cells / tissue resulting from injury. However, in some diseases, such as arthritis, inflammation occurs in the absence of injury. Prolonged inflammation can result in tissue destruction, fibrosis, and / or necrosis. Summary of the Invention [Means for solving the problem]

[0004] Abstract 1. A method of promoting tissue healing, treating tissue injury, or treating a condition associated with tissue injury, comprising administering to a subject an effective amount of: a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression administering to a subject in need thereof one or both of Here, provided herein is a method, wherein said effective amount is an amount that induces exosome production, secretion, and / or function in a subject.

[0005] In one embodiment, the CAMKK1 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 1 or 2, or their respective equivalents. In another embodiment, the SDF-1 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 3 or 4, or their respective equivalents. In a further embodiment, the CXRC4 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 5, or their respective equivalents.

[0006] The method may further comprise, or may consist essentially of, or may even consist of a step of determining circulating exosome levels in a sample isolated from the patient prior to administration. In another aspect, the method may further comprise, or may consist essentially of, or may even consist of a step of determining circulating exosome levels in a sample isolated from the patient after administration. The effective amount can be determined by the attending physician, medical professional, or veterinarian from the circulating exosome levels in the sample or based on past clinical information and data.

[0007] a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression Further disclosed herein are recombinant stem cells that overexpress one or both of the following: In one embodiment, the overexpression is by expression of a CAMKK1 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO:1 or 2 or their respective equivalents, an SDF-1 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO:3 or 4 or their respective equivalents, and a CXCR4 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO:5 or their equivalents.

[0008] The present disclosure further provides a recombinant stem cell that overexpresses one or both of CAMKK1 polypeptide or protein and / or SDF-1 polypeptide or protein or their respective equivalents. In one embodiment, the overexpression of CAMKK1 and / or SDF-1 is in the presence of expression or optional overexpression of Akt. In another embodiment, the overexpression of SDF-1 is in the presence of expression or optional overexpression of CXCR4. CXCR4 polypeptide expression can be endogenous or can be induced by exogenous expression of CXCR4 polynucleotide. The stem cell can be an embryonic stem cell, an induced pluripotent stem cell (iPSC), or a somatic stem cell (adult stem cell), and can be any specific type of stem cell, including, for example, adipose, mesenchymal, or neural. In another embodiment, the stem cell is a mesenchymal stem cell, such as a human mesenchymal stem cell.

[0009] In another embodiment, the stem cell is any type or any species of stem cell, for example, mammalian stem cell, such as human stem cell, in one embodiment, mesenchymal stem cell or CD34+ expressing stem cell.Further provided herein is recombinant stem cell derived differentiated cell that overexpresses one or both of polypeptide and protein.Stem cell is useful in the treatment and method disclosed herein.

[0010] A non-limiting example of a polynucleotide encoding a CAMKK1 polypeptide or protein comprises, or essentially consists of, or even consists of a polypeptide encoding SEQ ID NO: 1 or 2 or its equivalent. A polynucleotide encoding this sequence is also disclosed in ncbi.nlm.nih.gov / nuccore / NM_032294 (last accessed on April 13, 2018). A non-limiting example of a polynucleotide encoding an SDF-1 polypeptide or protein comprises, or essentially consists of, or even consists of a sequence encoding a polypeptide of SEQ ID NO: 4 or its equivalent (e.g., SEQ ID NO: 3 or its equivalent). These polynucleotides to be inserted into stem cells can further comprise, or essentially consist of, or even consist of a promoter sequence and / or enhancer sequence operably linked to the polynucleotide or its equivalent when in stem cells. These polynucleotides can be included in a vector (e.g., a prokaryotic or eukaryotic vector, such as a plasmid or a viral vector). Methods for determining whether a cell expresses appropriate levels of a polypeptide or protein are known in the art and include, for example, PCR and hybridization techniques (Northern and Southern techniques).

[0011] Methods for preparing such cells are known in the art (e.g., by inserting a gene encoding a protein or polypeptide into stem cells and culturing said cells under appropriate conditions). Expression of the gene can be determined using methods known in the art (e.g., PCR and / or ELISA technologies).

[0012] Also provided herein is an isolated cell culture medium produced by the above-mentioned stem cells and an exosome produced by the aforementioned stem cells. The exosome composition can be substantially purified or highly purified from the cells or cell culture medium.

[0013] The above-mentioned stem cells, cell culture medium and exosomes can be combined with carrier to prepare a composition.Non-limiting examples of carriers include pharmaceutically acceptable carriers or biocompatible matrices, and can further include preservatives, stabilizers, and / or cryoprotectants as necessary.In a further embodiment, the composition is a lyophilized composition and includes exosomes.

[0014] Also provided herein is a method for inducing exosome secretion and / or function in stem cells, comprising, or consisting essentially of, or even consisting of, upregulating the expression of CAMKK1 polynucleotide or protein or its respective equivalent in stem cells. In one embodiment, overexpression of CAMKK1 is in the presence of expression or optional overexpression of Akt. The stem cells can be embryonic stem cells, induced pluripotent stem cells (iPSCs), or somatic (adult) stem cells, and can be of any particular type, including, for example, adipose, mesenchymal, or neural. In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, the stem cells are of any type or species, for example, mammalian stem cells, such as human stem cells. Further provided herein are differentiated cells produced from these stem cells.

[0015] Non-limiting examples of polynucleotides encoding CAMKK1 polypeptides or proteins include, or consist essentially of, or even consist of, sequences encoding SEQ ID NO: 1 and 2 or equivalents. Polynucleotides encoding sequences are also disclosed in ncbi.nlm.nih.gov / nuccore / NM_032294 (last accessed: April 13, 2018). Polynucleotides in stem cells can further include, or consist essentially of, or even consist of promoter and / or enhancer sequences operably linked to the polynucleotide or equivalents. These polynucleotides can further be included in a vector (e.g., a prokaryotic or eukaryotic vector, such as a plasmid or viral vector). Methods for determining whether a cell expresses a polypeptide or protein at an appropriate level are known in the art and include PCR.

[0016] The method may further comprise culturing the cells for a time effective to allow the cells to secrete the exosomes into the culture medium, and then optionally separating the cells and / or the exosomes from the culture medium.

[0017] Also provided are cells, culture medium, and exosomes prepared by the method. In a further aspect, the method further comprises isolating the population of exosomes from the culture medium or stem cells.

[0018] As described above, the composition can be prepared by combining stem cells, cell culture medium and exosomes with carrier.Non-limiting examples of carrier include pharmaceutically acceptable carrier or biocompatible matrix, and can further include preservative, stabilizer, and / or cryoprotectant as necessary.In a further embodiment, the composition is a lyophilized composition and includes exosomes.

[0019] Still further provided is a method of inducing exosome secretion and function in stem cells, comprising, or consisting essentially of, or even consisting of, upregulating the expression of SDF-1 polypeptide or protein or its respective equivalent in stem cells, where overexpression is in the presence of CXCR4 expression. In one embodiment, overexpression of SDF-1 is in the presence of expression or optional overexpression of Akt. Also provided is a method of inducing exosome secretion and function in stem cells, comprising, or consisting essentially of, or even consisting of, upregulating the expression of SDF-1 polypeptide or protein or its respective equivalent in stem cells, where overexpression is in the presence of expression or optional overexpression of Akt. In one embodiment, overexpression of SDF-1 is in the presence of expression or optional overexpression of CXCR4. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), or somatic (adult) stem cells, and may be of any particular type, including, for example, adipose, mesenchymal, or neural. In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, the stem cells are of any type or species, for example, mammalian stem cells, such as human stem cells. Further provided herein are differentiated cells produced from stem cells.

[0020] A non-limiting example of the polynucleotide that encodes SDF-1 polypeptide or protein comprises, or essentially consists of, or even consists of SEQ ID NO:3 or its equivalent.These polynucleotides in stem cells can further comprise, or essentially consist of, or even consist of promoter and / or enhancer sequences operably linked to the polynucleotide or its equivalent.These polynucleotides can be contained in vectors (e.g., prokaryotic or eukaryotic vectors, such as plasmids or viral vectors).Methods for determining whether a cell expresses a polypeptide or protein at an appropriate level are known in the art and include PCR, Northern hybridization, and Southern hybridization.

[0021] The method may further comprise culturing the cells for a time effective to allow the cells to secrete the exosomes into the culture medium, then optionally separating the cells and / or the exosomes from the culture medium, and differentiating the cells into a lineage-specific cell type.

[0022] Also provided are cells, culture medium, and exosomes prepared by the method. In a further aspect, the method further comprises isolating the population of exosomes from the culture medium or stem cells.

[0023] As described above, the composition can be prepared by combining stem cells, cell culture medium and exosomes with carrier.Non-limiting examples of carrier include pharmaceutically acceptable carrier or biocompatible matrix, and can further include preservative, stabilizer, and / or cryoprotectant as necessary.In a further embodiment, the composition is a lyophilized composition and includes exosomes.

[0024] Also provided are methods of treatment. In one embodiment, a method is provided for treating one or more of ischemia, cardiomyopathy, and related disorders, or for promoting tissue healing. The method comprises, or consists essentially of, or even consists of administering to a subject in need of an effective amount of one or more of the stem cells described herein, the isolated culture medium described herein, the exosomes, the population described herein, or the composition described herein, wherein the overexpressed polypeptide or protein is a CAMKK1 polypeptide or protein or its equivalent. Further provided herein is a method for treating one or more of ischemic conditions, cardiomyopathy, and related disorders, or for promoting tissue healing, comprising, or consists essentially of, administering to a subject in need of an effective amount of the following: one or more of the exosome population of the present disclosure, or the composition described herein. The cells, culture medium, or exosomes can be allogeneic or autologous to the subject being treated. In one embodiment, the administration is local or systemic. In another embodiment, administration is systemic and distal to the site of action (eg, the heart). [Brief description of the drawings]

[0025] [Figure 1] 1A-1B show that SDF-1 and CAMKK1 individually had significant and equivalent effects on increasing ejection fraction compared to placebo. The combination of SDF-1 and CAMKK1 did not improve cardiac function any more than either of these molecules used alone.

[0026] [Diagram 2] FIG. 2 shows that the significant effect of SDF-1 and CAMKK1 is to enhance exosome release and function in the conditioned medium.

[0027] [Diagram 3]FIG. 3 shows that the effect of SDF-1 requires an autocrine or paracrine loop to induce Akt phosphorylation, whereas the CAMKK1 effect is direct.

[0028] [Figure 4] Figure 4 shows that CAMKK1 can still improve cardiac function when injected into normal tissues away from the heart. Conversely, SDF-1 injected into normal tissues away from the heart does not significantly improve cardiac function, because SDF-1 needs to bind to CXCR4 to induce Akt phosphorylation.

[0029] [Diagram 5] Figures 5A-5B show nanosight quantification of the number and size of exosomes derived from control MSCs and MSCs overexpressing CAMKK1. Figure 5A is a representative screenshot of the NTA video for Exo-control and Exo-CAMKK1. Exosome-control showed a significant number of particles (2.7×109±4.9 particles / ml). However, particle concentration of Exo-CAMKK1 was higher than control medium (3.76×109±2.2 particles / ml, p<0.05). Exosomes were estimated by NTA to be 10nm-1000nm in size. Figure 5B: Ejection fraction 2 weeks after left anterior descending artery ligation. Data are mean±SEM (n=4-8 animals / group). *, p<.05 CAMKK1 group vs. MSC group; Abbreviations; MSC, mesenchymal stem cell; CAMKK1, calcium calmodulin kinase kinase 1.

[0030] [Figure 6]Figure 6 shows that phosphorylation of Akt is required for MSC effects of CAMKK1 on cardiac function. Ejection fraction as a measure of cardiac function was measured 2 weeks after injection into the left anterior descending artery ligation in the heart (conditioned medium containing 2 million MSCs was collected and injected into the heart of animals immediately following induction of anterior myocardial infarction). In two treatments, MSCs were treated with one of two Akt inhibitors, and the presence of either Akt inhibitor reduced the benefit of cells overexpressing CAMKK1 over the benefit of control MSCs. Data show mean ± SEM (n = 4–8 / group) *, p < .05 cDNA-CAMKK1 vs. MSC control group. Abbreviations: AKT, protein kinase B; MSC, mesenchymal stem cell.

[0031] [Figure 7] Figure 7 shows that ejection fraction as a measure of cardiac function was measured 2 weeks after left anterior descending artery ligation in Exo-MSC control, Exo-MSC overexpressing CAMKK1, Exo overexpression, and siRNA AKT injected hearts. CAMKK1 is a kinase that directly phosphorylates akt to release exosomes from CAMKK1 overexpressing cells, and CAMKK1 can be injected into normal tissues away from the heart and still improve cardiac function.

[0032] [Figure 8]Figures 8A-8B show that CAMKK1 has an effect on chronic heart failure and how this compares to SDF-1. Injection of CAMKK1 at the time of AMI improved ejection fraction measurements 1 and 6 weeks later. It was expected that SDF-1 acted through its chemokine effect, thereby improving cardiac function through homing of tissue-specific stem cells and bone marrow-derived stem cells. CAMKK1 does not induce stem cell homing. That CAMKK1 and SDF-1 have equivalent effects on cardiac function, and the combination of the two does not further enhance the effect, thereby attempting to define a common pathway by which SDF-1 and CAMKK1 may act in a series of studies. CAMMKK1 levels 57.4%, 53.2%, 44.2%, and 40.1% (with placebo) and SDF-1 levels 54.7%, 52.5%, 50.4%, and 40.2% (with placebo) on days 1 and 6 after AMI. Data are presented as mean ± SEM (n = 5–8 animals / group) (**, p < .05 compared with saline control).

[0033] [Figure 9] Figure 9 shows that overexpression of CAMKK1 and SDF-1 enriches exosome amounts in MSCs. CAMKK1 and SDF-1 significantly improved EF 2 weeks after AMI in cardiac function (ejection fraction). The significant effect of SDF-1 and CAMKK1 is the release of exosomes into the conditioned medium and enhanced function. Cardiac function was significantly improved by CAMKK1 compared to saline control and SDF-1 injection. CAMKK1 significantly improved EF (21.8% ± 2.5, p < .01) and SDF-1 (16.8% ± 2.7, p > .05) compared to saline control group (1.9% ± 0.2) 2 weeks after AMI. Data are mean ± SEM (n = 7-8 / each group). The improvement of cardiac function induced by CAMKK1 overexpression in MSCs correlates with the increased vascular density in the cardiac border zone 2 weeks after AMI.

[0034] [Figure 10]Figure 10 shows cardiac function and echocardiography results 14 days after AMI. Animals underwent ligation of the LAD, immediately followed by thorax suturing and injection of CAMKK1 plasmid or SDF-1 plasmid or saline into the femur of animals receiving the drug. One week later, cardiac function in animals injected with CAMKK1 was significantly increased compared to saline, but not in SDF-1 treated animals. One week after AMI, CAMKK1 (30.3% ± 1.5, p < .01) and SDF-1 (15.7% ± 3.7, p > .05) were compared to the saline control group (12.9% ± 1.8). Data are mean ± SEM (n = 7-8 / each group).

[0035] [Figure 11] Figure 11 shows the effect of MSC-exosome function and overexpression of CAMKK1 and SDF-1 on cardiac function (ejection fraction). Exosomes were purified from conditioned medium (CM) containing 2 million control MSCs and MSCs overexpressing either SDF-1 (with or without AMD3100) or CAMKK1. The isolated exosomes were then injected into rat hearts immediately after AMI. Significant improvement in cardiac function was observed in the CAMKK1 group (21.8%) and the SDF-1 group (16.8%) compared to the saline control group 2 weeks after AMI. The benefit was lost in the SDF-1+AMD3100 group. Data are mean ± SEM (n=7-8 / each group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description definition The following abbreviations are used throughout this disclosure: CAMKK1 (Calcium / calmodulin-dependent protein kinase kinase 1); MSC (mesenchymal stem cell); and stromal cell-derived factor 1 alpha (SDF-1, (also known as CXCL12, pre-B cell growth-stimulating factor, PBSF, hIRH, chemokine (CXC motif) ligand 12, SDF1, SDF1A, TPAR1, SCYB12, SDF-1a, TLSF-a)), CXC chemokine receptor type 4 (CXCR-4), also known as fusin or CD184 (CXCR4).

[0037] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to the method and material described herein can be used in the practice or testing of this disclosure, the preferred method, device, and material are described herein.All technical publications and patent publications cited herein are incorporated herein by reference in their entirety.Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0038] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1:A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press (2002); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

[0039] All numerical designations including ranges (e.g., pH, temperature, time, concentration, and molecular weight) are approximate and vary in the (+) or (-) direction by 1.0 or 0.1 units, as appropriate. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0040] As used in this specification and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0041] As used herein, the term "comprising" shall mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other elements of essential importance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. "Consisting of" shall mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0042] The terms "polynucleotide", "nucleic acid", and "oligonucleotide" are used interchangeably and refer to either deoxyribonucleotides or ribonucleotides or analogs thereof that are polymeric forms of nucleotides of any length. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST tags, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted with non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present disclosure that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to be in double-stranded form.

[0043] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) in place of thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as functional genomics and homology searching.

[0044] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the aforementioned sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0045] That a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence means that, when aligned, the stated percentage of bases (or amino acids) in the two sequences being compared are the same. This alignment and percentage of homology or sequence identity can be calculated using software programs known in the art, such as Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. The alignment can be determined using those described in Biology. It is preferred to use default parameters for alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; display=50 sequences; sorting method=HIGH SCORE; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0046] An equivalent or biologically equivalent nucleic acid, polynucleotide, or oligonucleotide, or peptide is one that has at least 80% sequence identity, alternatively at least 85% sequence identity, alternatively at least 90% sequence identity, alternatively at least 92% sequence identity, alternatively at least 95% sequence identity, alternatively at least 97% sequence identity, or alternatively at least 98% sequence identity to the reference nucleic acid, polynucleotide, oligonucleotide, or peptide.

[0047] The term "amplification of polynucleotides" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR), and amplification methods. These methods are known and widely practiced in the art. See, for example, U.S. Patent Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR technique describes a gene amplification method that consists of (i) sequence-specific hybridization of primers to specific genes in a DNA sample (or library), (ii) subsequent amplification, including multiple rounds of annealing, extension, and denaturation using DNA polymerase, and (iii) screening of the PCR products for bands of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to initiate polymerization (i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified).

[0048] Reagents and hardware for performing PCR are commercially available. Primers useful for amplifying sequences from specific gene regions are preferably complementary to and specifically hybridize to sequences within the target region or adjacent regions. The nucleic acid sequences generated by amplification can be directly sequenced. Alternatively, the amplified sequence(s) can be cloned prior to sequence analysis. Methods for directly cloning and sequence analysis of enzymatically amplified genomic segments are known in the art.

[0049] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after transcription and translation.

[0050] The term "express" refers to the production of a gene product.

[0051] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0052] "Gene product" or "gene expression product" refers to the amino acids (eg, peptides or polypeptides) produced when a gene is transcribed and translated.

[0053] "Under transcriptional control" is a term well understood in the art and indicates that the transcription of a polynucleotide sequence, usually a DNA sequence, depends on the elements operably linked to said sequence that contribute to the initiation or promotion of transcription. "Operably linked" refers to the polynucleotide being placed in a manner that allows it to function in a cell. In one aspect, the present disclosure provides a promoter operably linked to a downstream sequence (e.g., CAMKK1, SDF-1, etc.).

[0054] The term "encode," as applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when the polynucleotide, in its native state or when manipulated by methods well known to those of skill in the art, can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0055] "Probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that serves as a reagent for detecting a target potentially present in a sample of interest by hybridization with the target. Usually, the probe contains a detectable label or a means that can attach the label either before or after the hybridization reaction. Alternatively, the "probe" can be a biological compound such as a polypeptide, an antibody, or a fragment thereof that can bind to a target potentially present in a sample of interest.

[0056] "Detectable labels" or "markers" include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to the polynucleotides, polypeptides, antibodies, or compositions described herein.

[0057] A "primer" is a short polynucleotide, generally with a free 3'-OH group, that binds to a target or "template" potentially present in a sample of interest by hybridization with the target, and subsequently promotes polymerization of a polynucleotide complementary to the target. "Polymerase chain reaction" ("PCR") is a reaction that uses a "primer pair" or "primer set" consisting of an "upstream" and a "downstream" primer, and a polymerization catalyst (such as a DNA polymerase, typically a thermostable polymerase enzyme) to generate replicate copies of a target polynucleotide. The PCR method is well known in the art and is taught, for example, in MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press). All processes of producing replicate copies of a polynucleotide (such as PCR or gene cloning) are collectively referred to herein as "replication". Primers can also be used as probes in hybridization reactions (such as Southern or Northern blot analysis). Sambrook and Russell (2001), infra.

[0058] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0059] Hybridization reactions can be carried out under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out in 10xSSC at about 40°C, or in solutions of equivalent ionic strength / temperature. Medium stringency hybridization is typically carried out in 6xSSC at about 50°C, and high stringency hybridization reactions are generally carried out in 1xSSC at about 60°C. Further examples of stringent hybridization conditions include: at low stringency, incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6xSSC to about 10xSSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 4xSSC to about 8xSSC. Examples of medium hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. In general, the hybridization incubation time is 5 minutes to 24 hours, including one, two, or more washing steps, and the wash incubation time is about 1, 2, or 15 minutes. SSC is 0.15M NaCl and 15 mM citrate buffer. It is understood that the equivalent of SSC using other buffer systems can be used. The hybridization reaction may be carried out under "physiological conditions," as known to those of skill in the art. Non-limiting examples of physiological conditions include temperature, ionic strength, pH, and Mg concentrations normally found within a cell. 2+ Concentration.

[0060] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, this reaction is called "annealing" and these polynucleotides are described as "complementary". A double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide if hybridization can occur between one of the first polynucleotide strand and the second polynucleotide strand. "Complementarity" or "homology" (the degree to which one polynucleotide is complementary to another) can be quantified in terms of the ratio of bases in opposite strands that are expected to form hydrogen bonds with each other according to commonly accepted base pairing rules.

[0061] The term "proliferate" or "expand" means that a cell or cell population grows. The term "growth" also refers to the proliferation of cells in the presence of a supporting medium, nutrients, growth factors, feeder cells, or any chemical or biological compounds necessary to obtain a desired cell number or cell type.

[0062] The term "culture" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0063] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains an intact replicon so that the vector can be replicated, for example, by a transformation process, when placed in a cell. A vector can be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpes viruses, bacculoviruses, modified bacculoviruses, papoviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles that contain DNA condensed with cationic polymers (such as heterologous polylysine, defined length oligopeptides, and polyethyleneimine), in some cases contained in liposomes; and ternary complexes that contain viruses and polylysine-DNA. Non-limiting examples of plasmid vectors include pHIV, pLVX, pLKO.1, pLemiR, pLV, pUC, pGEM, pSP73, pSP72, pSP64, pBR322, and pCCL plasmids.

[0064] A "viral vector" is defined as a virus or viral particle produced recombinantly, either in vivo, ex vivo, or in vitro, that contains a polynucleotide to be delivered to a host cell. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphaviral vectors. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0065] In the embodiment where gene transfer is mediated by lentiviral vector, vector construct refers to a polynucleotide that includes a lentiviral genome or a part thereof and a therapeutic gene. As used herein, "lentiviral-mediated gene transfer" or "lentiviral transduction" has the same meaning and refers to the process in which a gene or nucleic acid sequence is stably transferred into a host cell by a virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell by its normal infection mechanism or can be modified to bind to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, when the virus infects a cell, the RNA is reverse transcribed into the form of DNA and integrated into the genomic DNA of the infected cell. The integrated DNA form is referred to as a provirus. As used herein, lentiviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell via a viral or viral-like entry mechanism. "Lentiviral vector" is a type of retroviral vector well known in the art that has certain advantages in transducing non-dividing cells compared to other retroviral vectors. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

[0066] The lentiviral vectors of the present disclosure are based on or derived from oncoretroviruses (a subgroup of retroviruses including MLV) and lentiviruses (a subgroup of retroviruses including HIV). Examples include ASLV, SNV, and RSV, all of which are split into packaging and vector components of lentiviral vector particle production systems. The lentiviral vector particles of the present disclosure can be based on specific retroviral versions that have been genetically or otherwise altered (e.g., by specific selection of packaging cell lines).

[0067] A vector particle "based on" a particular retrovirus means that the vector is derived from said particular retrovirus. The genome of the vector particle contains components derived from said retrovirus as a backbone. The vector particle contains the essential vector components (including reverse transcription and integration systems) that fit into the RNA genome. Usually these include the gag and pol proteins from the particular retrovirus. Thus, most of the structural components of the vector particle are usually derived from said retrovirus, but the structural components may be genetically or otherwise modified to obtain desired useful properties. However, certain structural components, especially the env protein, may be derived from different viruses. The vector host range and the type of cells to be infected or transduced can be altered by using different env genes in the vector particle production system to obtain vector particles with different specificities.

[0068] The term "promoter" refers to a DNA region that initiates transcription of a particular gene. Promoters include the core promoter, which is the minimal portion of a promoter necessary to properly initiate transcription, and can also include regulatory elements, such as transcription factor binding sites. Regulatory elements may promote or inhibit transcription. Regulatory elements in a promoter can be binding sites for transcription activators or transcription repressors. Promoters can be constitutive or inducible. A constitutive promoter refers to a promoter that is always active and / or always directs transcription above basal transcription levels for a gene. Examples of such constitutive promoters include the phosphoglycerate kinase 1 (PGK) promoter; SSFV, CMV, MNDU3, SV40, Ef1a, UBC, and CAGG. An inducible promoter is a promoter that can be induced by a molecule or factor that is added to or expressed in a cell. An inducible promoter can still cause transcription at a basal level without induction, but typically induction produces significantly more protein. Promoters can also be tissue specific. A tissue-specific promoter can cause a protein to be produced in certain cell populations that have the appropriate transcription factors to activate the promoter.

[0069] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is juxtaposed to said gene by genetic engineering (i.e., molecular biology techniques) such that transcription of the gene is directed by the linked enhancer / promoter.

[0070] The term "isolated" as used herein refers to a molecule or biological or cellular material that is substantially free (e.g., 70%, or 80%, or 85%, or 90%, or 95%, or more than 98%) of other materials. In one embodiment, the term "isolated" refers to a nucleic acid (such as DNA or RNA), or a protein or polypeptide, or a cell or cell organelle, or a tissue or organ, respectively, that is separated from other DNA or RNA, or proteins or polypeptides, or cells or cell organelles, or tissues or organs present in the natural source, which allows for manipulation of the material (e.g., by recombinant replication or mutation) to achieve results that cannot be achieved when present in its native or natural state. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, culture medium when produced by recombinant DNA technology, or chemical precursors, other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that do not naturally occur as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to include both purified and recombinant polypeptides, for example, at greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98% purity. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues, and can include cells or tissues that have been cultured and / or manipulated.

[0071] As used herein, a "stem cell" is defined as a cell that has the ability to divide indefinitely in culture to give rise to specialized cells. For current convenience, stem cells are classified as somatic (adult), embryonic, or induced pluripotent stem cells. Somatic stem cells are undifferentiated cells found in differentiated tissues that can regenerate themselves (clonal) and differentiate (with certain limitations) to generate all specialized cell types of the tissue from which they originate. Embryonic stem cells are primitive (undifferentiated) cells derived from the embryo that have the potential to become a wide variety of specialized cell types. Non-limiting examples of embryonic stem cells are the HES2 (also known as ES02) cell line available from ESI, Singapore and the H1 or H9 (also known as WA01) cell lines available from WiCell, Madison, WI. Pluripotent embryonic stem cells can be distinguished from other cell types by the use of markers, including but not limited to Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, germ cell nuclear factor, SSEA1, SSEA3, and SSEA4. Induced pluripotent stem cells (iPSCs) are non-pluripotent cells, typically artificially induced stem cells derived from adult somatic cells, produced by inducing the expression of one or more stem cell-specific genes.

[0072] "Parthenogenetic stem cells" refers to stem cells resulting from parthenogenetic activation of eggs. Methods for producing parthenogenetic stem cells are known in the art. See, for example, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(Suppl.1)11911-6(2003).

[0073] "Embryoid bodies or EBs" are three-dimensional (3-D) aggregates of embryonic stem cells formed in culture that are amenable to subsequent differentiation. When grown in suspension culture, EB cells form small cellular aggregates surrounded by an outer layer of follicular endoderm. Upon growth and differentiation, EBs evolve into cystic embryoid bodies with a fluid-filled cavity and an inner layer of ectoderm-like cells.

[0074] The term "proliferate" means that a cell or cell population grows or changes phenotype. The term "growth" refers to the proliferation of cells in the presence of a supporting medium, nutrients, growth factors, supporting cells, or any chemical or biological compounds necessary to obtain a desired cell number or cell type. In one embodiment, the growth of cells regenerates tissue. In yet another embodiment, the tissue is composed of neural progenitor cells or neural cells.

[0075] The term "culture" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of cells grown in culture may not be completely (i.e., morphologically, genetically, or phenotypically) identical to the parent cell. "Expanded" refers to any proliferation or division of cells. A "cultured" cell is one that has been separated from its natural environment and grown under specific, predefined conditions.

[0076] "Differentiation" describes the process by which non-specialized cells acquire the characteristics of specialized cells, such as cardiac, hepatic, or muscular cells. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into specific cell types. "Dedifferentiated" defines cells that revert to a less committed state within a cell lineage. As used herein, the term "differentiating or differentiated" defines cells that assume a more committed ("differentiated") state within a cell lineage. As used herein, "cells that differentiate into mesodermal (or ectodermal or endodermal) lineages" defines cells that become committed to a specific mesodermal, ectodermal, or endodermal lineage, respectively. Examples of cells that differentiate into mesodermal lineages or give rise to specific mesodermal cells include, but are not limited to, adipogenic cells, smooth muscle-forming cells, chondrogenic cells, cardiogenic cells, skin-forming cells, hematopoietic cells, angiogenic cells, myogenic cells, nephrogenic cells, urogenital cells, osteogenic cells, pericardiogenic cells, or stromal cells.

[0077] Examples of cells that differentiate into ectodermal lineages include, but are not limited to, epidermal cells, neurogenic cells, and gliogenic cells.

[0078] "Bone marrow stromal cells", also called "mesenchymal stem cells" (i.e., MSCs), are multipotent stem cells that can differentiate into various cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchyme is fetal connective tissue that originates from the mesoderm and differentiates into hematopoietic and connective tissues, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supporting structure in which the functional cells of tissues reside. Methods for isolating such cells and for expanding and differentiating such cells are known in the technical and patent literature (e.g., U.S. Patent Application Publication Nos. 2007 / 0224171, 2007 / 0054399, and 2009 / 0010895, which are incorporated by reference in their entireties).

[0079] By CD34+ cells is intended cells expressing the cell surface CD34 marker. CD34 derives its name from the Classification of Cells by Differentiation protocol, which identifies cell surface antigens. CD34 was first described on hematopoietic stem cells and functions as an intercellular adhesion molecule. CD34 also mediates attachment of hematopoietic stem cells to bone marrow extracellular matrix or direct attachment to stromal cells. Clinically, CD34 is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplantation. CD34 is also found on cell types other than hematopoietic cells. Sidney LE, et al. (2014).”Concise review: evidence for CD34 as a common marker for diverse progenitors”.Stem Cells.32(6):1380-1389.

[0080] As used herein, the term "differentiating or differentiated" defines a cell that assumes a more committed ("differentiated") state within a cell lineage. "Dedifferentiated" defines a cell that reverts to a less committed state within a cell lineage.

[0081] CAMKK1 (calcium / calmodulin-dependent protein kinase kinase 1; human CAMKK1, known by its UniProtKB number Q8N5S9), belongs to the calcium-induced signaling cascade and is involved in several cellular processes. CAMKK1 is a transferase belonging to the Ser / Thr protein kinase family and the CamKK subfamily, and is expressed in the heart, pancreas, tonsil, hypothalamus, prostate, and lung. CAMKK1 activates CamK1 and CamK4 by phosphorylation of its amino acids Thr(177) and Thr(196), respectively. CAMKK1 activity is itself regulated by Ca2+ / calmodulin; its activity is decreased upon phosphorylation by PKA (cAMP-dependent protein kinase) and increased by incubation with PKA in the presence, but not in its absence, of Ca(2+) / calmodulin. This phosphorylation and inhibition of CAMKK1 by PKA is involved in modulating the balance between cAMP-dependent and Ca2+-dependent signaling pathways. The amino acid sequence of CAMKK1 is shown in SEQ ID NO: 1. Exemplary equivalent truncated and mutated versions of the CAMKK1 protein are described herein.

[0082] SDF-1 (stromal cell-derived factor-1) is a small cytokine that belongs to the chemokine family and is officially named chemokine (CXC motif) ligand 12 (CXCL12). It is produced in two forms, SDF-1α / CXCL12a and SDF-1β / CXCL12b, by alternative splicing of the same gene. Chemokines are characterized by the presence of four conserved cysteines that form two disulfide bonds. The CXCL12 protein belongs to the CXC chemokine group, with its first pair of cysteines separated by one intervening amino acid. CXCL12 exhibits strong chemotaxis for lymphocytes and is involved as an important cellular coordinator during development. The receptor for this chemokine is CXCR4, previously called fusin. This CXCL12-CXCR4 interaction was previously considered exclusive (unlike other chemokines and their receptors), but recently it has been suggested that CXCL12 is also bound by the CXCR7 receptor. The gene for CXCL12 is present on human chromosome 10. In humans and mice, both CXCL12 and CXCR4 show high sequence identity: 99% and 90%, respectively. A representative sequence for the human protein is mnakvvvvlv lvltalclsd gkpvslsyrc pcrffeshva ranvkhlkil ntpncalqiv arlknnnrqv cidpklkwiq eylekalnkr fkm (SEQ ID NO: 4) (Reproduced from NCBI Reference Sequence NP_000600.1, see ncbi.nlm.nih.gov / protein / NP_000600, last accessed on April 12, 2018). A representative coding polynucleotide is provided in SEQ ID NO:3, reproduced from NM_199168 (see ncbi.nlm.nih.gov / nuccore / NM_199168, last accessed April 12, 2018).

[0083] CXCR-4 is an α-chemokine receptor specific for stromal derived factor-1 (SDF-1). CXCR4 is one of several chemokine receptors that HIV can use to infect CD4+ T cells. SDF-1, the ligand of CXCR4, is known to be important in hematopoietic stem cell homing to bone marrow and hematopoietic stem cell quiescence.

[0084] A representative CXCR-4 protein sequence (SEQ ID NO:5) (reproduced from uniprot.org / uniprot / P61073 (last accessed April 12, 2018)) is as follows: [ka] [ka]

[0085] A representative human polynucleotide sequence (SEQ ID NO:6) (reproduced from ncbi.nlm.nih.gov / nuccore / NG_011587.1?from=5001&to=8807&report=genbank, last accessed April 12, 2018) is as follows: [ka] [ka] [ka]

[0086] As used herein, "overexpression of CAMKK1" means an amount of CAMKK1 expression product (mRNA or protein) in a patient's cells, organ, or tissue that is greater than the amount normally present in the patient's organ or tissue.

[0087] As used herein, "overexpression of SDF-1" means an amount of SDF-1 expression product (mRNA or protein) in a patient's cells, organ, or tissue that is greater than the amount normally present in the patient's organ or tissue.

[0088] The term "exosome" is well known in the art and refers to extracellular vesicles that are released from cells upon fusion with the plasma membrane of intermediate intracellular compartments, multivesicular bodies. See, e.g., Edgar JR (2016) BMC Biol. 13; 14: 46.

[0089] The term "exogenous" in relation to nucleic acid or protein refers to a polynucleotide sequence or polypeptide sequence that is artificially introduced into a cell, cell-derived vesicle, exosome, microvesicle, or a combination thereof. There may be an endogenous nucleic acid or protein in a cell-derived vesicle that has the same or substantially similar sequence as the polynucleotide or polypeptide sequence that encodes the exogenous nucleic acid or protein. For example, mesenchymal stem cells can be genetically modified to overexpress CAMKK1-encoding polynucleotides. It is expected that a purified cell-derived vesicle population isolated from the culture medium collected from MSCs genetically modified to overexpress CAMKK1 contains a higher level of CAMKK1 compared to cell-derived vesicles isolated from MSCs that are not genetically modified to overexpress CAMKK1-encoding polynucleotides.

[0090] "Composition" is also intended to include combinations of active agents and other carriers (e.g., compounds or compositions (inert (e.g., detectable agents or labels) or active (adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, or adjuvants, etc.)). Carriers also include biocompatible scaffolds, pharmaceutical excipients and additives such as proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; sugar derivatives (such as alditols, aldonic acids, and esterified sugars); and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins (such as human serum albumin (HSA)), recombinant human albumin (rHA), gelatin, and casein, etc. Representative amino acids / antibody components that can also function as buffers include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. Carbohydrate excipients are also contemplated within the scope of the present disclosure, examples of which include, but are not limited to, monosaccharides (such as fructose, maltose, galactose, glucose, D-mannose, and sorbose); disaccharides (such as lactose, sucrose, trehalose, and cellobiose); polysaccharides (such as raffinose, melezitose, maltodextrin, dextran, and starch); and alditols (such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol).

[0091] "Substantially homogeneous" describes a population of cells in which greater than about 50%, alternatively greater than about 60%, alternatively greater than 70%, alternatively greater than 75%, alternatively greater than 80%, alternatively greater than 85%, alternatively greater than 90%, alternatively greater than 95% of the cells are of the same or similar phenotype. Phenotype can be determined by preselected cell surface or other markers.

[0092] "Biocompatible matrix" refers to a scaffold or matrix for tissue engineering that can serve as a substrate to support appropriate cellular activity (including promotion of molecular or mechanical signaling systems) to generate a desired tissue without inducing any undesirable effects in cells or any undesirable local or systemic responses in the ultimate host. In other embodiments, the biocompatible scaffold is a precursor to an implantable device that can perform its intended function with a desired degree of host integration without inducing any undesirable local or systemic effects in the host. Biocompatible scaffolds are described in U.S. Patent Nos. 6,993,406 and 6,103,255.

[0093] As used herein and known to those of skill in the art, a "marker" refers to a receptor or protein expressed by or within a cell that can be used as an identification and / or discrimination factor. If a marker is marked with a ("+"), the marker is positively expressed. If a marker is marked with a ("-"), the marker is absent or not expressed. Variables of marker expression such as "high", "low", and relative terms are also used.

[0094] A "precursor" or "progenitor cell" is a cell that develops into a specific cell type. It is intended to mean a cell that has the ability to differentiate. A precursor cell can be a stem cell. A precursor cell can also be more specific than a stem cell. A precursor cell can be unipotent or multipotent. Compared to adult stem cells, a precursor cell can be at a later stage of cell differentiation. Examples of precursor cells include, but are not limited to, precursor neural cells.

[0095] A cell population intends a group of more than one cells that may or may not be phenotypically and / or genotypically identical (clonal).

[0096] The term effective amount refers to a concentration or amount of a reagent or composition (such as the compositions described herein), cell population, or other agent that is effective to obtain an intended result (including cell growth and / or differentiation in vitro or in vivo) or is effective in treating a neurodegenerative condition as described herein. It is recognized that the number of cells to be administered will vary depending on the characteristics of the disorder to be treated, including, but not limited to, the size or total volume / surface area to be treated and the proximity of the administration site to the location of the area to be treated, among other factors familiar to medicinal biologists.

[0097] The terms effective period (or effective time) and effective conditions refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods) required or preferred for an agent or composition to achieve its intended result (e.g., differentiation of cells into a given cell type).

[0098] The term patient or subject refers to an animal (including mammals such as bovine, canine, feline, ovine, equine, and preferably human) that is treated with a pharmaceutical composition or subject to the methods described herein.

[0099] The term pharma- ceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, refers to a reagent, cell, compound, material, composition, and / or dosage form that is not only compatible with cells and other reagents to be administered for therapeutic purposes, but also, within the bounds of sound medical judgment, suitable for use in contact with human and animal tissues commensurate with a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic response, or other complications. Pharmaceutically acceptable carriers suitable for use in the present disclosure include liquids, semi-solid (e.g., gels), and solid materials (e.g., cell scaffolds and matrices, tubes, sheets, and other materials known in the art and described in more detail herein). These semi-solid and solid materials can be designed to resist degradation in the body (non-biodegradable) or can be designed to degrade in the body (biodegradable, bioerodible). Biodegradable materials may further exhibit bioresorbability or bioabsorbability (i.e., they may be dissolved and absorbed into bodily fluids, either by conversion to other materials via natural pathways or by degradation and excretion (one example being a water-soluble implant), or may be broken down and ultimately excreted from the body).

[0100] A "control" is a counter subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative". For example, if the purpose of an experiment is to determine the correlation between changes in the expression level of a gene and a particular phenotype, it is generally preferable to use a positive control (a sample from a subject that has such changes and shows the desired phenotype) and a negative control (a subject or a sample from a subject that lacks changes in expression or phenotype). Furthermore, if the purpose of an experiment is to determine whether a drug affects the differentiation of stem cells, it is preferable to use a positive control (a sample that has an aspect known to affect differentiation) and a negative control (a drug known to have no effect or a sample without the addition of a drug).

[0101] Terms such as autologous transfer, autologous transplantation, and autologous graft refer to the treatment in which the cell donor is also the recipient of cell replacement therapy.Terms such as allogeneic transfer, allogeneic transplantation, and allograft refer to the treatment in which the cell donor is the same species as the recipient of cell replacement therapy, but is not the same individual.Cell transfer in which the donor's cells show histocompatibility with the recipient is sometimes called syngeneic transfer.Terms such as xenogeneic transfer, xenogeneic transplantation, and xenograft refer to the treatment in which the cell donor is a different species from the recipient of cell replacement therapy.

[0102] "Host cell" refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in later generations, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term as used herein.

[0103] "Treating" or "treatment" of a disease includes: (1) preventing the development of clinical symptoms of the disease in a patient who may be predisposed to being affected but who has not yet experienced or exhibited symptoms of the disease; (2) suppressing the disease (i.e., arresting or reducing the development of the disease or its clinical symptoms); or (3) ameliorating the disease (i.e., causing the disease or its clinical symptoms to recede). In one embodiment, treatment excludes prevention.

[0104] Treatment of cardiomyopathies and related disorders aims to alleviate the symptoms and associated clinical endpoints of these diseases or disorders, including, for example, myocardial disorders in which the myocardium is structurally and functionally abnormal. Most cardiomyopathies are extrinsic (external causes), of which ischemic cardiomyopathies are the most common. This term includes ischemic cardiomyopathies, which can refer to remodeled and hypertrophied myocardium as a result of inadequate oxygen delivery to the myocardium, with coronary artery disease being the most common cause.

[0105] The term "cardiomyopathy" is also sometimes used to specifically describe non-ischemic forms of cardiomyopathy, often intrinsic / idiopathic. Non-ischemic cardiomyopathy is associated with abnormalities in genes encoding sarcomeric proteins, the most common types of which are hypertrophic and dilated cardiomyopathy, in which a combination of myocardial hypertrophy and / or fibrosis occurs. Other types of non-ischemic cardiomyopathy include restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, endomyocardial fibrosis, and unclassified cardiomyopathy, including left ventricular compaction disorder (LVNC), and takotsubo cardiomyopathy.Further examples include arrhythmogenic right ventricular dysplasia (arrhythmogenic right ventricular cardiomyopathy); atrial arrest (atrial cardiomyopathy with heart block); sclerosing cardiomyopathy; congenital cataract-hypertrophic cardiomyopathy, mitochondrial myopathy; dilated cardiomyopathy; dilated cardiomyopathy-hypergonadotropic hypogonadism; dilated cardiomyopathy with ataxia; premature myopathy with fatal cardiomyopathy; encephalopathy-hypertrophic cardiomyopathy-renal tubular disease; familial dilated cardiomyopathy; familial dilated cardiomyopathy with conduction disorder due to LMNA mutations; familial hypertrophic cardiomyopathy; familial hypertrophic cardiomyopathy (familial Hypertrophic obstructive cardiomyopathy);Familial isolated arrhythmogenic right ventricular dysplasia (Familial isolated arrhythmogenic right ventricular cardiomyopathy);Familial isolated arrhythmogenic right ventricular dysplasia (Familial isolated arrhythmogenic ventricular cardiomyopathy);Familial isolated arrhythmogenic ventricular dysplasia (biventricular type) (Familial isolated arrhythmogenic ventricular cardiomyopathy, biventricular type);Familial isolated arrhythmogenic ventricular dysplasia (left ventricular type) (Familial isolated arrhythmogenic ventricular cardiomyopathy, left ventricular type);Familial isolated arrhythmogenic ventricular dysplasia (right ventricular type) (Familial isolated arrhythmogenic ventricular cardiomyopathy, classical form);Familial Isolated arrhythmogenic ventricular dysplasia (right ventricular type) (familial isolated arrhythmogenic ventricular cardiomyopathy, right ventricular type); familial isolated dilated cardiomyopathy; familial isolated dilated cardiomyopathy (familial or idiopathic dilated cardiomyopathy); familial isolated hypertrophic cardiomyopathy; familial isolated hypertrophic cardiomyopathy (familial isolated hypertrophic obstructive cardiomyopathy); familial isolated hypertrophic cardiomyopathy (familial or idiopathic hypertrophic obstructive cardiomyopathy); familial isolated hypertrophic cardiomyopathy (hypertrophic obstructive cardiomyopathy); familial isolated hypertrophic cardiomyopathy (primitive hypertrophic obstructive cardiomyopathy); familial isolated obstructive These conditions include: fascicular cardiomyopathy; familial isolated restrictive cardiomyopathy (familial or idiopathic restrictive cardiomyopathy); familial restrictive cardiomyopathy; familial restrictive cardiomyopathy types 1, 2, or 3; heart-hand syndrome; hypertrophic cardiomyopathy due to intensive athletic training; Leigh syndrome with cardiomyopathy; lipoatrophy, vitiligo-melanotic papule, fatty liver, and hypertrophic cardiomyopathy with diabetes; lysosomal disease with hypertrophic cardiomyopathy; lysosomal disease with restrictive cardiomyopathy; maternally inherited cardiomyopathy and hearing loss; and maternally inherited cardiomyopathy and hearing loss. Additional conditions are known in the art and are described, for example, in U.S. Patent Application Serial No. 14 / 893,920.

[0106] Non-limiting examples of associated disorders include, for example, secondary cardiomyopathies that may overlap with the above-mentioned forms of cardiomyopathies, the causes of which include metabolic storage diseases (e.g., amyloidosis, hemochromatosis), inflammatory (e.g., Chagas disease), endocrine (e.g., diabetic cardiomyopathy, hyperthyroidism, acromegaly), toxic (e.g., anthracycline chemotherapy, alcohol), neuromuscular (e.g., muscular dystrophies), and nutritional diseases (obesity-related). These may be in various combinations, either ischemic or non-ischemic, genetic or acquired (secondary).

[0107] "Promoting tissue healing" contemplates healing or reducing the effects of any tissue injury due to mechanical stress (e.g., puncture wounds), chemical or electrical stress, etc. The tissue can be internal or external (the outer skin of a patient or subject).

[0108] The term "suffering" in relation to the term "treatment" refers to a patient or individual who has been diagnosed with or is prone to an infection or a disease resulting from an infection. A patient may also be said to be "at risk" of a disease due to active or latent infection. The patient has not yet developed characteristic disease symptoms.

[0109] The term administration is intended to include, but is not limited to, administration orally, parenterally (e.g., intramuscularly, intraperitoneally, intravenously, ICV, intracisternal injection or infusion, subcutaneous injection, or implantation), by inhalation spray, or by nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), and may be formulated in suitable dosage unit formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration, either alone or in combination. The present disclosure is not limited by route of administration, formulation, or dosing regimen. MODES FOR CARRYING OUT THEINVENTION Recombinant stem cells

[0110] below: a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression Disclosed herein are recombinant stem cells that overexpress one or both of the following: In one embodiment, the overexpression is by expression of a CAMKK1 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO: 1 or 2 or their respective equivalents, by expression of an SDF-1 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO: 3 or 4 or their respective equivalents, and a CXCR4 polypeptide comprising, consisting essentially of, or even consisting of SEQ ID NO: 5 or their equivalents. Since some stem cells, such as mesenchymal stem cells or CD34+ stem cells, express CXCR4, in another embodiment, the stem cell of the present disclosure is a mesenchymal stem cell, a CD34+ cell, or a CD34+ stem cell, or a progenitor cell of each of these.

[0111] In another embodiment, the stem cell is selected from the group of embryonic stem cells, iPSCs, or somatic stem cells. In a further embodiment, the stem cell comprises, or consists essentially of, or even consists of a polynucleotide encoding a CAMKK1 polypeptide or its equivalent in an amount effective to induce exosome production, secretion, and / or function. The stem cell of the present disclosure may comprise, or consist essentially of, or even consist of one or more polynucleotides encoding CXCR4 and SDF-1 in an amount effective to induce exosome production, secretion, and / or function. The polypeptide may be expressed by one or more polynucleotides encoding CAMKK1, SDF-1, and / or CXCR4 polypeptides, or their respective equivalents, optionally contained within a vector. The stem cell may be a mammalian stem cell, such as a human stem cell. The vector may be a viral vector or a plasmid. Non-limiting examples of plasmid vectors include pHIV, pLVX, pLKO.1, pLemiR, pLV, pUC, pGEM, pSP73, pSP72, pSP64, pBR322, and pCCL plasmids.

[0112] The present disclosure provides an isolated stem cell or stem cell population that comprises, consists essentially of, or even consists of an isolated, exogenously added polynucleotide, viral particle, vector that encodes CAMKK1 polypeptide or protein and / or SDF-1 polypeptide or protein or their respective equivalents.The polynucleotide is introduced into the cell to overexpress CAMKK1 and / or SDF-1 protein or polypeptide or their respective equivalents.The polynucleotide can encode a mammalian polypeptide or protein (e.g., mouse, dog, cat, horse, or human), and for example, the species of the polypeptide can be the same or different from the species of the cell.In one embodiment, the polypeptide and the cell are derived from the same species (e.g., human).

[0113] The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), or somatic (adult) stem cells, and may be of any particular type, including, for example, adipose-derived, hematopoietic, mesenchymal, or neuronal. The cells may be derived from cultured cell lines available from a supplier or primary cells isolated from a subject. In one embodiment, the stem cells are mesenchymal stem cells, human stem cells, and the polynucleotide(s) encode human polypeptides and proteins.

[0114] In one embodiment, the stem cell population is substantially homogeneous or clonal. The stem cell population can be autologous or allogeneic to the subject being treated when used therapeutically.

[0115] In another embodiment, the stem cell is a stem cell of any type or species, for example a mammalian stem cell, such as a human stem cell.

[0116] Non-limiting examples of CAMKK1 polypeptides or proteins include, consist essentially of, or even consist of the sequences of SEQ ID NO: 1 or 2, or equivalents thereof. Non-limiting examples of equivalents are provided herein. Non-limiting examples of polynucleotides encoding SDF-1 polypeptides or proteins include, consist essentially of, or even consist of the sequences of SEQ ID NO: 3, or equivalents thereof, or polynucleotides encoding the polypeptide of SEQ ID NO: 4, or equivalents thereof.

[0117] These polynucleotides in stem cells can further comprise, or essentially consist of, or even consist of promoter and / or enhancer sequences operably linked to the polynucleotide.These polynucleotides can further be contained in vectors (e.g., prokaryotic or eukaryotic vectors, such as plasmid or viral vectors).Non-limiting examples of plasmid vectors include pHIV, pLVX, pLKO.1, pLemiR, pLV, pUC, pGEM, pSP73, pSP72, pSP64, pBR322, and pCCL plasmids.

[0118] The promoter may be a constitutive promoter or an inducible promoter that controls the expression of the polynucleotide(s). The polynucleotide(s) may further comprise a detectable label and / or a suicide gene and a promoter that controls the expression of the suicide gene. In a related embodiment, the suicide gene is a thymidine kinase gene. In a further embodiment, the polynucleotide(s) encoding the SDF-1 and / or CAMKK1 protein or polypeptide and the suicide gene are controlled by one promoter. In a further aspect, the promoter that controls the expression of the suicide gene is a constitutive promoter.

[0119] CAMKK1 can be overexpressed in stem cells by administering to the cells a polynucleotide encoding a wild-type CAMKK1 protein, for example, where the wild-type CAMKK1 protein is set forth in SEQ ID NO:1. In other embodiments, CAMKK1 levels can be increased in cells by administering a polynucleotide encoding a constitutively active CAMKK1. Constitutively active CAMKK1 can include, for example, the CAMKK1-413 truncation set forth in SEQ ID NO:2. Alternatively, equivalents can be provided. Non-limiting examples of equivalents include constitutively active CAMKK1, which can include a T108A mutation, an S459A mutation, or a T108A / S459A mutant CAMKK1. CAMKK1 levels can be increased in cells by administering to the cells a polynucleotide encoding a T108A mutant CAMKK1. CAMKK1 levels can also be increased in cells by administering to the cells a polynucleotide encoding a S459A mutant CAMKK1. CAMKK1 levels can be increased in cells by administering to the cells a polynucleotide encoding a T108A / S459A mutant CAMKK1. CAMKK1 levels can be increased in cells by administering to the cells a polynucleotide encoding a T108A / S459A mutant CAMKK1. This mutant is an example of an equivalent of wild-type CAMKK1.

[0120] SDF-1 can be overexpressed in stem cells by administering to the cells a polynucleotide encoding a wild-type SDF-1 protein, for example, where the wild-type SDF-1 protein is set forth in SEQ ID NO: 4 or an equivalent thereof. In other embodiments, SDF-1 levels can be increased in cells by administering a polynucleotide encoding a constitutively active SDF-1 polypeptide or protein, such as, for example, the polynucleotide of SEQ ID NO: 3 or an equivalent thereof.

[0121] In related embodiments, biological equivalents of SDF-1 and / or CAMKK1 polynucleotides include nucleic acids that hybridize under high stringency conditions to the complement of a polynucleotide encoding SEQ ID NO:3 or SEQ ID NO:1 or 2. In another embodiment, biological equivalents of the aforementioned polynucleotides include nucleic acids having at least 80% sequence identity, alternatively at least 85% sequence identity, alternatively at least 90% sequence identity, alternatively at least 92% sequence identity, alternatively at least 95% sequence identity, alternatively at least 97% sequence identity, alternatively at least 98% sequence identity to the complement or coding strand of the aforementioned polynucleotides.

[0122] The isolated cells described herein can be any cell of a species from the group of mouse, rat, rabbit, monkey, cow, sheep, pig, dog, cat, livestock, sport animals, pets, horses, and primates, particularly human cells. In one embodiment, the cell is a stem cell. In a related embodiment, the isolated cell is a mesenchymal stem cell. Expanded populations of stem cells having this phenotype are also provided, and the cells can be substantially homogenous for said phenotype. In one aspect, the cells are at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 97% homogenous for said phenotype.

[0123] In certain embodiments, the isolated cells described herein contain a certain level of SDF-1 and / or CAMKK1 protein or polypeptide. The aforementioned levels can be achieved by selecting an appropriate promoter and / or enhancer that produces the desired level of protein or by using an inducible system to control the amount of protein produced. These promoters and inducible systems have been previously described. In one embodiment, the isolated cells contain at least about 5×10 -6 In a further embodiment, the isolated cell comprises, or consists essentially of, or even consists of at least about 1×10 ng of a polypeptide or protein.-7 ng, approximately 3 × 10 -7 ng, approximately 5 × 10 -7 ng, approximately 7 × 10 -7 ng, approximately 9 × 10 -7 ng, approximately 1 × 10 -6 ng, approximately 2 × 10 -6 ng, approximately 3 × 10 -6 ng, approximately 4 × 10 -6 ng, approximately 6 × 10 -6 ng, approximately 7 × 10 -6 ng, approximately 8 × 10 -6 ng, approximately 9 × 10 -6 ng, approximately 10 × 10 -6 ng, approximately 12 × 10 -6 ng, approximately 14 × 10 -6 ng, approximately 16 × 10 -6 ng, approximately 18 × 10 -6 ng, approximately 20 × 10 -6 ng, approximately 25 × 10 -6 ng, approximately 30 × 10 -6 ng, approximately 35 × 10 -6 ng, approximately 40 × 10 -6 ng, approximately 45 × 10 -6 ng, approximately 50 × 10 -6 ng, approximately 55 × 10 -6 ng, approximately 60 × 10 -6 ng, approximately 65 × 10 -6 ng, approximately 70 × 10 -6 ng, approximately 75 × 10 -6 ng, approximately 80 × 10 -6 ng, approximately 85 × 10 -6 ng, approximately 90 × 10 -6 ng, approximately 95 × 10 -6 ng, approximately 10 × 10 -5 ng, approximately 20 × 10 -5 ng, approximately 30 × 10 -5 ng, approximately 40 × 10 -5 ng, approximately 50 × 10 -5 ng, approximately 60 × 10 -5 ng, approximately 70 × 10 -5 ng, approximately 80 × 10 -5 ng, or approximately 90 × 10 -5 ng of a polypeptide or protein, or alternatively consisting essentially of the same.

[0124] Vectors are convenient for introducing polynucleotides into stem cells. In one embodiment, the term "vector" intends a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of target cells. In some embodiments, the vector is derived from or based on a wild-type virus or a plasmid. In further embodiments, the vector is derived from or based on a wild-type adenovirus, adeno-associated virus (AAV), or lentivirus. Examples of such viruses include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Alternatively, it is expected that other retroviruses, such as murine leukemia virus (MLV), can be used as the basis for the vector backbone. It is clear that the viral vectors of the present disclosure need not be limited to components of a particular virus. A viral vector may include components from more than two different viruses and may also include synthetic components. Vector components can be engineered to obtain desired characteristics, such as target cell specificity.

[0125] The recombinant vectors of the present disclosure can be derived from primates and non-primates. Examples of primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human acquired immune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "slow virus" Visna-Maedi virus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), the more recently described Feline Immunodeficiency Virus (FIV), and Bovine Immunodeficiency Virus (BIV). Prior art recombinant lentiviral vectors are known in the art (see, for example, U.S. Patent Nos. 6,924,123; 7,056,699; 7,07,993; 7,419,829, and 7,442,551, which are incorporated herein by reference).

[0126] US Patent No. 6,924,123 discloses that certain retroviral sequences facilitate integration into the target cell genome. The patent teaches that each retroviral genome contains genes called gag, pol, and env that code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. LTRs also function as enhancer-promoter sequences. In other words, LTRs can regulate the expression of viral genes. Encapsidation of retroviral RNA occurs through the psi sequence present at the 5' end of the viral genome. The sequence of the LTRs themselves is identical, and this sequence can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The size of the three elements can vary considerably in different retroviruses. For the viral genome, a poly(A) addition (termination) site occurs to the right of the LTR, at the boundary of R and U5. U3 contains the majority of the proviral transcriptional regulatory elements, including the promoter and multiple enhancer sequences that are responsive to cellular and, in some cases, viral transcriptional activator proteins.

[0127] Regarding the structural genes gag, pol, and env themselves, gag codes for the internal structural protein of the virus. The gag protein is hydrolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene codes for the reverse transcriptase (RT) containing DNA polymerase, the associated RNase H, and integrase (IN), which mediate genome replication.

[0128] For the production of viral vector particles, the vector RNA genome is expressed in a host cell from a DNA construct encoding said genome. The components of the particle that are not encoded by the vector genome are provided in trans by additional nucleic acid sequences expressed in the host cell (usually a "packaging system" including either or both of the gag / pol and env genes). The set of sequences required for the production of viral vector particles can be introduced into the host cell by transient transfection, or said sequences can be integrated into the host cell genome, or said sequences can be provided in various ways. The techniques involved are known to those skilled in the art.

[0129] Viral vectors for use in this disclosure include, but are not limited to, Invitrogen's pLenti series versions 4, 6, and 6.2 "ViraPower" systems (from Lentigen Corp.); pHIV-7-GFP (generated in the laboratory and used by City of Hope Research Institute); the "Lenti-X" lentiviral vectors pLVX (from Clontech); pLKO.1-puro (from Sigma-Aldrich); pLemiR (from Open Biosystems); and pLV (generated in the laboratory and used by Charite Medical School, Institute of Virology (CBF), Berlin, Germany).

[0130] Non-viral vectors may include plasmids that contain exogenous polynucleotides that can be delivered to target cells either in vitro, in vivo, or ex-vivo. Non-limiting examples of plasmid vectors include pHIV, pLVX, pLKO.1, pLemiR, pLV, pUC, pGEM, pSP73, pSP72, pSP64, pBR322, and pCCL plasmids. Heterologous polynucleotides can contain sequences of interest and can be operably linked to one or more regulatory elements to regulate the transcription of the nucleic acid sequence of interest. As used herein, vectors may not be capable of replicating in the final target cell or target subject.

[0131] In one embodiment, the additional regulatory element is a promoter, an enhancer, and / or a promoter / enhancer combination. The promoter that controls the expression of the nucleic acid encoding the protein or polypeptide can be a constitutive promoter. In one aspect, the promoter that controls the expression of the suicide gene is a constitutive promoter. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG. In one aspect, the enhancer is a woodchuck posttranscriptional regulatory element ("WPRE") (see, e.g., Zufferey, R. et al. (1999) J. Virol. 73(4):2886-2992). The enhancer element can be downstream of the gene, immediately before the promoter and 3'LTR. However, the enhancer can be located anywhere and is not limited to the 3'LTR.

[0132] The promoter useful in the present disclosure can be constitutive or inducible.Some examples of promoters include T3 promoter, T7 promoter, SP6 promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter.In one embodiment, the promoter that controls the expression of tetracycline activator protein is a constitutive promoter.In other embodiments, the promoter is an inducible promoter, a tissue-specific promoter, or a promoter that controls expression transiently.In one embodiment, the promoter is a phosphoglycerate kinase promoter (PGK).

[0133] In one embodiment, the vector further comprises, consists essentially of, or even consists of a suicide gene and a promoter that controls the expression of the suicide gene. The suicide gene allows for negative selection of cells. In the methods described herein, the suicide gene is used as a safeguard that allows the introduction of a selection agent to kill cells expressing the gene. This is desirable in cases where the recombinant gene mutates and causes uncontrolled cell growth. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (tk or TK) gene, the cytosine deaminase gene, the varicella zoster virus thymidine kinase gene, the nitroreductase gene, the E. coli gpt gene, and the E. coli Deo gene (see, e.g., Yazawa K, Fisher WE, Brunicardi FC: Current progress in (See also suicide gene therapy for cancer. World J.Surg. 2002 July;26(7):783-9). In one embodiment, the suicide gene is a thymidine kinase (TK) gene. In one aspect, the TK gene is a wild-type TK gene. In another aspect, the TK gene is a mutant form of the aforementioned gene (e.g., sr23tk). Cells expressing the TK protein can be killed using ganciclovir. In another embodiment, the nucleic acid encoding the tetracycline activator protein and the suicide gene are controlled by one promoter.

[0134] In one aspect, the promoter that controls the expression of the suicide gene is a constitutive promoter. Nucleic acids encoding different proteins can be controlled by the same promoter to produce one mRNA and even two different proteins. This can be accomplished by various mechanisms. For example, a protein cleavage site can be encoded between the nucleic acids encoding the proteins within the nucleic acid. In this case, one mRNA is produced that encodes both the protein and the protein cleavage site. After mRNA transcription, the mRNA is translated into a chimeric polypeptide with both proteins linked by a protease cleavage site. This protein can then be cleaved by a protease that recognizes the cleavage site. The protease can be an endogenously expressed protease from a nucleic acid transferred into the cell by gene transfer methods, or an exogenously expressed protease. Thus, in one embodiment, the vector further comprises, consists essentially of, or even consists of a protease cleavage site between the suicide gene and the nucleic acid encoding the tetracycline activator protein. In one embodiment, the protease cleavage site is a 2A protease cleavage site. Two proteins can also be produced from the same promoter using an IRES, or internal ribosome entry site. In this case, a nucleic acid encoding an IRES is cloned between the nucleic acids encoding each protein. One mRNA is transcribed that encodes both proteins, but the IRES site allows for the separate translation of both proteins. Thus, two different proteins are produced by one mRNA.

[0135] In further embodiments, the vector further comprises a marker or detectable label, such as a gene encoding enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and the like, which are commercially available and described in the art.

[0136] Genes can be delivered to cells by various mechanisms commonly known to those skilled in the art. Viral constructs can be delivered by the production of viruses in suitable host cells. The viruses are then collected from the host cells and contacted with target cells. Viral and non-viral vectors capable of expressing genes of interest can be delivered to targeted cells via DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also contain targeting antibodies or fragments thereof can be used in the methods of the present disclosure. In addition to the delivery of polynucleotides to cells or cell populations, direct introduction of proteins described herein into cells or cell populations can be performed by protein transfection techniques (not limited to), or culture conditions that can enhance the expression and / or promote the activity of the proteins of the present disclosure are other non-limiting techniques.

[0137] Other methods for delivery of vectors encoding the genes of the present disclosure include, but are not limited to, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. Cell-derived vesicles

[0138] Cell-derived vesicles, also called extracellular vesicles, are membrane-enclosed structures released by cells in vitro and in vivo. Extracellular vesicles can contain proteins, lipids, and nucleic acids and can mediate intercellular communication between different cells (including different cell types) in the body. There are two types of extracellular vesicles: exosomes and microvesicles. Exosomes are small lipid-bound vesicles secreted by cells that mediate intercellular communication via intercellular transport of proteins and RNA (El Andaloussi, S. et al. (2013) Nature Reviews: Drug Discovery 12(5):347-357). The size of exosomes ranges from approximately 30 nm to about 200 nm. Exosomes are released from cells by fusion of multivesicular endosomes (MVEs) with the plasma membrane. Microvesicles, on the other hand, are released from cells upon direct budding from the plasma membrane (PM). Microvesicles are typically larger than exosomes, ranging from approximately 100 nm to 1 μm.

[0139] Cell-derived vesicles (e.g., exosomes and / or microvesicles) can be isolated from eukaryotic cells. Non-limiting examples of cells from which cell-derived vesicles can be isolated include stem cells. Non-limiting examples of such stem cells include adult stem cells, embryonic stem cells, embryonic-like stem cells, neural stem cells, or induced pluripotent stem cells. In some embodiments, the stem cells are adult stem cells, and optionally mesenchymal stem cells or CD34+ stem cells or CD34+ stem cells.

[0140] The stem cells of the present disclosure are modified, for example, by genetic modification. In some embodiments, the gene is modified to express at least one exogenous nucleic acid and / or at least one exogenous protein. The modification can be a transient modification. In other embodiments, the modification can be a stable modification. For example, the stem cells can be modified by transducing with a vector that comprises, or essentially consists of, or even consists of an effective amount of a polynucleotide encoding a CAMKK1 protein or polypeptide, an SDF-1 protein or polypeptide, and / or a CXCR4 protein or polypeptide, or its equivalent. Non-limiting examples of vectors include plasmid vectors and viral vectors. By modifying the cells prior to collection of cell-derived vesicles released by the modified cells, it is expected that exosomes containing different amounts and types of proteins, lipids, and nucleic acids will be collected compared to unmodified cells. Any cell modification method known to those skilled in the art can be used to modify the cells.

[0141] The cells of the present disclosure can be cultured in any culture medium known to those of skill in the art. For example, the cell culture medium can include 5%-40% fetal bovine serum (FBS), preferably about 20% FBS; 0.5%-5% L-glutamine, preferably about 1% L-glutamine; and 0.5%-1% penicillin and streptomycin (Penn-strep), preferably about 1% penn-strep, in a basal medium. In some embodiments, at least a portion of the FBS is replaced with a serum replacement, such as a platelet lysate (e.g., human platelet lysate (hPL)). In some embodiments, the amount of serum replacement (e.g., hPL) in the culture medium is 1%-20%. In some embodiments, the cells are cultured in the absence of FBS. In other embodiments, the cells are cultured in the presence of high levels of serum (e.g., 30% serum, 40% serum, 50% serum, or 60% serum).

[0142] The disclosed cells can be cultured under any condition known to those skilled in the art.Also provided herein is a method for inducing exosome secretion and / or function in stem cells, comprising, or consisting essentially of, or even consisting of upregulating the expression of CAMKK1 polynucleotide or protein or its respective equivalent in stem cells.In one embodiment, the overexpression of CAMKK1 is in the presence of expression or optional overexpression of Akt.Still further provided is a method for inducing exosome secretion and function in stem cells, comprising, or consisting essentially of, or even consisting of upregulating the expression of SDF-1 polypeptide or protein or its respective equivalent in stem cells, wherein the overexpression is in the presence of CXCR4 expression or optional expression.In one embodiment, the overexpression of SDF-1 is in the presence of expression or optional overexpression of Akt. Also provided is a method for inducing exosome secretion and function in stem cells, comprising, or consisting essentially of, or even consisting of upregulating the expression of SDF-1 polypeptide or protein or its respective equivalent in stem cells, where overexpression is in the presence of expression or optional overexpression of Akt. In one embodiment, overexpression of SDF-1 is in the presence of expression or optional overexpression of CXCR4. The stem cells can be embryonic stem cells, induced pluripotent stem cells (iPSCs), or somatic (adult) stem cells, and can be of any particular type, including, for example, adipose, mesenchymal, or neural. In another embodiment, the stem cells are mesenchymal stem cells, CD34+ cells, or CD34+ stem cells. In another embodiment, the stem cells are of any type or species of stem cells, for example, mammalian stem cells, such as human stem cells. Isolation of extracellular vesicles

[0143] The purified cell-derived vesicle populations (e.g., exosomes and / or microvesicles) of the present disclosure can be isolated using any method known to those of skill in the art and described herein. Non-limiting examples include differential centrifugation by ultracentrifugation (Thery et al. (2006) Curr. Protoc. Cell Biol. 30:3.22.1-3.22.29; Witmer et al. (2013) J. Extracellular v.2), sucrose gradient purification (Escola et al. (1998) J. Biol. Chem. 273:20121-20127), and combined filtration / concentration (Lamparski et al. (2002) J. Immunol. Methods 270:211-226). Formulations and Compositions

[0144] The present disclosure provides purified populations of cell-derived vesicles (e.g., exosomes and / or microvesicles). In some embodiments, the population of cell-derived vesicles is substantially homogeneous. In other embodiments, the population of cell-derived vesicles is heterogeneous.

[0145] In some embodiments, a substantially homogenous population is a purified population in which at least 90% of the cell-derived vesicles are less than 100 nm in diameter as determined by NanoSight LM10HS (available from Malvern Instruments Ltd, Amesbury, Mass., USA).

[0146] In some embodiments, the concentration of cell-derived vesicles in the population is approximately 10 6 In another embodiment, the concentration of cell-derived vesicles in the population is between about 10 and about 0.5 micrograms and 100 micrograms of exosomal and / or microvesicle protein recovered per cell. 6 In yet other embodiments, the concentration of cell-derived vesicles in the population is between about 10 and 20 micrograms of exosomal and / or microvesicle protein recovered per cell. 6In yet other embodiments, the concentration of cell-derived vesicles in the population is less than about 20 micrograms / 10 cell. 6 Less than a cell.

[0147] A purified population of cell-derived vesicles can be purified based on the average size of the cell-derived vesicles in the composition. Without being bound by theory, it is expected that cell-derived vesicles of different sizes may contain different types and / or amounts of nucleic acids, proteins, lipids, and other components. As such, it is expected that a composition comprising cell-derived vesicles of a certain average size may have different therapeutic efficacy compared to a composition comprising cell-derived vesicles of a different average size. In some embodiments, the average diameter of the cell-derived vesicles in the population is between about 0.1 nm and about 1000 nm. In other embodiments, the average diameter of the cell-derived vesicles in the population is between about 2 nm and about 200 nm. In other embodiments, the average diameter of the cell-derived vesicles in the population is less than 100 nm. In still other embodiments, the average diameter of the cell-derived vesicles in the population is less than 50 nm. In still other embodiments, the average diameter of the cell-derived vesicles in the population is less than about 40 nm.

[0148] The compositions disclosed herein may further comprise a carrier, for example, a pharma- ceutically acceptable carrier.In some embodiments, more than one pharma-ceutically acceptable carrier may be used.Any pharma-ceutically acceptable carrier known to those skilled in the art may be used.

[0149] In some embodiments, the pharma- ceutically acceptable carrier is a preservative, for example, a polymeric preservative.

[0150] In some embodiments, the pharma- ceutically acceptable carrier is selected from the group consisting of polyethylene glycol (PEG) (e.g., PEG 150 distearate), honey, high molecular weight proteins (e.g., bovine serum albumin or soy protein), polyvinyl alcohol, glyceryl monostearate, hyaluronic acid, glycerin, proteins, preferably from plants, preferably hydrolyzed proteins, (e.g., soy protein and silk protein), vasorin, citrocept, parabens, xanthan gum, i-karegaan, phytagel, Carbopol® polymers, and polyvinylpyrrolidone. In some embodiments, the stabilizer is selected from the group consisting of dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate. In other embodiments, the stabilizer is selected from the group consisting of DES mixtures, proteins, or high molecular weight molecules (such as human serum albumin or molecules such as dextran or poloxamer) described in U.S. Pat. No. 9,696,247.

[0151] In one aspect, a biocompatible gelling agent is an agent that is a liquid prior to application to a subject (e.g., at room temperature or below) and becomes a gel after application to a subject (e.g., at body temperature). In one embodiment, the biocompatible gelling agent is a hydrogel.

[0152] In another aspect, disclosed herein is a composition comprising exosomes and / or microvesicles and a poloxamer, wherein said composition is in a sol (liquid) phase at about 0° C. to about 20° C. and transitions to a gel (solid) phase at or near body temperature or above (such as about 25° C. to about 40° C. or about 30° C. to about 37° C.).

[0153] In some aspects, the pharmaceutically acceptable carrier is a pharmaceutically acceptable aqueous carrier (such as water or an aqueous carrier). Examples of pharmaceutically acceptable aqueous carriers include sterile water, saline, phosphate buffered saline, aqueous hyaluronic acid solution, Ringer's solution, dextrose solution, Hank's solution, and other physiologically balanced salt solutions. In some embodiments, the pharmaceutically acceptable aqueous carrier is Normosol®-R.

[0154] Nonaqueous pharma- ceutically acceptable carriers include fixed oils, vegetable oils (such as olive oil and sesame oil), triglycerides, propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate) can also be used.

[0155] Pharmaceutically acceptable carriers may also contain minor amounts of additives, such as substances that improve isotonicity, chemical stability, or cellular stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of preservatives include thimerosol, cresol, formalin, and benzyl alcohol. In certain embodiments, the pH can be modified depending on the mode of administration. In some embodiments, the composition has a pH within the physiological pH range, such as pH 7-9.

[0156] In one embodiment, depending on the type of pharma- ceutically acceptable carrier used, the compositions described herein can comprise about 0.1-100%, 0.1-50%, or 0.1-30% (such as 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the total weight of the composition, or any range between two values ​​(inclusive).

[0157] In some embodiments, any one of the above listed pharma- ceutically acceptable carriers is specifically excluded.

[0158] In some embodiments, the cell-derived vesicles described herein are frozen (e.g., flash frozen) or freeze-dried (e.g., lyophilized) to promote stability, protect against microbial activity, and extend shelf life. Those skilled in the art will understand how to reconstitute a lyophilized product prior to use.

[0159] In some embodiments, the cell-derived vesicle population described herein is used immediately after isolation. In other embodiments, the cell-derived vesicle population is cryopreserved (e.g., frozen), for example, using any cryopreservation technique known to those skilled in the art. In some embodiments, all or substantially all of the cells and / or cell debris are removed from the culture medium before cryopreservation. In some embodiments, all or substantially all of the cells and / or cell debris are removed from the culture medium after cryopreservation. Treatment method

[0160] 1. A method of promoting tissue healing, treating tissue injury, or treating a condition associated with tissue injury, comprising administering to a subject an effective amount of: a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression administering to a subject in need thereof one or both of Here, provided herein is a method, wherein said effective amount is an amount that induces exosome production, secretion, and / or function in a subject.

[0161] In one embodiment, the CAMKK1 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 1 or 2, or their respective equivalents. In another embodiment, the SDF-1 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 3 or 4, or their respective equivalents. In a further embodiment, the CXRC4 polypeptide may comprise, consist essentially of, or even consist of SEQ ID NO: 5, or their respective equivalents.

[0162] For the above method, the one or more polypeptides are administered by administration of a polynucleotide encoding the polypeptide or its equivalent. In one embodiment, the one or more polypeptides are administered in a vector that comprises, or consists essentially of, or even consists of the polynucleotide or its equivalent. The vector can be a viral vector or a plasmid vector. The polynucleotide or its equivalent or vector can be administered by administering a stem cell that comprises, or consists essentially of, or even consists of the polynucleotide or its equivalent. The cell can be autologous or allogeneic to the subject being treated. In one embodiment, the stem cell is a mesenchymal stem cell or a precursor cell thereof. For the SDF-1 polypeptide or its equivalent, it is administered locally to the injured tissue. For the CAMKK1 polypeptide or its equivalent, it is administered locally or distally to the injured tissue. In another embodiment, the tissue injury or tissue injury-associated condition is muscle or heart tissue injury or a condition associated with muscle or heart tissue injury. In a further embodiment, the associated condition comprises an ischemic condition or a cardiomyopathy. This method can be combined with other methods for treatment, etc., known in the art.

[0163] The method may further comprise, or may essentially consist of, or may even consist of, a step of determining circulating exosome levels in a sample isolated from a patient prior to administration. In another aspect, the method may further comprise, or may essentially consist of, or may even consist of, a step of determining circulating exosome levels in a sample isolated from a patient after administration. The effective amount may be determined from either the circulating exosome levels in the sample or from in-house testing or past clinical information.

[0164] It is known in the art that ischemic and inflammatory conditions can occur in several different organs or tissues. For example, ischemic conditions can occur in the heart, liver, kidney, brain, spine, lung, small intestine, large intestine, and arteries. Similarly, inflammatory conditions can occur in the heart, liver, kidney, brain, spine, lung, intestine, arteries, joints, cartilage, and skin. Thus, disclosed is a method of treating an ischemic or inflammatory condition in an organ or tissue of a patient, wherein said organ or tissue is the heart, liver, kidney, brain, spine, lung, small intestine, large intestine, arteries, joints, cartilage, skin, or any combination thereof. In some embodiments, the method can be used to treat an ischemic or inflammatory condition in the heart, such as cardiomyopathy. In some embodiments, the method can be used to treat an ischemic or inflammatory condition in the myocardium. In some embodiments, the method can be used to treat an ischemic or inflammatory condition in the liver. In some embodiments, the method can be used to treat an ischemic or inflammatory condition in the kidney. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the brain. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the spine. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the lung. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the small intestine. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the large intestine. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in an artery. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in a joint. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in cartilage. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in the skin. In some embodiments, the method may be used to treat an ischemic or inflammatory condition in any of the organs or tissues listed above.One of skill in the art can determine the duration of the practice of the method in order to obtain some clinical benefit from the reduction of clinical symptoms or clinical markers associated with inflammation, tissue injury, ischemic conditions, and / or cardiomyopathy.

[0165] Thus, the present disclosure provides a method for treating one or more of ischemic conditions, cardiomyopathies, and related disorders, or promoting tissue healing, comprising or consisting essentially of administering to a subject in need thereof an effective amount of one or more of the following: stem cells described herein, isolated culture medium described herein, cell population described herein, or composition described herein, wherein the cells and products prepared from the cells overexpress CAMKK1 polypeptide or protein or its equivalent. The cells may be autologous or allogeneic to the subject being treated. Further provided herein is a method for treating one or more of ischemic conditions, cardiomyopathies, and related disorders, or promoting tissue healing, comprising or consisting essentially of administering to a subject in need thereof an effective amount of one or more of the following: exosome population of the present disclosure, or composition described herein. Administration may be local or systemic. In one embodiment, administration is distal to the site of action, such as when the site of action is the heart. In another embodiment, the tissue injury or condition associated with tissue injury is muscle or cardiac tissue injury or a condition associated with muscle or cardiac tissue injury. The method can be combined with other methods for treatment, etc., known in the art.

[0166] Also provided herein is a method for treating one or more of ischemic conditions or cardiomyopathies and related disorders, comprising administering to a subject in need thereof an effective amount of one or more of the following: stem cells as described herein, isolated culture medium as described herein, cell population as described herein, or composition as described herein, wherein the cells and products prepared from the cells overexpress SDF-1 polypeptide or protein or its equivalent.Administration can be local or systemic. The cells or conditioned medium can be administered systemically, directly into the ischemic or inflamed tissue, or around the ischemic or inflamed tissue. Suitable systemic administration techniques include enteral or parenteral administration (injection, infusion, or implantation). The protein, vector, cell, or conditioned medium can be administered, for example, orally, epidurally, intracerebrally, intraventricularly, intraarterially, intraarterially, intraarterially, intracardially, intramuscularly, intralesionally, intraperitoneally, intrathecally, intravenously, subcutaneously, or any combination thereof. The ischemic or inflammatory condition can be acute myocardial infarction, cardiomyopathy, heart failure, chronic heart failure, peripheral arterial disease, stroke, liver disease, ischemic kidney disease, multiple sclerosis, traumatic brain injury, spinal cord injury, graft versus host disease (GVHD), diabetes, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, injury from solid organ transplantation, orthopedic injury, cartilage damage, wound, or any combination thereof.

[0167] The disclosed method may further comprise one or more additional regenerative medicines. Suitable additional regenerative medicines include, but are not limited to, mesenchymal stem cells derived from bone marrow, adipose tissue, placental tissue, umbilical cord, Wharton's jelly, menstrual blood, stem cells, M2 macrophages, monocytes, or any combination thereof. The stem cells may be neural progenitor cells, endothelial progenitor cells, organ-specific endogenous stem cells, or any combination thereof. In some embodiments, the method may further comprise administering neural progenitor cells. In some embodiments, the method may further comprise administering endothelial progenitor cells. In some embodiments, the method may further comprise administering endogenous stem cells (such as cardiac ckit+ cells). In some embodiments, the method may further comprise administering any combination of the above stem cells. The treatment method may further comprise, or may consist essentially of, or even consist of, determining circulating exosome levels in a sample isolated from the patient prior to administration. In another aspect, the method may further comprise, or may consist essentially of, or even consist of, determining the level of circulating exosomes in a sample isolated from the patient after administration. The effective amount may be determined from the level of circulating exosomes in the sample.

[0168] The administered stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), or somatic (adult) stem cells, and may be of any particular type, including, for example, adipose, mesenchymal, or neural. In one embodiment, the stem cells are mesenchymal stem cells, CD34+ cells, or CD34+ stem cells. In another embodiment, the stem cells are of any type or species, e.g., mammalian stem cells, such as human stem cells. Further provided herein are differentiated cells produced from stem cells. The cells may be allogeneic or autologous to the subject to be treated (e.g., a mammal, such as a dog, cat, horse, or human). As will be apparent to one of skill in the art, in most embodiments, the cells, polynucleotides, and the subject to be treated are of the same species (e.g., human cells transduced with human genes to treat a human patient).

[0169] As known to those skilled in the art, dosage, cells, and administration schedules are determined by the investigator and vary according to the condition being treated, the age and sex of the subject, and the species of the subject.Those skilled in the art can determine the period during which the method is successful by noting certain clinical or subclinical endpoints.Those skilled in the art can determine the period during which the method is performed to obtain some clinical benefit from the reduction of clinical symptoms or clinical markers related to inflammation, tissue injury, ischemic conditions, and / or cardiomyopathy by performing the method.The method can be combined with other appropriate treatments as determined by the treating physician.

[0170] The following examples are provided to illustrate, but not limit, the present disclosure. EXAMPLES

[0171] Experiment 1 As shown in FIG. 1, the purpose of this experiment was to determine whether CAMKK1 has an effect on chronic heart failure and how it compares to SDF-1. Based on applicants' mechanistic understanding of CAMKK1, the working hypothesis was that CAMKK1 would have no effect on cardiac function that SDF-1 would have, since applicants had previously determined that the combination of SDF-1 and CAMKK1 (not shown) was superior to SDF-1 alone. The rats used in the experiment had been through an anterior myocardial infarction for 9 months; thus, the rats had fully developed ischemic cardiomyopathy. The rats were administered either SDF-1 or CAMKK1 plasmid by opening the rats' chests and injecting the plasmid directly into the peri-infarct region. Echocardiography was performed at baseline (9 months after the heart attack) and then as a function of time after administration. FIG. 1 shows that SDF-1 and CAMKK1 individually had significant and equivalent effects on increasing ejection fraction compared to placebo. The combination of SDF-1 and CAMKK1 did not improve cardiac function any more than either of these molecules used alone. Experiment Number 2

[0172] Prior to carrying out the work described in Experiment No. 1, the applicant believed that SDF-1 acted through its chemokine effect, thereby improving cardiac function through the homing of tissue-specific stem cells and bone marrow-derived stem cells. CAMKK1 does not induce stem cell homing. This CAMKK1 and SDF-1 have equivalent effects on cardiac function, and the applicant attempted to define a common pathway through which SDF-1 and CAMKK1 may act. Exosomes were isolated from the conditioned medium of control mesenchymal stem cells (MSCs) and MSCs overexpressing either SDF-1 or CAMKK1. There were 2 million MSCs for each treatment. The isolated exosomes were then injected into the hearts of rats immediately after induction of anterior wall myocardial infarction by ligation of the left anterior descending branch of the coronary artery. As shown in Figure 2, the significant effect of SDF-1 and CAMKK1 is the release of exosomes into the conditioned medium and enhanced function. Experiment Number 3

[0173] Applicant then attempted to define the mechanism by which CAMKK1 enhances MSC effects on cardiac function. The results (see FIG. 3) show that phosphorylation of Akt is required for the enhancement of CAMKK1 MSC effects on cardiac function. In these studies, as above, conditioned medium from 2 million MSCs was collected and injected into the hearts of animals immediately after induction of anterior myocardial infarction. In two treatments, MSCs were treated with one of two Akt inhibitors, and the presence of either Akt inhibitor reduced the benefit of cells overexpressing CAMKK1 compared to that of control MSCs. Applicant also determined that SDF-1 requires Akt phosphorylation (data not shown). It is essential to note at this point that CAMKK1 is a kinase and directly phosphorylates Akt; in contrast, SDF-1 phosphorylates Akt via binding of the CXCR4 receptor. Thus, the effects of SDF-1 require an autocrine or paracrine loop to induce Akt phosphorylation, whereas the CAMKK1 effect is direct. Test Number 4

[0174] In experiment number 4, the effects of direct and indirect Akt phosphorylation were evaluated. The left anterior descending branch of the coronary artery of rats was ligated, the chest was immediately sutured, and the animals were injected with CAMKK1 plasmid, SDF-1 plasmid, or saline into the thigh. After one week, the cardiac function of animals injected with CAMKK1 was significantly increased compared to saline, but the cardiac function of SDF-1-treated animals was not increased. Test Number 5 animal

[0175] Eight-week-old male Lewis rats were maintained under standard conditions in the animal facility at Northeast Ohio Medical University. All animal procedures were approved by the Institutional Animal Care and Use Committee. Preparation of MSCs

[0176] MSCs were isolated from the bone marrow of adult Lewis rats. Cells were isolated by flushing the femur with 0.6 ml of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen, http: / / www.invitrogen.com, Carlsbad, CA, fishersci.com). Bone marrow clumps were gently minced with an 18-gauge needle, then centrifuged at 260 g for 5 min and washed with two changes of phosphate-buffered saline (Invitrogen). The washed cells were then resuspended and plated in DMEM-low glucose (Gibco, Invitrogen) containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen). Cells were incubated at 37°C, and non-adherent cells were removed by replacing the medium after 3 days. When the cultures were 70% confluent, the adherent cells were detached after a 5-minute incubation with 0.05% trypsin and 2 mM EDTA (Invitrogen) and then stained with 1 μl of primary phycoerythrin-conjugated mouse anti-rat CD45 (BD Biosciences, http: / / www.bdbiosciences.com, San Diego, CA) and CD34 antibodies (Santa Clara, Santa Fe, CA) per 10 × 6 cells using the EasySep PE selection kit (Stem Cell Technologies, Vancouver, Canada, https: / / www.stemcell.com / ). CD45+ and CD34+ cells were simultaneously depleted from MSC cultures by negative selection using each of the CD45+ and CD34+ antibodies (Cat. Cruz Biotechnology, https: / / www.scbt.com, Santa Cruz, Calif.). Finally, Icam-positive cells were selected by cell sorting. MSCs and MSC-conditioned medium

[0177] In preparation for transplantation, MSC transfection was performed by electroporation with the Neon Transfection system. Applicant reported that 2 × 10 6Cells were transfected with cDNA:CAMKK1 or siRNA:CAMKK1 or cDNA:SDF-1, cDNA AKT or siRNA:AKT, siRNA:CAMK4. Conditioned media was generated as follows: after 11 passages, 90% confluency was observed in both control and experimental groups. Conditioned media was generated by feeding the transfected cells with different treatments with serum-free DMEM and incubated for 48 hours. Control media was generated by using only untreated cells. Media was harvested and cells were counted for normalization: for each animal, applicants counted 2×10 6 The medium produced by the cells was used. After removal of cell debris, the supernatant was transferred to a 12 ml tube. Isolation of total exosomes (from conditioned medium MSCs) ExoQuick™ Precipitation

[0178] ExoQuick™ precipitation was performed according to the manufacturer's instructions (System Biosciences). Briefly, 500 μL of clarified MSC-CM was diluted to 5 mL with PBS and mixed with 1 mL of ExoQuick-TC™ solution by inverting the tube several times. Samples were incubated overnight at 4° C. and then centrifuged at 1,500 g for 30 and 5 minutes, respectively, to remove the supernatant. The supernatant was discarded and the pellet was resuspended in 200 μL of PBS. Isolated exosomes were injected into five different sites at the infarct border immediately after left anterior descending artery (LAD) ligation. Quantification and size determination of exosomes by nanoparticle tracking analysis (NTA)

[0179] Nanoparticle Tracking Analysis (NTA) is a powerful characterization technique that combines the properties of both laser light scattering microscopy and Brownian motion to obtain the size distribution of particles in suspension. The NanoSight instrument (Malvern, UK), currently the most widely used instrument for NTA in the exosome field, is equipped with an optical microscope connected to one or more lasers and a digital camera. According to the manufacturer, NanoSight is capable of characterizing particles between 10 and 2000 nm in solution. Particles are visualized by the light they scatter upon laser illumination and their Brownian motion is monitored. The NTA software is capable of classifying single particles by size by tracking their mean square displacement and thereby calculating the theoretical hydrodynamic diameter of the particle using the Stokes-Einstein equation. Based on knowledge of the analyzed sample volume, NTA is also capable of estimating the particle concentration. Size distribution analysis of homogenous particle preparations of identical or mixed sizes by NanoSight has previously been shown to be accurate. LAD ligation to induce MI

[0180] Myocardial infarction was induced in rats using a surgical microscope (LeicaMicrosystems M500) as previously described. Briefly, animals were anesthetized with intraperitoneal ketamine and xylazine, intubated, and ventilated with room air at 80 breaths / min using a pressure-controlled rodent ventilator (RSP1002; Kent Scientific Corporation). A sternotomy was performed and the proximal LAD was ligated with 7-0 prolene. LAD ligation was confirmed by anterior wall blanch and functional impairment. After LAD ligation, animals were evaluated using echocardiography and euthanized at different time points for organ harvesting (heart) and staining. Administration of exosomes after AMI

[0181] Immediately after AMI, rats were injected with saline (n=6), Exo-CAMKK1 (n=7), Exo-SDF-1 (n=7), Exo-AKT (n=6), siRNA-CAMKK1 and AKT, CAMK4 (n=7). In all groups, the injection volume was 600 μL exosome solution, which was injected into five different sites at the infarct border immediately after left anterior descending artery (LAD) ligation. Animals from each group received equal amounts of exosomes. The mortality rate of animals for this study was 10%. Echocardiographic assessment of cardiac function

[0182] As previously described ( XXXX ) Two-dimensional echocardiography was performed using a Sequoia C256 (Acuson) with a compatible 15 MHz linear array transducer. Briefly, baseline and left ventricular (LV) dimensions were quantified before LAD ligation and 2 and 6 weeks after LAD ligation. LV dimensions were quantified by digitally recorded two-dimensional clips and M-mode images from the mid-LV just below the papillary muscles to obtain consistent measurements from the same anatomical location in different rats. Ejection fraction (EF), fractional shortening, LV posterior wall diastolic thickness, LV posterior wall systolic thickness, diastolic LV internal dimensions, and systolic LV internal dimensions were measured. Echocardiograms were measured and analyzed by an investigator blinded to treatment and rat identity. statistics

[0183] Data are presented as mean±SEM. Comparisons between two groups were performed using a two-tailed Student's t-test with significance defined as p<.05. Analysis of variance (ANOVA) was used for multiple group comparisons. Characterization of MSC-derived exosomes

[0184] To investigate the effect of CAMKK1-exosome derived MSCs, applicants precipitated exosomes from conditioned medium containing 2 million control mesenchymal stem cells (MSCs) and MSCs overexpressing CAMKK1, as described above. The isolated exosomes were then injected into the hearts of rats immediately after LAD ligation. Figure 5A: The data show that CAMKK1 overexpression in MSCs increased the amount of exosomes (3.76 × 10 9 ±2.2 particles / ml, p < 0.05) compared with the control group (2.7 × 10 9 ±4.9 particles / ml), indicating that the exosome population was homogenous in terms of size. Nanoparticle tracking analysis estimated the size of exosomes to be between 10 nm and 1000 nm. Figure 5B: exo-CAMKK1 significantly improves cardiac function compared to the saline control group. A notable effect of CAMKK1 is the release of exosomes into the conditioned medium and enhanced function. Direct injection of exosomes isolated from MSCs overexpressing CAMKK1 significantly improves cardiac function 2 weeks after AMI compared to that observed with exosomes derived from control MSCs (Figure 5B). The MSC effect of CAMKK1 on cardiac function requires phosphorylation of Akt

[0185] Applicant wanted to determine the relevant phosphorylation target of CAMKK1 for the function and number of exosomes. Akt, CAMK1, and CAMK4 were inhibited. The data in Figure 6 demonstrates that the enhanced effect of CAMKK1 overexpression on MSC-derived exosomes is inhibited when Akt phosphorylation is inhibited. The data in Figure 7 further demonstrates that downregulation of CAMK1 also inhibits the enhanced effect of CAMKK1 on MSC-derived exosomes. Effects of CAMKK1 and SDF-1 on cardiac function in ischemic cardiomyopathy

[0186] Applicant has previously demonstrated that CAMKK1 does not induce SDF-1 and SDF-1 does not induce CAMKK1. Given the understanding of the mechanism of action of two different molecules, Applicant hypothesized that SDF-1 and CAMKK1 have different effects in ischemic cardiomyopathy that has developed. To test this hypothesis, Applicant injected DNA plasmids encoding SDF-1, CAMKK1, or both into the peri-infarct region of rats 9 months after AMI. As seen in Figure 8, in response to gene injection, identical improvement in cardiac function was observed between SDF-1 and CAMKK1, and no further improvement was observed with the combination (data not shown). CAMKK1 and SDF-1 mediate tissue repair through exosome release

[0187] Since the effects of SDF-1 and CAMKK1 in ischemic cardiomyopathy are similar, we investigated whether SDF-1 also mediates its effects by increasing and improving exosome function. To test this hypothesis, applicants isolated exosomes from MSCs overexpressing CAMKK1 or SDF-1. As seen in FIG. 9, the use of exosomes isolated from MSCs overexpressing CAMKK1 or SDF-1 improves cardiac function (ejection fraction) 2 weeks after AMI. Exosomes derived from MSCs overexpressing CAMKK1 or SDF-1 showed significant improvements in EF (21.8%±2.5, p<.01) and SDF-1 (16.8%±2.7, p>.05) compared to saline control group (1.9%±0.2) 2 weeks after AMI. Applicants repeated these experiments adding AMD3100, a CXCR4 inhibitor, to cell culture medium to inhibit paracrine and autocrine SDF-1 binding to its receptor. In these studies, exosomes derived from MSCs overexpressing SDF-1 in the presence of AMD-3100 did not improve cardiac function 2 weeks after AMI compared to saline controls (Figure 11, data not shown). The direct effect of CAMMK1 on exosome production has implications on distant gene expression.

[0188] The above data indicate that the effect of SDF-1 requires local CXCR4 expression; whereas the kinase CAMKK1 has no known receptor and acts directly through intracellular effects on AKt and CAMK4. Given some evidence that exosome injection improves tissue repair, applicants hypothesized that distal injection of CAMKK1 plasmid would repair myocardium, whereas SDF-1 injection into distal normal tissue would not. To test this hypothesis, applicants induced AMI by LAD ligation and injected animals with CAMKK1 or SDF-1 plasmid into the femur. As seen in FIG. 10, animals injected with AMI and CAMKK1 in the femur demonstrated significantly improved cardiac function compared to saline controls. SDF-1 plasmid injection into the femur did not significantly improve cardiac function compared to controls. Experimental Overview

[0189] Exosome release by stem cells is a growing area of ​​regenerative medicine. There is growing evidence that exosome release by MSCs mediates the tissue-preserving and tissue-healing effects of cell therapy. Over the past two decades, applicants have demonstrated in preclinical studies that overexpression of the chemokine stromal cell-derived factor-1 (SDF-1), and more recently the kinase calcium / calmodulin-dependent protein kinase kinase-1 (CAMKK1), reduces myocardial infarct size and improves cardiac function in acute myocardial infarction models. In clinical studies, applicants have demonstrated that direct injection of SDF-1 plasmid into the peri-infarct region improves cardiac function, but this improvement is limited to the most severely injured hearts. Applicants disclose herein that overexpression of CAMKK1 results in the same level of improvement in cardiac function as SDF-1 overexpression. We further demonstrate that the effects of both CAMKK1 and SDF-1 are mediated by enhanced exosome release and function. This is in direct opposition to the long-held notion that SDF-1 mediates tissue repair via stem cell homing. We further demonstrate that the effect of SDF-1 requires local tissue CXCR4 expression to release exosomes by an autocrine or paracrine loop, whereas the effect of CAMKK1 is direct and requires phosphorylation of AKT and CAMK4. Myocardial infarction was induced in an animal model, and SDF-1 or CAMKK1 plasmids were injected into the thigh of the animals. Overexpression of CAMKK1 in the thigh improved cardiac function (CAMKK1 30.3% ± 1.5, p < .01) compared to saline control group (12.9% ± 1.8); whereas overexpression of SDF-1 had no effect, whereas SDF-1 (15.7% ± 3.7, p > .05). These data demonstrate unique induction pathways for the potential repair effects of CAMKK1 and SDF-1 overexpression; suggesting that CAMKK1 overexpression is a more direct strategy to induce tissue repair. These differences are therapeutically important and help explain the failure of previous trials with SDF-1. Furthermore, to the extent that the efficacy of cell therapy is attributable to SDF-1 release, these data provide insight into why the efficacy of cell therapy has been shown to correlate with the degree of tissue injury, as greater tissue injury results in greater amounts of CXCR4 expression, thereby leading to greater production of exosomes in response to stem cell release of SDF-1.

[0190] Part of the sequence listing SEQ ID NO:1 CAMKK1 full-length protein NM_032294 (ncbi.nlm.nih.gov / nuccore / NM_032294; last accessed April 13, 2018) [ka] SEQ ID NO:2 CAMKK1 1-413 truncated polypeptide [ka] Equivalent

[0191] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative and not limiting of the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0192] Furthermore, when features or aspects of the disclosure are described in terms of Markush groups, those of skill in the art will recognize that the disclosure is also described with respect to any individual element or subgroup of elements of the Markush group.

[0193] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of promoting tissue healing, treating tissue injury, or treating a condition associated with tissue injury, comprising administering to a subject an effective amount of: a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression to a subject in need thereof, wherein the effective amount is an amount that induces exosome production, secretion, and / or function in the subject. (Item 2) The method of claim 1, wherein the CAMKK1 polypeptide comprises SEQ ID NO: 1 or 2 or their respective equivalents. (Item 3) 2. The method of claim 1, wherein the SDF-1 polypeptide comprises SEQ ID NO: 3 or 4 or their respective equivalents. (Item 4) 4. The method of claim 1 or 3, wherein the CXRC4 polypeptide comprises SEQ ID NO: 5 or an equivalent thereof. (Item 5) The method of any of the preceding items, wherein the one or more polypeptides are administered by administration of a polynucleotide encoding said polypeptide, or an equivalent thereof. (Item 6) 6. The method of claim 5, wherein the one or more polypeptides are administered in a vector containing the polynucleotide or its equivalent. (Item 7) 6. The method of claim 5, wherein the vector is a viral vector or a plasmid vector. (Item 8) 8. The method according to any one of items 5 to 7, wherein the polynucleotide or its equivalent or vector is administered by administration of stem cells comprising the polynucleotide or its equivalent. (Item 9) 9. The method of claim 8, wherein the stem cells are mesenchymal stem cells or progenitor cells thereof. (Item 10) The method according to Item 1, wherein in item 1 b, the compound is administered locally to the damaged tissue. (Item 11) The method according to item 1, wherein in item 1a, the compound is administered locally or distally to the damaged tissue. (Item 12) 20. The method of any of the preceding items, wherein the tissue is muscle tissue or cardiac tissue. (Item 13) The method of any of the preceding items, wherein the associated condition comprises an ischemic condition or a cardiomyopathy. (Item 14) The method of any of the preceding items, further comprising the step of determining circulating exosome levels in a sample isolated from the patient prior to administration. (Item 15) The method of any of the preceding items, further comprising the step of determining circulating exosome levels in a sample isolated from the patient after administration. (Item 16) 16. The method of claim 14 or 15, wherein the effective amount is determined from the level of circulating exosomes in the sample. (Item 17) a. a CAMKK1 polypeptide or its equivalent; or b. SDF-1 polypeptide or its equivalent in the presence of CXCR4 polypeptide expression Recombinant stem cells that overexpress one or both of the following: (Item 18) 18. The stem cell of item 17, wherein the overexpression is due to expression of a CAMKK1 polypeptide comprising SEQ ID NO: 1 or 2 or their respective equivalents, or due to expression of an SDF-1 polypeptide comprising SEQ ID NO: 3 or 4 or their respective equivalents, and a CXCR4 polypeptide comprising SEQ ID NO: 5 or their equivalents. (Item 19) 19. The stem cell according to item 17 or 18, wherein the stem cell is a mesenchymal stem cell, a CD34+ stem cell, or a progenitor cell of each of these. (Item 20) 19. The stem cell according to item 18, wherein the stem cell is selected from the group of embryonic stem cells, iPSCs, or somatic stem cells. (Item 21) 21. The stem cell according to any one of items 17 to 20, wherein the stem cell comprises a polynucleotide encoding a CAMKK1 polypeptide or an equivalent thereof in an amount effective to induce exosome production, secretion, and / or function. (Item 22) 22. The stem cell according to any one of items 17 to 21, wherein the stem cell comprises one or more polynucleotides encoding CXCR4 and SDF-1 in an amount effective to induce exosome production, secretion, and / or function. (Item 23) 23. The stem cell according to any one of items 17 to 22, wherein the stem cell is a mammalian stem cell. (Item 24) 24. The stem cell according to item 23, wherein the mammalian stem cell is a human stem cell. (Item 25) 25. The stem cell according to any one of items 17 to 24, wherein the polypeptide is expressed by one or more polynucleotides encoding a CAMKK1 polypeptide, an SDF-1 polypeptide, and / or a CXCR4 polypeptide, or an equivalent of each of these, optionally contained in a vector. (Item 26) 26. The stem cell of any one of items 21, 22, or 25, wherein the vector is a viral vector or a plasmid. (Item 27) 27. A method of producing therapeutic exosomes, comprising growing the stem cells according to any one of items 17 to 26 under conditions promoting exosome production and isolating exosomes produced from said stem cells. (Item 28) An isolated cell culture medium produced by the stem cells according to any one of items 17 to 26. Earth. (Item 29) 27. A population of exosomes produced by the cell of any one of claims 17 to 26. (Item 30) 29. A population of exosomes isolated from the cell culture medium of item 28. (Item 31) A composition comprising a carrier and the isolated stem cell according to any one of items 17 to 26, the isolated cell culture medium according to item 26, or one or more of the groups of populations according to items 29 or 30. (Item 32) 32. The composition according to item 31, wherein the carrier is a pharma- ceutically acceptable carrier or a biocompatible matrix. (Item 33) 33. The composition according to item 31 or 32, further comprising a preservative, a stabilizer, and / or a cryoprotectant. (Item 34) 31. A lyophilized composition comprising the population of exosomes according to item 29 or 30. (Item 35) 35. The composition according to item 34, further comprising a cryoprotectant. (Item 36) A method of treating one or more of an ischemic condition, a cardiomyopathic disorder, or promoting tissue healing, comprising administering to a subject in need thereof an effective amount of one or more of the following: the exosome population of items 29 or 30; or the composition of items 32 or 33. (Item 37) 37. The method of claim 36, wherein the administration is local or systemic. (Item 38) 37. The method of claim 36, wherein the administration is distal to the site of the tissue. (Item 39) 39. The method according to any one of items 36 to 38, wherein the tissue is muscle tissue or cardiac tissue. (Item 40) 40. The method of any one of items 36 to 39, further comprising a step of determining circulating exosome levels in a sample isolated from the patient prior to administration. (Item 41) 40. The method of any one of items 36 to 39, further comprising a step of determining circulating exosome levels in a sample isolated from said patient after administration. (Item 42) 42. The method of claim 40 or 41, wherein the effective amount is determined from the level of circulating exosomes in the sample. (Item 43) A method for inducing exosome secretion and function in a cell, the method comprising upregulating expression of a CAMKK1 polypeptide or protein or its equivalent in the cell. (Item 44) 44. The method of claim 43, wherein the cell is a stem cell. (Item 45) 45. The method of claim 44, wherein the stem cells are selected from the group of embryonic stem cells, iPSCs, or somatic stem cells. (Item 46) 45. The method of claim 44, wherein the stem cells are mesenchymal stem cells or progenitor cells thereof. (Item 47) 47. The method according to any one of items 44 to 46, wherein the stem cells are mammalian stem cells. (Item 48) 48. The method of claim 47, wherein the mammalian stem cells are human stem cells. (Item 49) 49. The method according to any one of items 44 to 48, wherein the CAMKK1 protein or polypeptide comprises the sequence of SEQ ID NO: 1 or 2 or an equivalent thereof, and expression of CAMKK1 is upregulated by transducing the stem cell with an effective amount of a polynucleotide encoding the CAMKK1 protein or polypeptide or an equivalent thereof. (Item 50) 50. The method of claim 49, wherein the polynucleotide further comprises a detectable label. (Item 51) 51. The method of claim 49 or 50, wherein the polynucleotide further comprises a promoter sequence and / or an enhancer sequence operably linked to the polynucleotide or its equivalent. (Item 52) 52. The method of claim 50 or 51, wherein the polynucleotide further comprises a vector. (Item 53) 53. The method of item 52, wherein the vector is a prokaryotic vector or a eukaryotic vector. (Item 54) 54. The method of claim 53, wherein the prokaryotic vector is a plasmid. (Item 55) 54. The method of claim 53, wherein the eukaryotic vector is selected from the group of adenovirus vectors, adeno-associated virus vectors, or retrovirus vectors. (Item 56) 56. The method of any one of items 43 to 55, further comprising culturing the cells for a time effective for the cells to secrete exosomes into the culture medium, and then optionally separating the cells from the culture medium. (Item 57) 57. The method of claim 56, further comprising isolating a population of exosomes from the culture medium or the stem cells. (Item 58) 1. A method for inducing exosome secretion and function in stem cells, comprising upregulating expression of SDF-1 protein or its equivalent in said stem cells, wherein said expression is in the presence of CXCR4 expression. (Item 59) 59. The method of claim 58, wherein the stem cells are selected from the group of embryonic stem cells, iPSCs, or somatic stem cells. (Item 60) 60. The method of claim 59, wherein the stem cells are mesenchymal stem cells or progenitor cells thereof. (Item 61) 61. The method according to any one of items 58 to 60, wherein the stem cells are mammalian stem cells. (Item 62) 62. The method of item 61, wherein the mammalian stem cells are human stem cells. (Item 63) 58 to 6, wherein the SDF-1 protein comprises the sequence of SEQ ID NO: 4 or 5 or their respective equivalents, and the expression of SDF-1 is upregulated by transducing the stem cells with an effective amount of a polynucleotide encoding the SDF-1 protein or its equivalent. 3. The method according to any one of claims 2 to 21. (Item 64) 64. The method of claim 62 or 63, wherein the SDF-1 protein is expressed by a polynucleotide, the polynucleotide further comprising a promoter sequence and / or an enhancer sequence operably linked to the polynucleotide or its equivalent. (Item 65) 65. The method of claim 64, wherein the polynucleotide further comprises a vector. (Item 66) 66. The method of claim 65, wherein the vector is a prokaryotic vector or a eukaryotic vector. (Item 67) 67. The method of item 66, wherein the prokaryotic vector is a plasmid. (Item 68) 67. The method of claim 66, wherein the eukaryotic vector is selected from the group of adenovirus vectors, adeno-associated virus vectors, or retrovirus vectors. (Item 69) 69. The method of claim 67 or 68, further comprising culturing the cells for a time effective to allow the cells to secrete exosomes into the culture medium, and then optionally separating the cells from the culture medium. (Item 70) 70. The method of claim 69, further comprising isolating a population of exosomes from the culture medium or the stem cells.

Claims

1. A composition for treating an inflammatory condition, an ischemic condition, or a cardiomyopathy, the composition comprising an effective amount of a vector comprising a polynucleotide encoding an SDF-1 polypeptide; The effective amount is an amount that induces exosome production, secretion and / or function in a subject, The composition is for local administration to damaged tissue.

2. The composition described in claim 1, wherein the damaged tissue expresses CXCR4.

3. The composition described in claim 1, further comprising a polynucleotide encoding a CXCR4 polypeptide.

4. The composition of claim 3, wherein the vector comprises a polynucleotide encoding the SDF-1 polypeptide and a polynucleotide encoding the CXCR4 polypeptide.

5. The composition of any one of claims 1 to 4, wherein the SDF-1 polypeptide comprises SEQ ID NO: 4 or an equivalent thereof.

6. The composition according to claim 1, wherein the polynucleotide encoding the SDF-1 polypeptide comprises SEQ ID NO:

3.

7. The composition of any one of claims 2 to 6, wherein the CXRC4 polypeptide comprises SEQ ID NO:5 or an equivalent thereof.

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