Extracellular vesicle microRNA and uses thereof

Abstract

Disclosed herein are compositions comprising mesenchymal stromal cell-derived extracellular vesicle-related miRNA (MSC-EV-miRNA) and their use in diagnosing subjects suffering from acute respiratory distress syndrome (ARDS) and other inflammatory diseases. Also disclosed herein are methods for creating a differential MSC-EV-miRNA profile for any disease of interest (e.g., ARDS), as well as the use of the miRNA profile in creating miRNA-based therapies. Further aspects of the disclosure relate to a primer mixture for rapid detection of a specific miRNA profile and a kit thereof.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 349,551, filed on June 6, 2022, under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety. Background of the Invention

[0004] Cell-based therapies utilizing mesenchymal stromal cells (MSCs) are increasingly being investigated as potential treatments for a variety of diseases, including, for example, ischemic stroke, myocardial infarction, and acute respiratory distress syndrome (ARDS), including ARDS caused by SARS-CoV-2 infection. However, despite significant preclinical success, results from recent human clinical studies remain variable, suggesting that new approaches are needed to improve the therapeutic efficacy of MSC-based therapies (e.g., MSC-EVs). Summary of the invention

[0005] Each limitation of the present invention may include various embodiments of the present invention. Therefore, it is contemplated that each limitation involving any one element or combination of elements in the present invention may be included in each aspect of the present invention. The application of the present invention is not limited to the construction details and component arrangements set forth in the following description or shown in the accompanying drawings. The present invention can have other embodiments and can be practiced or implemented in various ways. The details of one or more embodiments of the present invention are set forth in the accompanying detailed description, examples, and claims. Other features, objects, and advantages of the present invention will be apparent from the description and from the claims.

[0006] In some aspects, the present disclosure relates to a method for generating an EV-miRNA profile, comprising adding an activator to a culture of mesenchymal stromal cells (MSCs), determining the presence or absence of at least one MSC extracellular vesicle-related miRNA (MSC-EV-miRNA) in the culture of the MSCs using a miRNA sequencing platform, and creating an EV-miRNA profile for MSCs exposed to the activator based on the presence or absence of each miRNA relative to a control. In some embodiments, the method further comprises using the MSC-EV-miRNA profile to identify subjects receiving the MSCs for treatment of a disease. In some embodiments, the miRNA comprises hsa-miR-7107-5p. In some embodiments, the miRNA comprises hsa-miR-6803-5p. In some embodiments, the miRNA comprises hsa-miR-6798-5p. In some embodiments, the miRNA comprises hsa-miR-760. In some embodiments, the miRNA comprises hsa-miR-6727-5p. In some embodiments, the miRNA comprises hsa-miR-4763-3p. In some embodiments, the miRNA comprises hsa-miR-3652. In some embodiments, the miRNA comprises hsa-miR-885-3p. In some embodiments, the miRNA comprises hsa-miR-766-3p. In some embodiments, the miRNA comprises hsa-miR-3175. In some embodiments, the miRNA comprises hsa-miR-6893-5p. In some embodiments, the miRNA comprises hsa-miR-6875-5. In some embodiments, the miRNA comprises hsa-miR-6799-5p. In some embodiments, the miRNA comprises hsa-miR-6787-5p.

[0007] Some aspects of the present disclosure relate to methods, comprising obtaining a first biological sample from a healthy first subject and obtaining a second biological sample from a second subject suspected of having a disease, culturing the first biological sample in a first MSC culture and culturing the second biological sample in a second MSC culture, detecting the presence of at least one MSC-EV-miRNA in the first and / or second MSC cultures using a next-generation sequencing platform, creating a differential EV-miRNA profile using the next-generation sequencing platform, and determining whether the subject suspected of having the disease is a candidate for MSC-based therapy based on the differential EV-miRNA profile.

[0008] Other aspects of the present disclosure relate to pharmaceutical compositions, comprising at least one mesenchymal stromal cell-derived extracellular vesicle-related miRNA (MSC-EV-miRNA) and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition further comprises a lipid nanoparticle (LNP) encapsulating at least one MSC-EV-miRNA. In certain embodiments, the pharmaceutical composition comprises an LNP encapsulating at least four MSC-EV-miRNAs. However, in some other embodiments, the pharmaceutical composition further comprises an LNP encapsulating at least 14 MSC-EV-miRNAs. In some cases, the LNP may include a targeting moiety. In some embodiments, the miRNA includes hsa-miR-7107-5p. In some embodiments, the miRNA includes hsa-miR-6803-5p. In some embodiments, the miRNA includes hsa-miR-6798-5p. In some embodiments, the miRNA includes hsa-miR-760. In some embodiments, the miRNA includes hsa-miR-6727-5p. In some embodiments, the miRNA comprises hsa-miR-4763-3p. In some embodiments, the miRNA comprises hsa-miR-3652. In some embodiments, the miRNA comprises hsa-miR-885-3p. In some embodiments, the miRNA comprises hsa-miR-766-3p. In some embodiments, the miRNA comprises hsa-miR-3175. In some embodiments, the miRNA comprises hsa-miR-6893-5p. In some embodiments, the miRNA comprises hsa-miR-6875-5. In some embodiments, the miRNA comprises hsa-miR-6799-5p. In some embodiments, the miRNA comprises hsa-miR-6787-5p.

[0009] Some aspects of the present disclosure relate to a cell therapeutic agent comprising a culture of engineered mesenchymal stromal cells configured to release a plurality of extracellular vesicles (MSC-EVs), wherein the plurality of MSC-EVs include hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, and hsa-miR-6787-5p. In some embodiments, the cell therapeutic comprises MSC-EVs including hsa-miR-760, hsa-miR-3175, hsa-iR-885-3p, and hsa-miR-776-3p.

[0010] Certain aspects of the present disclosure also relate to a method for producing therapeutic mesenchymal stromal cells (MSCs), the method comprising obtaining a first biological sample from a healthy first subject and a second biological sample from a second subject suspected of having a disease, culturing the first biological sample in a first MSC culture and culturing the second biological sample in a second MSC culture, isolating hMSC-related extracellular vesicles (hMSC-EVs) from cell cultures, using a next-generation sequencing platform to determine whether at least one EV-related miRNA (EV-miRNA) is present in the hMSC-EVs, using the next-generation sequencing platform to create a differential EV-miRNA spectrum, and engineering the therapeutic MSCs to overexpress one or more miRNAs identified in the EV-miRNA spectrum. In some embodiments, the method further comprises delivering the therapeutic MSCs to the subject. In some cases, the method may include therapeutic MSCs that overexpress hsa-miR-760, hsa-miR-3175, hsa-miR-885-3p, and hsa-miR-766-3p.

[0011] Aspects of the present disclosure also relate to an oligonucleotide primer mixture comprising one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single target miRNA, and wherein the target miRNA is selected from hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, and hsa-miR-6787-5p.

[0012] Some aspects of the present disclosure also relate to a kit for detecting the presence of at least one mesenchymal stromal cell-derived extracellular vesicle-associated miRNA (MSC-EV-miRNA) in a biological sample, comprising a primer mixture comprising one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single target miRNA, and wherein the target miRNA is selected from the group consisting of hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-m iR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, hsa-miR-6787-5p; Reagents for performing nucleic acid assays using nucleic acid pairs to detect at least one MSC-EV-related miRNA and instructions for performing the assays to detect at least one MSC-EV-related miRNA. In some embodiments, the kit includes at least four oligonucleotides for detecting at least four of the MSC-EV-related miRNAs. In certain other embodiments, the kit includes at least eight oligonucleotides for detecting at least four of the MSC-EV-related miRNAs.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by the same numeral. For clarity, not every component may be labeled in every figure. In the drawings:

[0015] Figure 1 Schematic diagram of the experimental design. Human mesenchymal stromal cells (hMSCs) were exposed in vitro to bronchoalveolar lavage fluid (BALF) from healthy volunteers (HV) or patients with acute respiratory distress syndrome (ARDS) for 5 hours. Extracellular vesicles (EVs) contained in the supernatant were isolated, and the number of particles, size distribution, and expression of characteristic cell surface markers were characterized. MiRNAs from purified EVs were sequenced and analyzed. MiRNAs that met the statistical cutoff for differential expression were further analyzed to identify their predicted gene targets and putative functional enrichment. At the same time, partial least squares discriminant analysis (sPLS-DA) was used to identify miRNAs that classified samples into specific treatment groups. Overlap analysis was used to overlay the results from DEA and sPLS-DA to identify 14 differentially expressed miRNAs that were able to classify samples into treatment response groups. Network analysis identified 4 miRNAs as putative "hub" regulators involved in top-ranked biological pathways. EVs contained in BALF samples from ARDS patients and HV were isolated and characterized for particle number, size distribution, and expression of tetraspanin markers. miRNAs from purified BALF-derived EVs were sequenced and analyzed for differential expression.

[0016] FIG. 2A to FIG. 2D It was shown that EVs from BALF-stimulated hMSCs differed in size distribution and expression of tetraspanins compared with EVs isolated directly from BALF. Figure 2A It was shown that EVs secreted by hMSCs exposed to serum-free medium alone (control) were similar in size (50 μm to 200 μm) and number (n=3, 4, and 4 for control, HV, and ARDS, respectively) to those secreted by hMSCs exposed to BALF collected from HV or ARDS patients. As detailed in the methods, data for each condition are mean ± SEM per 100 μm bin. The AUC for each condition was not statistically significant, p>0.05. Figure 2B Shows Figure 2A Figure 2 Flow cytometry images of the same EV samples characterized in Figure 2 and shows that HV and ARDS BALF exposures overall reduced CD63-positive EVs compared to controls, with HV BALF exposures resulting in lower CD63 expression relative to ARDS BALF exposures. There were no significant differences in CD81 expression between groups, and CD9 expression was not detected in any group. * Different from control and different from HV. Figure 2CEVs present in BALF alone vs. control (serum-free medium) show a different pattern from EVs obtained from BALF-stimulated hMSCs: larger EVs are more prevalent, and ARDS BALF has a higher number of EVs (n=2, 3, 3 for control, HV, and ARDS, respectively). Data for each condition are mean ± SEM per 100 μm bin. As indicated by the asterisk, the AUC of ARDS was significantly higher than that of HV and control, p<0.05. Figure 2D Shows Figure 2B Image flow cytometry of the same EV samples characterized in and shows the presence of CD9 in addition to CD63 and CD81, but the expression of these tetraspanins did not differ between groups. Abbreviations: ARDS, acute respiratory distress syndrome; AUC, area under the curve; EV, extracellular vesicle; HV, healthy volunteers; NTA, nanotracking analysis.

[0017] FIG. 3A to FIG. 3E We show that EVs derived from quiescent control hMSCs contain miRNAs involved in cell cycle regulation. Figure 3A is the output of significance analysis of microarrays (SAM), showing over-represented miRNAs in EVs derived from control hMSCs. The delta value in SAM was set to 7 (the best delta was chosen by the software for the lowest false discovery rate [FDR]), and the FDR was 0 (%). A total of 770 miRNAs were observed to be over-represented (above the upper delta line) at a higher than expected ratio compared to the average expression of all miRNAs in replicates. There was no over-representation of miRNAs between the upper and lower delta lines. A total of 48 miRNAs were significantly over-represented (score ≥ 20, above the cutoff, in the square dashed line). Median false positives = 0; Tail Strength (%) = 99.4; se (%) = 153.7. Figure 3B Results of a plot showing functional enrichment analysis of 48 miRNAs with scores >20 (expected vs observed) performed in miRNet are shown. Putative relationships were obtained using DIANA-TarBase v8 (a collection of experimentally supported miRNA-gene interactions). Enriched terms (screened by hypergeometric test cutoff = adjusted p-value ≤ 0.05). Figure 3C The top 10 REACTOME terms are shown. The Log10 q value (FDR) of the enrichment score is plotted. The top network of miRNAs and putative targets involved in the cell cycle were generated using the "shortest path finder", a filter based on degrees (cutoff value = 3) and a minimal network algorithm. Figure 3DmiRNAs are shown, and Figure 3E Their known targets are shown. Square nodes are miRNAs (D, N=24), and circles are genes (N=61). Cell cycle related targets are highlighted (circles, N=24). Edges represent empirically proven direct interactions between nodes (TarBase v8.0). The size of the node reflects the number of direct connections. MiRNAs are labeled by their miRNA database IDs, and genes are labeled using their official HUGO symbols.

[0018] FIG. 4A to FIG. 4C Figure 3 shows miRNAs differentially expressed in EVs derived from hMSCs exposed to HV or ARDS BALF. Volcano plot of differentially expressed miRNAs for pairwise comparison (fold change (FC) in normalized counts > 2 and false discovery rate (FDR) < 0.05): Figure 4A Control versus healthy volunteers (HV) are shown; Figure 4B ARDS vs. control is shown, and Figure 4C ARDS vs. HV is shown. The total number of miRNAs that meet the differential expression criteria is illustrated in each figure. Individual differentially expressed miRNAs are drawn as circles and colored according to FC. In the volcano plot, non-differentially expressed miRNAs are shown as gray dots, and the dotted lines correspond to the FC and FDR cutoffs. The concordant regulated pathways predicted by TarBase v8 for overexpressed and underexpressed miRNAs are shown as horizontal bar graphs and colored according to the expected direction of change. The top 10 enrichment items arranged according to the hypergeometric test by adjusted p-value <0.05 (expressed as -Log10 q value) are presented.

[0019] FIG. 5A to FIG. 5G It was shown that different miRNA expression patterns were observed between the control, HV and ARDS treated groups. Figure 5A is a Venn diagram showing the overlap of miRNA expression patterns in all three pairwise comparisons. FIG. 5B to FIG. 5D Shows Figure 5B Overlap of differential expression patterns identified in over-represented miRNAs in all comparisons with control-derived EVs, Figure 5C Overlap of differential expression patterns identified in over-represented miRNAs in comparison with HV, and Figure 5D Overlap of differential expression patterns identified in the differentially expressed miRNAs after exposure to BALF from ARDS patients. Figure 5E to Figure 5Gare the consensus regulated pathways predicted by MiRNet and are shown as horizontal bar graphs of the top 10 enriched terms filtered by hypergeometric test and adjusted p-value < 0.05 (expressed as -Log10 q-value). ARDS, acute respiratory distress syndrome; C, serum-free medium control; FC, fold change; HV, healthy volunteers.

[0020] 6A to 6F We showed that 52 miRNAs were commonly found in EVs derived from hMSCs exposed to different experimental conditions. FIG. 6A to FIG. 6B is a Venn diagram showing the overlap of the 13 and 52 miRNAs differentially expressed in all treatment groups—even when considering the combined effect of control plus HV (ARDS vs [control+HV]). Figure 6C (N=13) and Fig.6D (N=52; Table 2) The top 10 pathways predicted to be affected by miRNA regulation from overlapping miRNAs are shown, ranked by hypergeometric test cutoff = adjusted p-value ≤ 0.05 (miRNet Tarbase v8). Log10 q values ​​(FDR) of the enrichment scores are plotted. FIG. 6E to FIG. 6F In Figure , the Log2 fold change (FC) of miRNAs by statistical cutoff (FC ≥ 2.0; FDR ≤ 0.05) is plotted for each miRNA in each comparison in addition to the heatmap to emphasize the consistency of these miRNA expression patterns in EVs derived from hMSCs. Individual miRNAs are shown. The color legend is Log2 FC. ARDS, acute respiratory distress syndrome; C, control; control, serum-free medium; EV, extracellular vesicles; hMSC, human mesenchymal stromal cells; HV, healthy volunteers; miR, microRNA.

[0021] 7A to 7C The top EV-mRNAs over-represented in hMSC-derived EVs were shown to be involved in inflammatory and cellular injury pathways. Fig. 7A Figure 2 is a Venn diagram showing 52 miRNAs present in EVs derived from control hMSCs that were also differentially expressed in all comparisons: ARDS vs control, ARDS vs HV, and ARDS vs HV+control. Functional enrichment analysis showed that these 52 miRNAs are involved in various pathways important in the regulation of inflammation and injury, including but not limited to Figure 7B The regulation of inflammasomes shown, as well as Figure 7C Cellular stress pathways shown. FIG. 7B to FIG. 7CNetwork analysis of the two pathways in Figure 4 shows the miRNAs associated with the network as well as their putative target genes. ARDS, acute respiratory distress syndrome; control, serum-free medium; EV, extracellular vesicles; hMSC, human mesenchymal stromal cells; HV, healthy volunteers.

[0022] Fig. 8A The top 20 (out of 52) hMSC-derived EV miRNAs are shown to exhibit different expression patterns after HV or ARDS BALF exposure. Box plots show changes in normalized miRNA counts (Log2) in EVs derived from control hMSCs (red), EVs treated with BALF from HV and ARDS patients. The line in the box is the median, the squares are the upper and lower quartiles and the whiskers are the maximum and minimum ranges. ARDS, acute respiratory distress syndrome; control, serum-free medium; EVs, extracellular vesicles; hMSCs, human mesenchymal stromal cells; HV, healthy volunteers; miR, microRNA.

[0023] FIG. 8B to FIG. 8C Figure 3 shows differentially expressed miRNAs in EVs derived from BALF-exposed hMSC-derived EVs vs EVs isolated directly from BALF. Boxplots show representative changes (Log2) in normalized counts of miRNAs considered differentially expressed in BALF versus those found to be differentially expressed in EVs derived from control hMSCs, EVs treated with BALF from HV and ARDS patients. The line in the box is the median, the squares are the upper and lower quartiles and the whiskers are the maximum and minimum range. BALF, bronchoalveolar lavage fluid sample; control, serum-free medium; HV, healthy volunteers; ARDS, acute respiratory distress syndrome; miR, microRNA; hMSC, human mesenchymal stromal cells; EV, extracellular vesicle.

[0024] 9A to 9C miRNAs identified by supervised discriminant analysis that were able to classify samples into experimental treatment groups are shown. Fig.9A A sparse PLS-DA plot is shown, where each sample is a point colored by class, and lines extend from the centroid of the group to each sample. 20 miRNAs were selected for maximum discrimination between classes. Fig. 9B ROC curves and AUC for all-vs-one comparisons are shown, where the AUC is averaged across all cross validations and is based on the maximum distance of prediction. Fig. 9Cis a box plot showing the normalized counts of all 20 miRNAs selected as classifiers by sPLS-DA. The median and upper and lower quartiles are shown; whiskers are the maximum and minimum range. ARDS, acute respiratory distress syndrome; control, serum-free medium; HV, healthy volunteers; miR, microRNA.

[0025] FIG. 10A to FIG. 10C The overlap of 14 miRNAs between discriminant and differential analysis is shown. Fig. 10A is a Venn diagram showing the overlap between the discriminant analyses. FIG. 10B to FIG. 10C The interaction network of the cellular response to stress or to Wnt signaling is shown. miRNA 766-3p, miRNA-760, miR885-3p and miRNA-3175 are shown. Genes that are in the network but not associated with the cellular response to stress or the cellular response in Wnt signaling are shown as small circles. Edges represent direct interactions between nodes (Tarbase v8). DETAILED DESCRIPTION OF THE INVENTION

[0027] Cell-based therapies utilizing mesenchymal stromal cells (MSCs) are increasingly being investigated as potential treatments for a variety of diseases, including, for example, ischemic stroke, myocardial infarction, and acute respiratory distress syndrome (ARDS), including ARDS caused by SARS-CoV-2 infection. These approaches are based on the paracrine anti-inflammatory effects of MSCs, which in the case of ARDS can reduce intra-alveolar inflammation, promote endothelial repair, increase alveolar fluid clearance, and modulate lung epithelial and endothelial permeability.

[0028] For example, during disease-mediated inflammatory responses, one mechanism by which MSCs can regulate paracrine signaling is the release of extracellular vesicles (EVs), including exosomes and microvesicles. EVs are particles that are bounded by a lipid bilayer, cannot replicate, and encapsulate cargo including a variety of signaling molecules (i.e., proteins, lipids, and nucleic acids such as mRNA, microRNA or miRNA, long non-coding RNA, DNA, and various other metabolites). Without wishing to be bound by theory, it is generally believed that MSC-EVs function by transferring cargo that communicates between adjacent and / or distant cells as paracrine signaling agents.

[0029] Several preclinical studies have consistently shown that systemic or intratracheal MSC administration, and more recently systemic administration of MSC-EVs, produces improved clinical outcomes (e.g., improved lung injury in ARDS models). However, despite the success of MSC-based cell-based therapies in treating human disease in various preclinical models (e.g., mouse models of ARDS), results from recent human clinical studies remain variable, indicating that new approaches are needed to improve the therapeutic efficacy of MSC-based therapies (e.g., MSC-EVs).

[0030] As described herein, MSC-based paracrine responses and the potential therapeutic effects resulting therefrom can differ depending on the cellular microenvironment encountered by the MSC. For example, as shown in Example 1, human MSCs (hMSCs) cultured with bronchoalveolar lavage fluid (BALF) obtained from subjects diagnosed with ARDS (ARDS BALF) differ in hMSC-EV release, expression of characteristic EV cell surface tetraspanin protein markers, and miRNA content compared to hMSCs cultured with BALF (HV BALF) obtained from healthy subjects. This discovery allows the creation of differential EV-miRNA profiles (e.g., using target prediction, functional analysis, and visual exploration), which highlights changes in miRNAs known to be associated with hMSC therapeutic effects (e.g., paracrine effects), including those involved in the regulation of inflammation, cell cycle, proliferation, and apoptosis.

[0031] Therefore, in some aspects, the present disclosure solves the aforementioned problems by providing a method for determining disease-specific MSC-EV-miRNA profiles. In some embodiments, the method can be used to identify miRNAs that are regulators of MSC paracrine responses. For example, in some embodiments, the method can be used to identify miRNAs that regulate MSC paracrine responses in ARDS.

[0032] In other aspects, the present disclosure solves the aforementioned problems by providing disease-specific MSC-EV-miRNA spectra, which can be used, for example, to identify diseases that are most likely to benefit from MSC-based cell therapeutics. For example, in some embodiments, the method includes obtaining a biological sample from a subject with and without a disease, adding it to an MSC culture, and analyzing the miRNA expression pattern, and creating a differential MSC-EV-miRNA spectrum. For example, the disease-specific differential MSC-EV-miRNA spectrum produced by exposing MSC cultures to ARDS BALF or HV BALF contains a variety of miRNAs involved in Wnt signaling and other cellular responses to stress, indicating that MSC-based cell therapeutics may have therapeutic benefits in the context of ARDS. On the other hand, MSC-EV-miRNA spectra (generated for specific purpose diseases) showing limited changes in miRNA expression patterns may indicate that MSC-based cell therapeutics may not have therapeutic benefits for specific purpose diseases.

[0033] In some aspects, the present disclosure further solves the aforementioned problems by providing a method for customizing the contents of MSC-EVs, such as using genetic engineering. In some embodiments, the method includes creating a differential MSC-EV-miRNA profile, and identifying miRNAs that counteract the pathophysiology of various diseases (such as, for example, proinflammatory responses associated with ARDS). In some cases, MSCs can be genetically modified, for example, to overexpress the identified miRNAs and package them into EVs, thereby improving the therapeutic efficacy of MSCs after administration to subjects in need.

[0034] In another aspect, the present disclosure solves the aforementioned problems by identifying factors (e.g., miRNAs) in EVs that can be used as cell-free therapeutics. This may, for example, be particularly useful where the EV-miRNA profile identifies miRNA subpopulations that regulate multiple paracrine responses. In some cases, miRNA subpopulations can be encapsulated in microcarriers such as lipid nanoparticles (optionally containing targeting moieties) and administered intravenously, intraosseously, intramuscularly, intraperitoneally, or by subcutaneous injection. In some cases, EVs can be used as drug delivery vehicles. Methods for generating MSC-EV-miRNA profiles

[0035] Aspects of the present disclosure relate to methods for generating differential MSC-EV-miRNA profiles. In some embodiments, the method includes adding an activator to a culture of mesenchymal stromal cells (MSC). In some cases, the activator may include various disease-related stimuli, such as chemical, mechanical, temperature, light, etc. For example, in some embodiments, the activator includes a biological sample (i.e., chemical stimulation) obtained from a subject suffering from a disease; in other cases, the biological sample may be obtained from a healthy subject. Exemplary embodiments of biological samples include, but are not limited to, blood, serum, urine, semen, synovial fluid, interstitial fluid (i.e., lymphatic fluid), bile, pus, phlegm, saliva, inflammatory secretions (rheum), cerebrospinal fluid, plasma, transudate, tears, gastric juice, amniotic fluid, aqueous humor, breast milk, cerumen, chyle, exudates, mucus, pericardial fluid, peritoneal fluid, pleural effusion, sebum, serous fluid, sputum, sweat, vomitus, bronchoalveolar lavage fluid (BALF), etc.

[0036] According to other embodiments, the biological sample can include all human tissues (e.g., fresh, frozen, fixed or processed) and / or all human blood (e.g., peripheral blood and / or umbilical cord blood) and blood byproducts (e.g., serum, plasma, buffy coat) and / or all human biological fluids (e.g., sputum, urine, bile) and / or human primary cells derived from a human biological sample, and / or any DNA derived from an individual donor.

[0037] In other embodiments, the activator may include a solution containing one or more reagents related to a disease (e.g., inflammation) known to those skilled in the art. In certain embodiments, the activator includes one or more immune and / or inflammatory cells, such as Th1 cells, CD4+ cells, macrophages, dendritic cells, or any combination thereof. In other cases, the activator includes one or more types of cytokines, such as, for example, lymphokines (cytokines produced by lymphocytes), monokines (cytokines produced by monocytes), chemokines (cytokines with chemotactic activity) and / or interleukins (cytokines produced by a kind of leukocytes but acting on other leukocytes). Non-limiting examples include but are not limited to interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-17 (IL-17), interleukin-18 (IL-18), IFN-γ and TNF-α.

[0038] In some embodiments, the activator includes mechanical stimulation. For example, many solid tumors (e.g., glioblastoma, prostate, breast) show high solid stress driven by hypoxic tumor microenvironment. In these cases, the activator may include, for example, the oxygen tension of MSC culture. For example, in some cases, the method includes reducing the oxygen tension of MSC culture from ~20% oxygen to 1% oxygen to 5% oxygen (percentage is volume percentage). In some embodiments, the method includes reducing the oxygen tension to greater than or equal to 1% of the total gas volume, greater than or equal to 2%, greater than or equal to 3%, greater than or equal to 4%, greater than or equal to 5%, etc. In other embodiments, the method includes reducing the oxygen tension to less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, etc. of the total gas volume.

[0039] Other mechanical stresses, such as shear stress or cyclic mechanical strain, can also be used as activators. For example, in some embodiments, MSC can be grown on microcarriers and amplified in a stir-batch bioreactor, where the stirring behavior induces fluid shear stress on the cultured MSC. Other types of mechanical stimulation can include acoustic activation (e.g., subjecting MSC cultures to ultrasonic treatment) and / or repeated exposure to tensile or compressive forces (e.g., after growing MSCs on a flexible substrate). Exposure to various temperature cycles and wavelengths of light can also be used to enhance the MSC microenvironment and stimulate changes in EV miRNA content. Combinations are also possible, for example, MSC cultures can be exposed to activator solutions and low oxygen tension.

[0040] After MSC culture is exposed to activator, mesenchymal stromal cell extracellular vesicle (MSC-EV) can be harvested, and the miRNA content in the MSC-EV is determined. Commercially available package products such as Exoseme Isolation Kits (MiltenyliBiotec) and exoEasy Maxi Kit (Qiagen) can be used, or by any other technology known to those skilled in the art, such as, for example, magnetic separation, ultracentrifugation, differential ultracentrifugation, sequential centrifugation, fractionation based on size (such as tangential flow filtration and size exclusion chromatography) harvest MSC-EV from cell culture medium. After separating EV, related miRNA can be separated (for example, using standard laboratory specifications). In most cases, commercially available package products can be purchased and used to extract miRNA (for example, Qiagen's exoRNeasy Midi and Maxi Kits). The miRNAs can then be sequenced using nucleic acid-based detection assays (e.g., using pyrosequencing on the 454 Life Sciences platform, polymerase-based synthesis sequencing on the Illumina platform, sequencing by ligation on the ABI solid sequencing platform, or the HTG EdgeSeq-miRNA whole transcriptome assay) to identify the miRNAs present in the EVs. The latter can include the identification of known miRNAs and the identification of novel miRNAs (e.g., by performing miRNA alignment analysis).

[0041] In some embodiments, the miRNA sequencing analysis measures the expression of 1000 to 5000 human miRNA transcripts. In some embodiments, the sequencing analysis measures greater than or equal to 1000, greater than or equal to 2000, greater than or equal to 3000, greater than or equal to 4000, greater than or equal to 5000 human miRNA transcripts. In other embodiments, the sequencing analysis measures less than or equal to 5000, less than or equal to 4000, less than or equal to 3000, less than or equal to 2000, or less than or equal to 1000 human miRNA transcripts.

[0042] After miRNA sequencing of MSC-EV-miRNA, miRNA differential expression analysis can be performed. Differential expression analysis can be used, for example, to compare the effects of two different activators on MSC-EV-miRNA content (e.g., ARDSBALF vs. HV BALF) or the effects of a single activator at different time points. For example, in some embodiments, relative to MSCs cultured with HV BALF, cultures of MSCs treated with ARDS BALF induce overexpression of hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, hsa-miR-6787-5p, etc.

[0043] Differential expression analysis (DEA) can be performed using any software known to those skilled in the art, for example, DESeq2 in R (R package "DESeq2"). Without wishing to be bound by theory, in some cases, a variance stabilizing transformation can be used to prepare miRNA sequence data for differential expression analysis. In some embodiments, the differential expression analysis includes running a microarray significance analysis (SAM) of standardized read counts using a single-class analysis method to identify miRNAs that are over-represented in EVs derived from control samples. In other embodiments, the differential expression analysis includes setting the delta value to 7 (i.e., to ensure the lowest false discovery rate [FDR]) using 1000 permutations. In some cases, miRNAs considered to be differentially expressed (DE) exhibit expression changes greater than or equal to 1.2 times, expression changes greater than or equal to 1.5 times, expression changes greater than or equal to 1.7 times, expression changes greater than or equal to 2 times, expression changes greater than or equal to 2.5 times, expression changes greater than or equal to 3 times, and the like. In other embodiments, miRNAs considered to be differentially expressed exhibit less than or equal to a 3-fold change in expression, less than or equal to a 2.5-fold change in expression, less than or equal to a 2-fold change in expression, less than or equal to a 1.7-fold change in expression, less than or equal to a 1.5-fold change in expression, less than or equal to a 1.2-fold change in expression, etc. In some cases, miRNAs considered to be differentially expressed (DE) can be selected based on a greater than or equal to 2-fold change in expression (i.e., mean expression of samples >50 read counts) and an adjusted P value (or FDR) <0.05 after correction for multiple comparisons.

[0044] In some embodiments, the method further comprises performing target prediction and enrichment analysis. Target prediction analysis identifies miRNA target mRNAs and helps provide an understanding of the genes or gene networks whose expression is regulated. Without wishing to be bound by theory, target prediction analysis can be performed using commercially available software (e.g., using software such as Reactome, RNA22, TargetScan, miRanda, PicTar, miRNet, etc.), and generally involves: (1) determining miRNA: mRNA binding pairs. This can be accomplished by identifying the complementarity between miRNA sequences at the 3'-UTR of the mRNA sequence. (2) determining the conservation of miRNA: mRNA binding pairs across species, and (3) observing evidence of miRNA targeting in mRNA-seq or protein expression data, where if miRNA expression is high, the gene and protein expression of its target gene should be low.

[0045] According to some embodiments, after identifying the target mRNA, gene set enrichment analysis can be performed. As will be appreciated by those skilled in the art, this analysis can be used for, for example, identifying the categories of genes or proteins that are over-represented in a large number of genes or proteins, and can be associated with disease phenotypes. As with other "omics platforms", there are many commercially available tools for performing gene enrichment analysis. Exemplary tools can include but are not limited to NASQAR, PlantRegMap, Blas2Go, GREAT, MSigDB, etc.

[0046] Other aspects of the method include performing sparse partial least squares discriminant analysis (sPLS-DA). In the case of not wishing to be bound by theory, sPLS-DA is a statistical method that uses a linear regression model to find the basic relationship between a response (e.g., y variable, ARDS positive or negative patient) and an independent variable (e.g., x variable, miRNA expression). This is achieved by using a hidden variable method, which allows the categorical response variable (e.g., ARDS positive or negative patient sample) to be analyzed as a continuous variable. This allows the model to perform variable selection and classification in a one-step method. In other words, sPLS-DA is performed to sequencing data (e.g., miRNA sequencing data) and can be used to create a mathematical equation to associate x variables (e.g., EV-miRNA) and y variables (e.g., ARDS positive, ARDS negative, control). For example, this equation can therefore be used to classify the disease of interest as having responsiveness or non-responsiveness to a therapy based on MSC cells. For example, by providing the identity of therapeutic miRNA and an estimate of its relative concentration (e.g., for encapsulation in lipid nanoparticles), the equation can also be used to formulate acellular drug therapy.

[0047] In some embodiments, performing the sPLS-DA analysis includes obtaining a library of miRNA sequences. In some embodiments, the library of miRNA sequences may include 1000 to 5000 human miRNA transcripts. In some embodiments, the library includes greater than or equal to 1000, greater than or equal to 2000, greater than or equal to 3000, greater than or equal to 4000, greater than or equal to 5000 human miRNA transcripts. In other embodiments, the library includes less than or equal to 5000, less than or equal to 4000, less than or equal to 3000, less than or equal to 2000 or less than or equal to 1000 human miRNA transcripts.

[0048] In some embodiments, performing sPLS-DA analysis includes randomly splitting the miRNA sequencing data into a training set and a test set using a 0.7 / 0.3 split. In other embodiments, the ratio of the training set / test set can be greater than or equal to 0.1 / 0.9, greater than or equal to 0.2 / 0.8, greater than or equal to 0.3 / 0.7, greater than or equal to 0.4 / 0.6, greater than or equal to 0.5 / 0.5, greater than or equal to 0.6 / 0.4, greater than or equal to 0.7 / 0.3, greater than or equal to 0.8 / 0.2, greater than or equal to 0.9 / 0.1. In some embodiments, the ratio of miRNAs in the training set can be less than or equal to 0.9 / 0.1, less than or equal to 0.8 / 0.2, less than or equal to 0.7 / 0.3, less than or equal to 0.6 / 0.4, less than or equal to 0.5 / 0.5, less than or equal to 0.4 / 0.6, less than or equal to 0.3 / 0.7, less than or equal to 0.2 / 0.8, or less than or equal to 0.1 / 0.9.

[0049] In some embodiments, the method includes performing overlapping analysis and / or network analysis. In the case of not wishing to be bound by theory, for example, when comparing the differentially expressed genes between different experimental groups and / or analytical techniques (e.g., differential expression analysis vs. sPLS-DA), overlapping analysis can be useful. For example, the "direct" interaction predicted between the differentially expressed target miRNA and its target gene can be visualized using network analysis. These methods can allow identification of one or more subgroups of miRNA, for example, as a master regulator or "center" of MSC paracrine signaling (e.g., for a given activator, such as ARDS BALF).

[0050] ARDS-specific MSC-EV-miRNA profile

[0051] In some cases, aspects of the present disclosure relate to using the aforementioned methods to identify MSC-EV-miRNA profiles for a disease of interest, such as, for example, ARDS. For example, Examples 1 to 8 highlight the use of the methods to determine differential MSC-EV-miRNA profiles for subjects with acute respiratory distress syndrome (ARDS). Thus, in some cases, the MSC-EV-miRNA profile of ARDS includes, but is not limited to, the following 14 differentially regulated miRNAs: hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, hsa-miR-6787-5p, wherein these miRNAs co-regulate processes such as inflammation, cell cycle, proliferation, apoptosis, and Wnt signaling. In some embodiments, differentially regulated miRNAs can be further screened for reduced miRNA groups that work together to form putative in silico regulatory networks as putative central regulators of the entire gene target group (in the case of the ARDS spectrum, which is ~1259 putative targets). In the case of the MSC-EV-miRNA spectrum (generated from ARDS BALF and HV BALF), in some embodiments, the reduced miRNA group includes miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p.

[0052] Other target diseases can also be analyzed using the aforementioned methods. For example, other pulmonary diseases (e.g., chronic lung disease, chronic obstructive pulmonary disease, emphysema, asthma, etc.), nervous system diseases (e.g., epilepsy, post-traumatic brain injury, premature brain injury and stroke), ischemic diseases (e.g., myocardial infarction, chronic renal failure respiratory failure), arthritis diseases (e.g., osteoarthritis and rheumatoid arthritis), infectious diseases, various ophthalmic diseases and cancer MSC-EV-miRNA spectra can be generated. In some embodiments, it is also possible to analyze MSC-EV contents during the regeneration process (e.g., regeneration of bone, liver, heart, muscle, blood cells).

[0053] Methods using MSC-EV-miRNA profiling

[0054] In some cases, aspects of the present disclosure relate to methods for identifying diseases that may benefit from MSC therapy (such as, for example, cancer immunotherapy). For example, in some embodiments, the method includes obtaining a first biological sample from a healthy first object, and obtaining a second biological sample from an object suspected of having a disease (e.g., cancer, ARDS, myocardial infarction). In other embodiments, the method includes adding the first biological sample and the second biological sample to the first MSC culture and the second MSC culture, respectively, and using, for example, next-generation sequencing (e.g., microRNA-Seq) to detect whether there is at least one MSC-EV-miRNA in the first and / or second MSC culture. As described elsewhere herein, a series of analyses (e.g., functional prediction, overlap analysis, and discriminant analysis) can be completed after miRNA sequencing to create disease-specific differential EV-miRNA profiles based on the presence or absence of each miRNA (relative to a control). According to certain embodiments, the differential MSC-EV-miRNA profile indicating the overexpression of the relevant miRNA can indicate that the object suspected of having the disease may be a candidate for MSC-based therapy. Likewise, according to other embodiments, a miRNA profile indicative of limited changes in the differential miRNA expression profile may indicate that a subject suspected of having the disease may not be a candidate for MSC-based therapy.

[0055] Pharmaceutical composition

[0056] In some cases, aspects of the present disclosure relate to pharmaceutical compositions for delivering one or more miRNAs from MSC-EV-miRNA spectra, wherein the pharmaceutical composition does not include complete MSC or MSC-EV. In some embodiments, the pharmaceutical composition includes at least one naked miRNA (e.g., identified using MSC-EV-miRNA spectra). As used herein, "naked miRNA" refers to miRNA that is not compounded with another compound (e.g., not encapsulated in lipid nanoparticles). In some embodiments, the pharmaceutical composition includes at least one miRNA (e.g., identified using MSC-EV-miRNA spectra), and a pharmaceutically acceptable excipient (e.g., carrier). As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmacologically inactive material used to prepare a composition together with a pharmacologically active material. Pharmaceutically acceptable excipients include various materials known in the art, including but not limited to sugars (e.g., glucose, lactose, etc.), preservatives such as antibacterial agents, reconstruction aids, colorants, saline (e.g., phosphate buffered saline) and buffers.

[0057] In some embodiments, the pharmaceutical composition comprises at least one miRNA (identified using MSC-EV-miRNA spectrum), and a delivery vehicle encapsulating the at least one miRNA, for example, a lipid nanoparticle. In some cases, the pharmaceutical composition comprises greater than or equal to 1 miRNA, greater than or equal to 2 miRNAs, greater than or equal to 3 miRNAs, greater than or equal to 5 miRNAs, greater than or equal to 7 miRNAs, greater than or equal to 10 miRNAs, greater than or equal to 12 miRNAs, greater than or equal to 14 miRNAs, greater than or equal to 16 miRNAs or greater than or equal to 20 miRNAs. In other embodiments, the pharmaceutical composition comprises less than or equal to 20 miRNAs, less than or equal to 16 miRNAs, less than or equal to 12 miRNAs, less than or equal to 10 miRNAs, less than or equal to 7 miRNAs, less than or equal to 5 miRNAs, less than or equal to 3 miRNAs or less than or equal to 1 miRNA.

[0058] The at least one miRNA may include hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, hsa-miR-6787-5p or any combination thereof.

[0059] In some embodiments, the delivery vehicle may include lipid nanoparticles and / or liposomes. "Liposome" is a general term, including various single and multi-layer lipid vehicles formed by producing a closed lipid bilayer or aggregate. The feature of liposomes may be a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multi-layer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess aqueous solution, they form spontaneously. The lipid component undergoes self-rearrangement before forming a closed structure, and captures water and dissolved solutes (Ghosh et al., 1991 Glycobiology 5: 505-10) between lipid bilayers. However, compositions having a structure different from a normal vesicle structure in a solution are also included. For example, lipids may present a micellar structure, or exist only as an inhomogeneous aggregate of lipid molecules.

[0060] The at least one miRNA can be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome by a linker molecule associated with the liposome and the oligonucleotide, captured in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, or otherwise associated with lipids. The lipid or lipid / nucleic acid composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, such as a micelle, or have a "collapsed" structure. They may also be simply dispersed in a solution, possibly forming aggregates of uneven size or shape.

[0061] In another embodiment, the liposome includes a transfection reagent (e.g., a cationic and / or anionic lipid). In another embodiment, the liposome increases intracellular stability, increases uptake efficiency, and improves biological activity. In another embodiment, the liposome is a hollow spherical vesicle composed of lipids, and the arrangement of the lipids is similar to those lipids that constitute the cell membrane. In some embodiments, the liposome includes an internal aqueous space for capturing water-soluble compounds. In another embodiment, the liposome can deliver the at least one MSC-EV-miRNA to the cell in an active form.

[0062] In one embodiment, the composition comprises lipid nanoparticles (LNPs) and at least one miRNA.

[0063] The term "lipid nanoparticle" (LNP) refers to a particle having at least one dimension at the nanoscale (e.g., 1 nm to 1000 nm) comprising one or more lipids. In some embodiments, the LNP comprises at least one agent organized in a reverse lipid micelle and encapsulated in a lipid monolayer envelope, or inserted between adjacent lipid bilayers (e.g., lipid bilayer-agent-lipid bilayer). In some embodiments, the morphology of the LNP is unlike conventional liposomes, characterized in that the lipid bilayer surrounds an aqueous core, because they have an electron-dense core in which cationic / ionizable lipids are organized into reverse micelles surrounding an encapsulated agent.

[0064] In some embodiments, the lipid nanoparticles are substantially non-toxic.In certain embodiments, the at least one agent is resistant to degradation by intracellular or intercellular enzymes in aqueous solution when present in the lipid nanoparticles.

[0065] LNP can include any lipid that can form a particle, and the at least one miRNA is attached to the particle, or the at least one miRNA is encapsulated in or compounded in the particle.Term " lipid " refers to a group of organic compounds, which are derivatives (e.g., esters) of fatty acids, and their general characteristics are insoluble in water but soluble in many organic solvents.Exemplary lipids are shown elsewhere herein.

[0066] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids, anionic lipids and pegylated lipids.

[0067] In one embodiment, LNP includes cationic lipids. As used herein, the term "cationic or ionizable lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH value is reduced to below the pKa of the lipid ionizable group, but at higher pH values, it gradually becomes more neutral. At a pH value lower than pKa, the lipid is therefore able to associate with negatively charged nucleic acids. In certain embodiments, cationic lipids include zwitterionic lipids that present positive charges when the pH is reduced.

[0068] In various embodiments, the LNPs comprise a cationic or ionizable lipid, a stabilizing lipid, a sterol, and a lipid-anchored polyethylene glycol (ie, PEGylated lipid).

[0069] In certain embodiments, the LNP comprises one or more stabilizing lipids (eg, neutral or anionic lipids) that aid in encapsulation of the cargo and stabilize particle formation during particle formation.

[0070] In various embodiments, the LNP further comprises a steroid or a steroid analog.

[0071] In certain embodiments, LNP comprises one or more targeting moieties that target LNP to a cell or cell population. For example, in one embodiment, the targeting domain is a ligand that directs LNP to a receptor found on the cell surface. Exemplary targeting domains include but are not limited to Toll-like receptors or other damage or pathogen-related molecule receptors.

[0072] In certain embodiments, LNPs are formed by co-injecting an aqueous solution of mRNA and an ethanolic solution of lipids through a microfluidic device, resulting in spontaneous vesicle formation.

[0073] In certain embodiments, LNP comprises one or more internalization domains. For example, in one embodiment, LNP comprises one or more domains, which bind cells to induce the internalization of LNP. For example, in one embodiment, the one or more internalization domains bind receptors found on the cell surface to induce receptor-mediated LNP uptake. In certain embodiments, LNP can bind biomolecules in vivo, wherein the biomolecules bound by LNP can therefore be recognized by cell surface receptors to induce internalization. For example, in one embodiment, LNP binds systemic ApoE, which results in the uptake of LNP and related goods.

[0074] Other exemplary LNPs and their manufacture are described in the art, for example, in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7):1357-1364; Basha et al., 2011, Mol Ther, 19(12):2186-2200; Leung et al., 2012, JPhys Chem C Nanomater Interfaces, 116(34):18440-18450; Lee et al., 2012, Int J Cancer., 131(5):E781-90; Belliveau et al., 2012, Mol Ther nucleic acid Acids, 1:e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34):8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8):1570-1578; and Tam et al., 2013, Nanomedicine, 9(5):665-74, each of which is incorporated by reference in its entirety.

[0075] Engineering MSCs for cell therapeutics

[0076] In some cases, aspects of the present disclosure relate to cell therapeutic agents, comprising genetically engineered MSC cells, which are configured to overexpress one or more miRNAs identified using MSC-EV-miRNA profiles and packaged into EVs. In certain embodiments, the cell therapeutic agent comprises engineered MSCs (i.e., MSCs and EVs); however, in some cases, secreted EVs can be isolated and used to deliver the miRNA of interest directly to a subject (e.g., similar to LNPs).

[0077] In some embodiments, MSCs can be genetically engineered to overexpress hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, hsa-miR-6787-5p, or any combination thereof. In other embodiments, MSCs can be genetically engineered to overexpress hsa-miR-760, hsa-miR-3175, hsa-miR-885-3p, has-miR-766-3p, or any combination thereof. Other miRNAs (and combinations thereof) are also possible.

[0078] In some embodiments, the cell therapy comprises a MSC culture that overexpresses a single target miRNA, such as a miRNA identified from a MSC-EV-miRNA profile. In certain embodiments, the cell therapy comprises a mixture of MSC subpopulations, each of which overexpresses a single target miRNA. The cell therapy may also comprise one or more MSC subpopulations that are engineered to overexpress multiple miRNAs (see below).

[0079] In some cases, aspects of the present disclosure relate to methods for producing therapeutic MSCs, wherein the therapeutic MSCs are genetically engineered MSC cells configured to overexpress one or more miRNAs determined from the MSC-EV-miRNA spectrum. In some embodiments, the method includes obtaining a first biological sample from a healthy first subject, and obtaining a second biological sample from a subject suspected of having a disease (e.g., cancer, ARDS, myocardial infarction). In other embodiments, the method includes adding the first biological sample and the second biological sample to the first MSC culture and the second MSC culture, respectively, and using, for example, next-generation sequencing (e.g., microRNA-Seq) to detect the presence or absence of at least one MSC-EV-miRNA in the first and / or second MSC culture. As described elsewhere herein, a series of analyses (e.g., functional prediction, overlap analysis, and discriminant analysis) can be completed after miRNA sequencing to create differential EV-miRNA profiles based on the presence or absence of each miRNA relative to the first healthy subject (or other appropriate controls). Then, the miRNA overexpressed in the MSC-EV-miRNA spectrum can be used as a therapeutic target overexpressed in clinical-grade MSCs.

[0080] Any technology known to those of ordinary skill in the art can be used to engineer MSCs to overexpress the target miRNA, such as, for example, infection with a virus carrying a target gene (e.g., a recombinant gene) or by directly transferring a plasmid DNA carrying a target gene (e.g., a recombinant gene). For example, any target miRNA, e.g., hsa-miR-760, can be cloned into, for example, a commercially available lentiviral vector system (e.g., XMIRXpress cloned lentiviral vector, systemBiosciences), the system comprising an RNA sequence tag, e.g., XMotif, which targets small RNA to exosomes for packaging. In some embodiments, engineered MSCs can express the target recombinant gene indefinitely (i.e., the effect is similar to a cell line), but in some cases, engineered MSCs can only express the target recombinant gene transiently.

[0081] In some embodiments, the method includes engineering MSC to overexpress more than one target gene. Any technology known to those of ordinary skill in the art can be used for engineering MSC, and the MSC overexpresses multiple target genes and packages them into EV. For example, in some cases, different expression vectors can be used, each carrying different target miRNA genes. In some cases, a single vector can be constructed, which contains multiple genes, each with its own promoter. Some other options may include using a translation fusion method, in which two target genes are genetically connected in the framework, which can ensure the stoichiometric production of two miRNAs. Another strategy can include using an internal ribosome entry site (IRES), which promotes ribosome binding to the second and subsequent transcription units. Other strategies and methods are also possible. According to certain embodiments, the method also includes delivering therapeutic MSC to a subject in need (e.g., a subject diagnosed as ARDS).

[0082] Primers for rapid detection of miRNA profiling

[0083] In some cases, aspects of the present disclosure relate to oligonucleotide primer mixtures. For example, primer mixtures can be used to quantify the expression of miRNA in MSC-EV (i.e., biomarkers of QC) to ensure the correct combination of miRNA (e.g., miRNA identified in the MSC-EV-miRNA spectrum) to produce. In some cases, the primer mixture includes a forward primer and / or a reverse primer. In some embodiments, the oligonucleotide primer mixture includes a stem-loop reverse transcriptase (stem-loop RT) primer and / or a linear primer. In certain embodiments, the primer mixture comprises one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single target miRNA, wherein the target miRNA is selected from hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, and hsa-miR-6787-5p.

[0084] In some embodiments, an oligonucleotide primer mixture comprising one or more oligonucleotide primers can be used to detect the presence (or absence) of a target miRNA in a sample (e.g., during a quality control check of an MSC cell therapeutic). For example, in some embodiments, a stem-loop hairpin RT can be used to bind a target miRNA at the 3' end, followed by reverse transcription using a reverse transcriptase (e.g., MultiScribe reverse transcriptase), and quantification of the RT product using conventional PCR (e.g., Taqman PCR), the conventional PCR comprising a miRNA-specific forward primer, a reverse primer, and a dye-labeled probe (e.g., Taqman probe).

[0085] Quantitative RT-PCR using DNA primers is another example in which a primer mixture can be used to quantify the target miRNA in a sample. Without wishing to be bound by theory, the method relies on poly (A) tailing of the miRNA followed by reverse transcription (RT) with a labeled poly (T) primer. The RT product can then be quantified using standard PCR with a primer set (e.g., forward and reverse primers) specific to the target miRNA transcript (e.g., 5' tag and 3' tag).

[0086] In certain embodiments, the number of primers in the primer mixture can be greater than or equal to 1 primer, greater than or equal to 2 primers, greater than or equal to 4 primers, greater than or equal to 6 primers, greater than or equal to 8 primers, greater than or equal to 10 primers, greater than or equal to 12 primers, greater than or equal to 14 primers, greater than or equal to 20 primers or more. In other embodiments, the number of primers in the primer mixture is less than or equal to 20 primers, less than or equal to 14 primers, less than or equal to 12 primers, less than or equal to 10 primers, less than or equal to 10 primers, less than or equal to 8 primers, less than or equal to 4 primers, less than or equal to 2 primers, less than or equal to 2 primers, less than or equal to 1 primer, etc.

[0087] Primer Set Products

[0088] In some cases, aspects of the present disclosure relate to kits for detecting in a biological sample the presence of at least one miRNA identified using a MSC-EV-miRNA profile. In some embodiments, the kit comprises a primer mixture comprising one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single miRNA of interest, wherein the miRNA of interest is selected from the group consisting of hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p, and hsa-miR-6787-5p. According to certain embodiments, the package product may also include a reagent (e.g., a fluorescent probe) for performing a nucleic acid assay (e.g., PCR) to detect at least one MSC-EV-miRNA using a nucleic acid pair, and instructions for performing the assay to detect at least one MSC-EV-miRNA. According to some embodiments, the primer mixture comprises 1 to 14 primer pairs for detecting up to 14 different miRNAs. In other embodiments, the primer mixture comprises 1 to 28 primers for detecting 1 to 14 target miRNAs. In some embodiments, the package product can be used to screen for the presence of therapeutic miRNAs (identified by MSC-EV-miRNA profiles) for the treatment of ARDS and other inflammatory diseases (e.g., COPD, stroke, myocardial infarction) in MSC cultures (intended for cell therapy applications).

[0089] MSC cell source

[0090] In some aspects, the present disclosure relates to obtaining mesenchymal matrix / stem cells (MSC). In some embodiments, MSC is of human origin. In some embodiments, MSC is of animal origin (e.g., dog, cat or monkey). Primary MSC can be obtained from any suitable source (i.e., tissue), such as, for example, bone marrow (BM), adipose tissue (AD) and / or placental membrane / cord blood (UC), and cultured using, for example, a cell culture device. In some embodiments, MSC culture may include BM-MSC; in some cases, MSC culture may include AD-MSC. In other embodiments, MSC culture may include UC-MSC. MSC can also be obtained from the differentiation of various progenitor cells, such as embryonic stem cells, induced pluripotent stem cells, etc. Combinations are also possible (e.g., a culture may include 50% BM-MSC and 50% AD-MSC, wherein the percentage is relative to the total number of MSC in the culture).

[0091] In some embodiments, the MSC culture comprises BM-MSCs. In some cases, BM-MSCs account for greater than or equal to 10%, greater than or equal to 25%, greater than or equal to 50%, greater than or equal to 75%, greater than or equal to 100% of the total number of MSCs in the culture. In other embodiments, BM-MSCs account for less than or equal to 100%, less than or equal to 75%, less than or equal to 50%, less than or equal to 25%, less than or equal to 10% of the total number of MSCs in the culture.

[0092] In some embodiments, the MSC culture comprises AD-MSCs. In some cases, AD-MSCs account for greater than or equal to 10%, greater than or equal to 25%, greater than or equal to 50%, greater than or equal to 75%, greater than or equal to 100% of the total number of MSCs in the culture. In other embodiments, AD-MSCs account for less than or equal to 100%, less than or equal to 75%, less than or equal to 50%, less than or equal to 25%, less than or equal to 10% of the total number of MSCs in the culture.

[0093] In some embodiments, the MSC culture comprises UC-MSCs. In some cases, UC-MSCs account for greater than or equal to 10%, greater than or equal to 25%, greater than or equal to 50%, greater than or equal to 75%, greater than or equal to 100% of the total number of MSCs in the culture. In other embodiments, UC-MSCs account for less than or equal to 100%, less than or equal to 75%, less than or equal to 50%, less than or equal to 25%, less than or equal to 10% of the total number of MSCs in the culture.

[0094] One of ordinary skill will appreciate that the confluence of MSC cultures can negatively impact the production of EVs. Thus, according to some embodiments, MSC cultures can be grown to greater than or equal to 25% confluence, greater than or equal to 50% confluence, or greater than or equal to 75% confluence. In certain embodiments, MSC cultures can be grown to less than or equal to 75% confluence, less than or equal to 50% confluence, or less than or equal to 25% confluence.

[0095] It is also generally understood that the number of cell passages (i.e., replicative senescence) of MSC cultures may also negatively affect the production of MSC EVs. Therefore, according to some embodiments, MSC cultures may be passaged 3 to 8 times before showing morphological abnormalities. In some embodiments, MSC may be passaged greater than or equal to 3 times, greater than or equal to 4 times, greater than or equal to 5 times, greater than or equal to 6 times, greater than or equal to 7 times, or greater than or equal to 8 times. In other embodiments, MSC may be passaged less than or equal to 8 times, less than or equal to 7 times, less than or equal to 6 times, less than or equal to 5 times, less than or equal to 4 times, or less than or equal to 3 times.

[0096] In some cases, the MSC cells can be immortalized MSC lines. For example, immortalized MSCs can be used to ensure batch reproducibility, avoid inter-individual donor variability, and maintain biological activity during culture expansion. MSCs can be immortalized using any technique known to those of ordinary skill in the art. For example, in some embodiments, MSCs derived from embryonic stem cells can be immortalized by transfecting a lentivirus carrying the c-Myc oncogene.

[0097] In some aspects, the present disclosure relates to culturing mesenchymal matrix / stem cells (MSC). MSC can be cultured using any technology known to those skilled in the art, such as, for example, bioreactors or other in vitro cell culture devices (e.g., culture dishes, multilayer cell culture bottles, hollow fiber bioreactors, stirred tank bioreactors, and spherical aggregates of MSC). In some embodiments, compared with a 2D culture system (e.g., culture dish), MSC is cultured using a 3D system (e.g., bioreactor) to increase EV production by 40 to 100 times. In some cases, compared with a 2D culture system, MSC is cultured using a 3D system to increase EV production by greater than or equal to 40 times, greater than or equal to 60 times, greater than or equal to 80 times, or greater than or equal to 100 times. In other embodiments, compared with a 2D culture system, MSC is cultured using a 3D system to increase EV production by less than or equal to 100 times, less than or equal to 80 times, less than or equal to 60 times, or less than or equal to 40 times.

[0098] In some embodiments, MSC culture can be grown in cell growth medium. In some cases, cell growth medium includes defined cell growth medium. In certain embodiments, the defined growth medium is xeno-free and / or EV-free, for example, for clinical applications that focus on MSC source variability and animal contamination. In other embodiments, cell growth medium includes undefined culture medium, which includes, for example, human serum or human platelet lysate. Human platelet lysate includes a variety of growth factors, cytokines, hormones, proteins, carbohydrates, lipids that stimulate cell proliferation, and is often used as a substitute for fetal bovine serum.

[0099] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the greatest extent based on the above description. Therefore, the following specific embodiments are interpreted as merely illustrative and do not limit the remainder of the present disclosure in any way. For the purpose or subject matter cited herein, all publications cited herein are incorporated by reference. Example

[0100] In order that the invention described herein may be more fully understood, the following examples are set forth.The examples described in this application are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein, and are not to be construed in any way as limiting the scope thereof.

[0101] Example 1. Extracellular vesicle (EV) characteristics after bronchoalveolar lavage fluid (BALF) exposure

[0102] Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) can be used as therapeutic agents for acute respiratory distress syndrome (ARDS). As described herein, MSC gene and protein expression is regulated by the ARDS lung environment. The effects of this environment on MSC-EV characteristics and miRNA content were studied as described below.

[0103] BALF exposure did not significantly affect number or size distribution but reduced CD63 expression in hMSC-secreted EVs

[0104] Figure 1 An overall schematic diagram of the study is shown in , summarizing the experimental protocol and analytical methods used. First, to determine whether exposure of hMSCs to different lung environments alters the number and size of EVs, hMSC-EVs were prepared from hMSCs exposed to BALF from ARDS patients or HV or to control medium, using BALF as a clinical surrogate. NTA analysis revealed that 90% of the EVs secreted by hMSCs were 50 μm to 200 μm in size, regardless of whether the cells were exposed to control medium or BALF, and there was no significant difference in the number of EVs ( Figure 2A). HV and ARDS BALF exposures overall reduced CD63-positive EVs compared with controls, with HV BALF exposure resulting in lower CD63 expression compared with ARDS BALF exposure ( Figure 2B , left). EV CD81 expression did not differ between experimental conditions. The difference in CD63 expression between HV and ARDS conditions translated into a significant difference in double-positive (CD63 / CD81) EV expression ( Figure 2B , middle and right). No CD9 expression was detected (data not shown).

[0105] EV number but not tetraspanin expression differs in EVs directly derived from ARDS vs HV BALF samples

[0106] Although EVs isolated from BALF-exposed hMSCs showed a narrow size distribution and similar area under the curve (AUC) measurements, the size distribution of EVs isolated from separate BALF samples covered a wider range, with approximately 20% of EVs from ARDS and HV being larger than 200 μm. In addition, ARDS BALF samples had significantly more particles ( Figure 2C Flow imaging revealed two major findings that contrasted with the analysis of EVs isolated from hMSCs. Specifically, expression of CD63 and CD81 was increased in HV and ARDS BALF compared with controls. In addition, there was detectable expression of CD9 in both HV and ARDS BALF EVs, compared with the lack of expression in EVs derived from hMSCs exposed to HV or ARDS BALF ( Figure 2D ).

[0107] Example 2. EVs derived from resting control hMSCs contain miRNAs predicted to downregulate inflammatory pathways

[0108] Prior to investigating the impact of the inflammatory lung environment on EV miRNA cargo, miRNAs were profiled in EV preparations obtained from control hMSCs (exposed only to serum-free medium). A total of 770 miRNAs were found to be present, as defined by counts significantly above the mean counts of all miRNAs in all biological replicates (Significance Analysis of Microarrays [SAM] score ≥ 20, delta [δ] set to 7 (default), false discovery rate [FDR] = 0.0, Figure 3A and Figure 3B), 48 were significantly over-represented (observed / expected). These 48 miRNAs were enriched in miRNet based on target prediction (DIANA-miRTarbase v8.0), and over-representation of miRNA-regulated genes involved in pathways such as cell cycle, gene expression regulation, immune system regulation, immune system cytokine signaling, and transforming growth factor β (TGFβ) receptor complex signaling was identified ( Figure 3C ). Network analysis was used to visualize the predicted “direct” interactions between the top 48 over-represented miRNAs and experimentally identified gene targets. The top-ranked network-predicted miRNAs and gene targets were those involved in cell cycle regulation ( Figure 3D and Figure 3E ).

[0109] Example 3. miRNAs in hMSC-EVs are not randomly packaged and can be altered by environmental factors

[0110] This experiment examined whether exposure of hMSCs to BALF from different environments altered EV miRNA cargo. EVs were isolated from conditioned medium of hMSCs exposed to serum-free medium (control, N=16), BALF of ARDS patients (ARDS, N=12), or healthy volunteers (HV, N=14). Whole transcriptome sequencing of miRNA was used to analyze the expression levels of 2083 miRNAs contained in EVs isolated from these 42 samples. Principal component analysis (PCA) of normalized unfiltered miRNA counts revealed clustering of samples by treatment assignment, indicating that miRNA content is not randomly packaged and can change due to exposure of hMSCs to different environments.

[0111] Example 4. hMSCs differentially package miRNAs into EVs in response to different inflammatory environments

[0112] To determine the impact of different inflammatory environments on EV miRNA cargo, three pairwise comparisons were performed: HV vs control (DE = 153 miRNAs), ARDS vs control (DE = 97DE miRNAs), and ARDS vs HV (DE = 126 miRNAs). MiRNAs were considered differentially expressed if they met a statistical cutoff of more than a two-fold expression change (Log2 fold change [FC]) and FDR ≤ 0.05. FIG. 4A to FIG. 4C The volcano plot in shows the over- and under-represented miRNAs for each comparison. FIG. 4A to FIG. 4CThe top 10 pathways predicted to be consistently regulated for each comparison are shown in Figure 4. EVs from hMSCs exposed to HVBALF had more miRNAs associated with decreased expression of genes involved in the cell cycle and in TGFβ, VEGF, and EGFR signaling compared to controls. Figure 4A ). In contrast, EVs from hMSCs exposed to HV BALF had fewer miRNAs involved in cellular responses to stress, including extracellular matrix organization and activation of hypoxia-inducible factor (HIF), compared with controls. Some groups, such as genes involved in platelet activation, signaling, and aggregation, had individual genes that were both over- and under-represented. Compared with controls, EVs from hMSCs exposed to ARDS BALF had over-representation of miRNAs predicted to suppress genes involved in cellular responses to stress and interferon signaling, and under-representation of miRNAs predicted to enhance expression of genes involved in BH3-selective triggering of canonical mitochondrial apoptosis in response to developmental cues or stress signals like DNA damage ( Figure 4B ). Comparing the differentially expressed miRNAs in ARDS vs HV EVs, surprisingly, the number of underrepresented miRNAs was greater than in previous comparisons of control vs HV or ARDS, respectively. After exposure to ARDS BALF, affected genes included those involved in the cell cycle, DNA synthesis, TLR4, and NF-κB activation. In contrast, overrepresented miRNAs in ARDS exposed hMSCs compared to HV were enriched for miRNAs regulating genes involved in interferon, activin (related to TGF-β), and FGFR signaling ( Figure 4C ).

[0113] Overlap analysis revealed consistent differential expression of EV miRNAs between experimental groups

[0114] Venn diagrams were used to visualize the overlap between the differentially regulated miRNA lists in each comparison ( Figures 5A-5D ). Consistent differential expression in more than one comparison was used to screen for miRNAs that were more likely to be altered by each of the three conditions (i.e., control versus exposure to BALF from HV or ARDS patients, FIG. 4B to FIG. 4C ). The miRNA expression pattern was visualized by plotting the Log2 FC of miRNA expression for each overlapping miRNA in each comparison ( Figure 5E to Figure 5G ). Enrichment analysis of miRNAs in each overlap analysis is shown in Figure 5E to Figure 5GFunctional analysis showed that differentially expressed miRNAs in all comparisons with control hMSC-EVs (N=23) were predicted to be involved in the regulation of gene expression, cellular stress, RNA processing, alternative splicing, and TGF-β receptor complex signaling (the top 10 pathways enriched in the analysis are shown in Figure 5E miRNAs regulated in all comparisons with samples exposed to HV BALF (N=57) were of particular interest because they were regulated in opposite directions in EVs from hMSCs exposed to ARDS BALF. Depending on the direction of change in the miRNAs, these miRNAs were predicted to increase or decrease pathways involved in cell cycle, infectious diseases, and TGF-β-regulated signaling ( Fig. 5F ). The miRNAs that responded to ARDS BALF were predicted to be involved in cytokine signaling, interferon β and α signaling, and programmed cell death, etc. ( Figure 5G ).

[0115] Example 5. Identification of 52 miRNAs that may be highly relevant to the biological and therapeutic effects of hMSCs

[0116] It is thought that miRNAs that are differentially present in all comparisons may have biological and / or therapeutic significance, as they appear to be packaged within hMSC-derived EVs in all experimental conditions. Therefore, if hMSCs exert a beneficial effect, these miRNAs may be mechanistically involved in producing this effect, rather than being found nonspecifically in hMSC-derived EVs. The overlap between the 3 pairwise comparisons indicated that 13 miRNAs were differentially regulated in all hMSC-EV preparations ( Fig. 6A To highlight the effect of ARDS-BALF on differential miRNA packaging, separate pairwise comparisons were performed in conjunction with the effects of control and HV (control+HV). A total of 52 miRNAs were found to be robustly differentially expressed in all three comparisons, including 13 overlapping miRNAs ( Figure 6B ; Table 1).

[0117] is predicted to be caused by overlap 1 ( Fig. 6A ) of the 13 commonly differentially expressed miRNAs regulated pathways that affected genes involved in Wnt signaling and mRNA processing and stability ( Figure 6C ). Overlap 2 (52 miRNAs, Figure 6B ) interact with genes known to be involved in interferon and cytokine signaling, Wnt, Toll-like receptor (TLR3 / 4) and nerve growth factor (NGF) signaling, and programmed cell death ( Fig.6D). Similar patterns of miRNA expression changes in each comparison are visualized as horizontal bar graphs. A heat map summarizing the consistency of miRNA expression changes in all 42 biospecimens is shown in Fig. 6E and Fig. 6F middle.

[0118] To identify miRNAs that may be associated with the biological and therapeutic effects of hMSCs, miRNAs present in EVs were isolated from control hMSCs and differentially expressed under all treatment conditions; including the consistent differential expression shown when the effects of unstimulated and healthy volunteers (control + HV vs ARDS) were combined. Fig. 7A ) showed that co-regulated miRNAs and their gene targets formed functionally relevant interaction networks. Two of these networks, which were of particular interest in acute lung injury, involved inflammasome activation and cellular stress ( Figure 7B and Figure 7C ). Sample-level data are shown as the top 20 miRNAs from both networks (sorted by FDR, Fig. 8A ) box plot of Log2-normalized counts, indicating the differential expression trend of EV-derived miRNAs.

[0119] Table 1. LIMMA overlap analysis output for combined HV+control vs ARDS

[0120]

[0121]

[0122] Example 6. Differential expression of the top 20 miRNAs contained in BALF-EVs strongly suggests a biological effect of exposure on hMSC EV miRNAs rather than cross-contamination from BALF samples

[0123] The miRNAs contained in EVs isolated directly from BALF of HV (N=4), ARDS patients (N=4), and directly from serum-free medium used to incubate hMSCs in BALF exposure experiments (N=5) were evaluated to determine whether the hMSC EV miRNA content might reflect carryover of EVs in the BALF, which adhere to hMSCs and are not washed away before collecting BALF-exposed hMSC-EVs. EV and miRNA isolation and miRNA sequencing were performed as described for the hMSC-EV study. Differential expression analysis was performed in two different ways, first using DESeq2 to determine differential expression and comparing the list of differentially expressed miRNAs in the BALF with the list of differentially expressed miRNAs in the EVs. In addition, given the small sample size, group medians were compared using the Mann-Whitney test.

[0124] Among the top 20 target miRNAs differentially expressed in EVs derived from hMSCs exposed to ARDS BALF ( Fig. 8A ), 13 miRNAs were not differentially expressed in EVs obtained directly from ARDS BALF compared with HV BALF. Of the 7 miRNAs differentially expressed in BALF and differentially present in hMSC-derived EVs, 3 had opposite directions of change ( FIG. 8B to FIG. 8C ), suggesting different biological effects of HV and ARDS BALF exposure rather than cross-contamination from EVs found in HV or ARDS BALF samples.

[0125] Example 7. Supervised classification analysis generates overlapping features for discriminating miRNAs

[0126] Discriminant analysis was used to determine whether HV or ARDS BALF-exposed and control hMSC-derived EVs could be classified using a reduced miRNA signature. The samples were divided into training and test sets (70% vs. 30%, respectively), and a classification model was developed on the training data using sparse partial least squares discriminant analysis (sPLS-DA). A two-component model of 15 and 5 miRNAs selected on the first two components, respectively, was found to have the lowest balanced discrimination error rate (0.0883). The 20-miRNA signature was found to distinguish samples into their appropriate treatment groups (ARDS BALF-exposed, HV BALF-exposed, or control), with the first component separating ARDS from other categories and the second component providing resolution between HV and controls ( Fig.9AIn the test cohort, the ability of the 20 miRNAs to accurately classify samples into treatment groups was validated using leave-one-out cross-validation (LOOCV). The area under the receiver operating characteristic curve for ARDS versus HV+ controls was 0.9643 ( Fig. 9B ), which is based on the prediction distance and averaged across all cross validations. The accuracy of the feature in predicting each class in the test set was 0.92, 0.85, and 0.92 for ARDS, HV, and control groups, respectively.

[0127] A total of 14 of the 20 classifiers identified using sPLSDA were also found to be differentially expressed between treatment groups. Functional analysis and visual exploration of miRNA-target interactions in miRNet revealed that 4 of the 14 differentially expressed classifiers (miRNA-760, miRNA-3175, miRNA-855-3p, and miRNA-766-3p) formed putative in silico regulatory networks as putative "hub" regulators of 1259 putative targets, 75 of which were involved in cellular responses to stress and Wnt signaling (enrichment scores = 9.7E-9 and 3.2E-9, Fig. 10B and Fig. 10C ).

[0128] To understand how pathways predicted to be regulated by their gene targets are involved in acute lung injury, these 14 miRNAs were analyzed in more detail (Table 2). miRNA-766-3p shared direct connections with genes involved in cellular stress response, while miRNA-885-3p, miRNA-3175, and miRNA-760 each contributed to the enrichment of genes involved in Wnt signaling ( Fig. 10A and Fig. 10B ). MiRNA-885-3p has direct interactions with genes involved in proinflammatory cytokine regulation, apoptosis, chemoresistance, proliferation, and metastasis. MiRNA-766-3p regulates genes that suppress inflammation by acting on NF-κB signaling. MiRNA-664b-3p, miRNA-4644, miRNA-6803-5p, miRNA-6869-5p, miR-3940-5p, and miRNA-766-3p are associated with proliferation regulation. MiRNA-3175 promotes epithelial-mesenchymal transition by targeting Smad 7. MiRNA-760 is considered a possible tumor suppressor as it negatively regulates oncogenic proteins and reduces proliferation, cell cycle progression, migration, and differentiation. A summary of the miRNA level results for the top 14 differentially expressed miRNA classifiers and overlapping target miRNAs as well as functional enrichment that may be associated with acute lung injury is shown in Table 2.

[0129] Table 2: Functional prediction - focus on possible role in acute lung injury

[0130]

[0131]

[0132]

[0133] Example 8. Discussion

[0134] There is still a lack of basic understanding of the fate and role of exogenously administered hMSCs and their EV products in the inflammatory environment of clinical lung diseases. It has been previously shown that the ARDS inflammatory environment has a profound impact on hMSC gene and protein expression, recognition ability of the host immune system, and downstream effects on relevant immune effector cells using clinical BALF as a surrogate. In some embodiments, it is shown that the ARDS inflammatory environment also affects hMSC-EV four-transmembrane protein expression and related miRNA content. A data-driven approach was used to identify the most abundant and consistently represented miRNAs in hMSC-EVs. Analysis of differential expression patterns found that EVs isolated from ARDS-exposed hMSCs contained more miRNAs predicted to inhibit genes involved in cellular responses to stress and interferon signaling. In contrast, EVs isolated from hMSCs exposed to a healthy non-inflammatory environment contained more miRNAs predicted to inhibit genes involved in the cell cycle and in TGF-β, VEGF, and EGFR signaling. Finally, EV miRNA signatures were identified and samples were classified into treatment groups using the signatures. In the future, these signatures may provide biological activity of hMSCs and may become efficacy markers. Of the 14 miRNAs that passed the differential and discriminant analysis filters, 10 were virtually novel and there were few data in the literature available to elucidate their possible functions. In contrast to the empirical determination, in silico predictions indicated important roles in the regulation of cell-cell, cell-matrix, oxidative stress, fatty acid metabolism, programmed cell death, and endothelial cell activation (Table 1). For the remaining four (miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p), they clearly formed an interaction network with their known target genes predicted to regulate key processes in acute lung injury—for example, responsive to cellular stress and Wnt signaling, strongly suggesting a biological role for the miRNAs contained in hMSC-derived EVs.

[0135] EVs are increasingly being recognized to mediate the anti-inflammatory and other effects of their parental hMSCs. This includes preclinical models of acute lung injury and other lung diseases, where EVs are as effective as, and sometimes more effective than, the parental EVs themselves. This has provided a platform for initial clinical studies of hMSC-derived EVs in patients with ARDS, bronchopulmonary dysplasia, and other lung diseases. However, the mechanisms by which hMSC-EVs affect the inflammatory lung environment remain to be elucidated. So far, the initial focus has been on EV-associated miRNAs. For example, miRNA-27 and its target gene VAV3 have been shown to play a role in cell infiltration and cell adhesion during acute lung injury.

[0136] In this study, substantial and significant differences were observed in the miRNA profiles associated with hMSC-EVs obtained under different BALF exposure conditions. Interestingly, a reduced number of miRNAs were isolated from ARDS-exposed hMSC-EVs compared to HV-exposed hMSC-EVs. One possible explanation is that the disease leads to a loss of variability. Variability determines plasticity, i.e., the extent to which a gene can change its expression in response to environmental fluctuations. Under pathological stress, this plasticity may be lost. Another interesting finding was that hMSCs exposed to ARDS and HV BALF samples produced EVs containing miRNAs with general anti-inflammatory activities, such as inhibition of cellular responses to stress, as well as inhibition of IFN, TGF-β, VEGF, and EGFR signaling. However, these pathways were predicted to be less affected in EVs isolated from control hMSCs exposed to serum-free medium. Furthermore, EVs isolated from hMSCs exposed to a healthy, non-inflammatory environment contained miRNAs that led to activation of proliferation, platelet activation, HIF signaling, and extracellular matrix—all pathways involved in wound healing and tissue remodeling.

[0137] In an attempt to identify key miRNAs, two different bioinformatics strategies, DEA and sPLS-DA, identified 14 miRNAs of interest. This list was further narrowed by exploring miRNA-target interactions in miRNet using network analysis and visualization to find miRNAs that work together. Using this strategy, four “hub” regulator miRNAs were identified: miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p, which are known to be involved in cellular responses to stress and Wnt signaling. Interestingly, Wnt is an evolutionarily conserved pathway that regulates key aspects of cell fate determination, cell migration, cell polarity, neural patterning, and organ formation during embryonic development. The Wnt / β-catenin pathway has been implicated in the induction, promotion, and aberrant repair of acute lung injury. An advantage of these observations is that any functional predictions are limited to miRNA-mRNA interactions that have been experimentally demonstrated to occur based on a large experimental compendium of miRNA-mRNA interaction data.

[0138] Although the hMSCs used in this study were carefully washed after BALF exposure, it is possible that some BALF-derived EVs were internalized or remained "stuck" on the cells. There is little available literature on this issue, and to address this issue, comparable analyses were performed on EVs isolated from the same BALF sample and serum-free medium used to incubate control hMSCs. Despite the presence of overlapping miRNAs in the original BALF samples as well as the control medium, a considerable number of significantly differentially expressed miRNAs were identified. For example, miRNA-885-3p, miRNA3652, and miRNA-4763-3p were not found to be differentially expressed between the original BALF samples and the control medium, yet significant differences were observed in EVs isolated from exposed hMSCs. These observations strongly suggest a biological effect of BALF exposure, rather than cross-contamination of EVs found in the BALF samples.

[0139] The studies described here provide evidence for the plasticity of hMSCs and their EVs and, importantly, offer a number of mechanistic hypotheses to be evaluated regarding the different hMSC EV-associated miRNAs. These observations provide a growing understanding of the complex interactions of inflammatory and other pathways involved in the effects of hMSCs in the lung and provide important information for the development of more effective hMSC-based cell therapeutics for ARDS and other lung diseases.

[0140] Example 9. Materials and Methods

[0141] Human BALF samples

[0142] Collection and processing of BALF samples from healthy volunteers (HV) and ARDS patients were performed as described previously. Briefly, HV underwent standard fiberoptic bronchoscopy of the right middle lobe at Dartmouth University (exclusion criteria for HV were history of cardiopulmonary disease, regular smoking or e-cigarette use, and use of immunomodulatory medications). BALF samples from ARDS patients without sepsis were prospectively collected as part of an unrelated clinical study. For HV lavage, 20 cc of sterile saline was used, the samples were centrifuged, and the supernatant was stored at −70°C. For ARDS patient lavage, a standard 40 ml mini-BALF containing sterile saline was used in intubated ARDS patients, and BALF samples were similarly centrifuged and stored.

[0143] hMSCs exposed to BALF in vitro

[0144] hMSCs were obtained and cultured in MEM / EBSS medium supplemented with 1% penicillin / streptomycin and 20% fetal bovine serum in a standard tissue culture incubator. hMSCs were obtained from multiple donors and have been previously characterized according to the standards of the International Society for Cell and Gene Therapy. hMSCs were used at passages 3 to 5 and were identical to those used in recent hMSC administration trials in non-COVID ARDS patients and previous studies of the effects of BALF on hMSC action.

[0145] miRNA and NTA analysis

[0146] To prepare EVs for miRNA and nanoparticle tracking analysis (NTA), hMSCs were seeded into 6-well plates (2 × 10 5 cells / well, 2 wells / BALF sample or control) and seeded overnight. The next day, the cells were washed twice with PBS and synchronized in serum-free medium for 24 hours. After synchronization, the serum-free medium was replaced with 1 ml of serum-free medium containing a single ARDS or single HV BALF sample at a concentration of 20% (v / v). Control hMSCs were exposed only to serum-free medium. After incubation at 37°C for 5 hours in a standard tissue culture incubator, the cell culture medium was removed, the cells were washed once with PBS, and 2 ml of serum-free medium was added to each well. After 48 hours of incubation (37°C), the conditioned medium was collected, passed through a 0.8μm syringe filter, and processed for EV preparation, NTA, and miRNA evaluation as described below.

[0147] Tetraspanin expression

[0148] To evaluate the expression of characteristic cell surface tetraspanins (CD9, CD63, and CD81) using imaging flow cytometry, 2 × 10 6 Cells (for each condition) were exposed to ARDS or HV BALF samples or to serum-free medium (control) as described above. Given the large BALF volumes required for exposure (20% v / v) and the limited amount of BALF available, these studies used pooled rather than individual HV or ARDS samples.

[0149] Preparation of extracellular vesicles (EVs) and isolation of miRNA

[0150] EV isolations from conditioned media of BALF-exposed hMSCs or directly from BALF samples themselves were prepared according to the latest recommendations of the International Society for Extracellular Vesicles. EVs and EV-derived miRNAs were isolated from conditioned media and BALF samples using the exoRNeasy Serum / Plasma Maxi / Maxi Midi Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. Sample preparations were stored at −20°C until miRNA sequencing. For NTA and imaging flow cytometry analysis, ExoQuick- EVs were isolated using a 50 μM elution column (cat#EXOTC50A; System Biosciences, Palo Alto, CA) to avoid interference of the elution column with the NTA protocol. This was because, when performing preliminary NTA on samples isolated using the exoRNeasy kit, background contamination from the kit elution column was noted. As this contamination interfered with the proper assessment of particle number and size distribution using NTA, a subsequent precipitation-based separation method that does not rely on an elution column (i.e., ExoQuick- ) for characterization by NTA and imaging flow cytometry. This approach yielded robust, reproducible results without concern for contaminants.

[0151] Nanoparticle Tracking Analysis (NTA)

[0152] NTA( Particle Metrix Inc, Germany; 488 nm laser) was used to measure EV particle size and concentration. All samples were analyzed at 25 °C after daily instrument calibration according to the manufacturer's recommendations. Samples were diluted to appropriate concentrations in ultrapure water before analysis. Video acquisition with fixed settings was performed for all samples (scattering mode: sensitivity 85, shutter 75; fluorescence mode: sensitivity 95, shutter 32; both: minimum brightness 20, minimum size 5, and maximum size 200). Videos of all 11 positions were recorded with 5 cycles per sample at each position (1 cycle equals 1 s) and were acquired using Analyze using the ELISA software (version 8.03.08.02).

[0153] Imaging flow cytometry

[0154] In AMNIS Imaging flow cytometry was performed on a Mark II flow cytometer (AMNIS / Luminex, Seattle, WA, USA). Briefly, antibodies were added to the samples and incubated for 1 hour at room temperature. Unstained EV samples (uEV), NaCl-HEPES buffer containing antibodies but no EV samples, and stained samples supplemented with 1% NP40 (Calbiochem, San Diego, CA, USA) were analyzed as controls according to the recommendations of the MIFlowCyt-EV guide. After staining, the samples were diluted with PBS without any washing and analyzed using the built-in autosampler of a 96-well round bottom plate. The acquisition time was selected to be 5 minutes per well. The data were acquired at 60x magnification, low flow rate, and disabled with the removed beads option. The data were analyzed as described previously using IDEAS software (version 6.2). Fluorescence events were plotted against side scatter (SSC). Combined mask features (MC and NMC) were used to improve the detection of fluorescent images. Image consistency (population detection) was analyzed by using a point counting feature. Each data point with multiple objects was excluded from the analysis. Events with low side scatter values ​​(<500) and fluorescence intensity above 300 were considered uEVs. The average concentration was calculated based on the acquisition volume and time.

[0155] MiRNA sequencing and differential expression analysis

[0156] As described above, EVs were isolated from hMSCs exposed to serum-free medium (control, n = 16) or from hMSCs exposed to separate BALF samples obtained from ARDS patients (N = 16) or HV, or directly from pure BALF samples obtained from HV (N = 4) and ARDS patients (N = 4). RNA was isolated from each EV preparation as described above, and only samples with quality control of A260 / A280 above 1.80 were used for RNA sequencing (control = 16; ARDS = 12; HV = 14). A total of 35 ng of eRNA (EV-derived RNA) preparations were used for miRNA sequencing using the HTG EdgeSeq miRNA whole transcriptome assay (miRNA WTA, according to the manufacturer's instructions), which measured the expression of 2083 human miRNA transcripts using next-generation sequencing (NGS). The raw read counts for each of the 42 samples were input into DESeq2 for differential expression analysis. Variance stabilization transformations were used to prepare data for analysis. Significance analysis of microarrays (SAM) with normalized read counts was run using a single-class analysis approach to identify miRNAs that were over-represented in EVs derived from control hMSCs. 1000 permutations were used, the delta value was set to 7 (the best delta was selected by the software with the lowest false discovery rate [FDR]), and the FDR cutoff was 0 (%). Differential expression analysis (DEA) was performed using DESeq2 in R (R package "DESeq2"). Four comparisons were performed: (1) control vs HV, (2) ARDS vs control, (3) ARDS vs HV, and (4) ARDS vs combined control + HV. Stringent selection criteria were used to reduce the FDR rate. MiRNAs considered differentially expressed (DE) were selected based on ≥2-fold expression change (average expression of samples >50 read counts) and adjusted p-value (or FDR) <0.05 after correction for multiple comparisons. Consistently over-represented miRNAs were identified by combination with pairwise comparisons and visualized using Venn diagrams.

[0157] Target prediction and functional analysis were performed using miRNet (miRNet.ca). Enriched pathways were selected based on the hypergeometric test of miRNAs with FDR ≥ 0.05 in the reaction set. Putative relationships were obtained using DIANA TarBase v8, a collection of experimentally supported miRNA-gene interactions.

[0158] Supervised classification analysis of ARDS, HV and control hMSCEV miRNAs

[0159] Sparse partial least squares (PLS) discriminant analysis (sPLS-DA) (R package “mixOmics”) was performed to identify the most discriminative features from ~2000 miRNAs to classify samples into these categories using normalized expression profiles of ARDS or HV BALF-exposed or control hMSC-derived EVs. Briefly, samples were divided into training and test sets using a 0.7 / 0.3 split. Leave-one-out cross-validation (LOOCV) was used to select the best parameters for the model: the number of components required to distinguish the classes (up to 4 components were considered as the number of classes (K-1) and generally performed best) and the number of features (discriminatory miRNAs) per component (5 to 20 features were tested per component). The final model selected the combination of components and features with the lowest balanced error rate (BER) (2 components with 15 and 5 features, respectively). The prediction distance for measuring the classification error was calculated using the maximum distance. The performance of the training set model and the prediction of the test set results were then evaluated using the accuracy, sensitivity, and specificity of each class assignment. The area under the receiver operating characteristic curve (AUROC) for many-to-one comparisons of PLS-DA was based on the maximum distance of predictions averaged across all cross-validations and was intended to supplement the analysis rather than evaluate model performance.

[0160] Statistical analysis

[0161] NTA and image cytometry data

[0162] use The particle size obtained and counting are drawn with 100 μ m intervals in the range of 2.5 μ m to 903 μ m on the X-axis, and the area under the curve (AUC) of each sample is calculated. One-way ANOVA is used to analyze data, with group (control, HV, ARDS) as factor, and Bonferroni post-hoc test is performed to identify the difference between the group mean value. The number of positive staining particles of each tetraspanin antibody (CD9, CD63 and CD81) and the number of double positive (CD63 / CD81) are similarly analyzed in each tetraspanin type. All analyses and charts are completed using Prism (version 9.3, GraphPad software).

[0163] miRNA data

[0164] To determine the differential expression of miRNAs found in BALF-derived EVs and hMSC-derived EVs, unpaired T-tests with Welch correction (not assuming equal standard deviations) were performed for those that passed the normality (Kolmogorov–Smirnov) test. For those that were not normally distributed, two-tailed Mann–Whitney tests were performed on a miRNA-by-miRNA basis (p = 0.05). Statistical analysis was performed using GraphPad Prism software. Mann–Whitney tests were used to assess differences between the two groups. P values ​​≤ 0.05 were considered significant, except for RNA sequencing data analyzed in DESeq, in which case a multiple hypothesis-corrected FDR of less than 0.05 was significant. Spearman correlations were calculated in Base R, and for cases involving rank ties, P values ​​were calculated using the t distribution.

[0165] Table 3: miRNA sequences

[0166]

[0167]

[0168]

[0169]

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[0220] Other embodiments

[0221] In the claims, articles such as "a", "an", and "the" may mean one or more, unless indicated to the contrary or obvious from the context. Claims or descriptions that include "or" between one or more members of the group are considered satisfied if one, more, or all of the group members are present in, used in, or otherwise related to a given product or method, unless indicated to the contrary or obvious from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or method. The invention includes embodiments in which more than one or all of the group members are present in, used in, or otherwise related to a given product or method.

[0222] In addition, the present invention includes all variations, combinations and arrangements, wherein one or more restrictions, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim subordinate to another claim can be modified to include one or more restrictions found in any other claim subordinate to the same basic claim. In the case where an element is presented as a list, for example, in the Markush group format, each subgroup of the element is also disclosed, and any element can be removed from the group. It should be understood that, in general, in the case where the present invention or aspects of the present invention are referred to as including specific elements and / or features, some embodiments of the present invention or aspects of the present invention are made up of or mainly made up of these elements and / or features. For simplicity, these embodiments are not specifically described in this article with these words (in haec verba). It should also be noted that the terms "comprising" and "containing" are intended to be open, and allow the inclusion of other elements or steps. In the case of a given range, endpoints are included. In addition, unless otherwise indicated or obvious from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may, in different embodiments of the present invention, present any specific value or sub-range within the range, up to one-tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0223] This application relates to various authorized patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, this specification shall prevail. In addition, any specific embodiment of the present invention that falls into the prior art can be explicitly excluded from any one or more claims. Because these embodiments are considered to be known to those of ordinary skill in the art, they can also be excluded even if they are not explicitly stated to be excluded in this article. Any specific embodiment of the present invention can be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[0224] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments described herein using only routine experiments. The scope of the current embodiments described herein is not intended to be limited to the above description, but as set forth in the appended claims. It will be appreciated by those of ordinary skill in the art that various changes and modifications may be made to this description without departing from the spirit or scope of the invention (as defined by the appended claims).

Claims

1. A method for generating an EV-miRNA profile, comprising: adding an activating agent to a culture of mesenchymal stromal cells (MSCs); Determining whether at least one MSC extracellular vesicle-related miRNA (MSC-EV-miRNA) is present in the culture of the MSCs using a miRNA sequencing platform; Based on the presence or absence of each miRNA relative to the control, EV-miRNA profiles were created for MSCs exposed to the activating agents.

2. The method of claim 1, further comprising using the MSC-EV-miRNA profile to identify a subject to receive the MSC for treatment of a disease.

3. The method of any one of claims 1 or 2, wherein the miRNA comprises hsa-miR-7107-5p.

4. The method of any one of claims 1-3, wherein the miRNA comprises hsa-miR-6803-5p.

5. The method of any one of claims 1-4, wherein the miRNA comprises hsa-miR-6798-5p.

6. The method of any one of claims 1-5, wherein the miRNA comprises hsa-miR-760.

7. The method of any one of claims 1-6, wherein the miRNA comprises hsa-miR-6727-5p.

8. The method of any one of claims 1-7, wherein the miRNA comprises hsa-miR-4763-3p.

9. The method of any one of claims 1-8, wherein the miRNA comprises hsa-miR-3652.

10. The method of any one of claims 1-9, wherein the miRNA comprises hsa-miR-885-3p.

11. The method of any one of claims 1-10, wherein the miRNA comprises hsa-miR-766-3p.

12. The method of any one of claims 1-11, wherein the miRNA comprises hsa-miR-3175.

13. The method of any one of claims 1-12, wherein the miRNA comprises hsa-miR-6893-5p.

14. The method of any one of claims 1-13, wherein the miRNA comprises hsa-miR-6875-5.

15. The method of any one of claims 1-14, wherein the miRNA comprises hsa-miR-6799-5p.

16. The method of any one of claims 1-15, wherein the miRNA comprises a miR selected from the group consisting of miR-6787-5p, miR-200b, miR-101, miR-145, miR-223, miR-494, miR-509-3p, miR-1246, miR-9, miR-138, miR-384, miR-600, miR-146a, miR-93, miR-17, miR-155, miR-199a-3p, miR-199a-5p, miR-126, miR-221, miR-31, miR-16 and / or miR-1343.

17. A method comprising: obtaining a first biological sample from a healthy first subject and a second biological sample from a second subject suspected of having a disease; culturing the first biological sample in a first MSC culture and culturing the second biological sample in a second MSC culture; detecting the presence of at least one MSC-EV-miRNA in the first and / or second MSC culture using a next generation sequencing platform; The next generation sequencing platform was used to create differential EV-miRNA profiles. and determining whether the subject suspected of having the disease is a candidate for MSC-based therapy based on the differential EV-miRNA profile.

18. A pharmaceutical composition comprising: At least one mesenchymal stromal cell-derived extracellular vesicle-associated miRNA (MSC-EV-miRNA); and Pharmaceutically acceptable excipients.

19. The pharmaceutical composition of claim 18, further comprising lipid nanoparticles (LNPs) encapsulating the at least one MSC-EV-miRNA.

20. The pharmaceutical composition of claim 19, wherein the LNP encapsulates at least four MSC-EV-miRNAs.

21. A pharmaceutical composition as described in any one of claims 19 or 20, wherein the LNP encapsulates at least 14 MSC-EV-miRNAs.

22. A pharmaceutical composition as described in any one of claims 18-21, wherein the LNP comprises a targeting moiety.

23. The pharmaceutical composition of any one of claims 18-22, wherein the miRNA comprises hsa-miR-7107-5p.

24. The pharmaceutical composition of any one of claims 18-23, wherein the miRNA comprises hsa-miR-6803-5p.

25. The pharmaceutical composition of any one of claims 18-24, wherein the miRNA comprises hsa-miR-6798-5p.

26. The pharmaceutical composition of any one of claims 18-25, wherein the miRNA comprises hsa-miR-760.

27. The pharmaceutical composition of any one of claims 18-26, wherein the miRNA comprises hsa-miR-6727-5p.

28. The pharmaceutical composition of any one of claims 18-27, wherein the miRNA comprises hsa-miR-4763-3p.

29. The pharmaceutical composition of any one of claims 18-28, wherein the miRNA comprises hsa-miR-3652.

30. The pharmaceutical composition of any one of claims 18-29, wherein the miRNA comprises hsa-miR-885-3p.

31. The pharmaceutical composition of any one of claims 18-30, wherein the miRNA comprises hsa-miR-766-3p.

32. The pharmaceutical composition of any one of claims 18-31, wherein the miRNA comprises hsa-miR-3175.

33. The pharmaceutical composition of any one of claims 18-32, wherein the miRNA comprises hsa-miR-6893-5p.

34. The pharmaceutical composition of any one of claims 18-33, wherein the miRNA comprises hsa-miR-6875-5.

35. The pharmaceutical composition of any one of claims 18-34, wherein the miRNA comprises hsa-miR-6799-5p.

36. A pharmaceutical composition as described in any one of claims 18-35, wherein the miRNA includes miR-6787-5p, miR-200b, miR-101, miR-145, miR-223, miR-494, miR-509-3p, miR-1246, miR-9, miR-138, miR-384, miR-600, miR-146a, miR-93, miR-17, miR-155, miR-199a-3p, miR-199a-5p, miR-126, miR-221, miR-31, miR-16 and / or miR-1343.

37. A cell therapy agent comprising: A culture of engineered mesenchymal stromal cells configured to release a plurality of extracellular vesicles (MSC-EVs), wherein the plurality of MSC-EVs comprises hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6 875-5p、hsa-miR-6799-5p、hsa-miR-6787-5p、miR-200b、miR-101、miR-145、miR-223、miR-494、miR-509-3p、miR-1246、miR-9、miR-138 ,miR-384,miR-600,miR-146a,miR-93,miR-17,miR-155,miR-199a-3p,miR-199a-5p,miR-126,miR-221,miR-31,miR-16 and / or miR-1343.

38. The cell therapeutic agent of claim 37, wherein the MSC-EV comprises hsa-miR-760, hsa-miR-3175, hsa-iR-885-3p and hsa-miR-776-3p.

39. A method for producing therapeutic mesenchymal stromal cells (MSCs), the method comprising: obtaining a first biological sample from a healthy first subject and a second biological sample from a second subject suspected of having a disease; culturing the first biological sample in a first MSC culture and culturing the second biological sample in a second MSC culture; Isolation of hMSC-associated extracellular vesicles (hMSC-EVs) from cell culture; Determine whether at least one EV-associated miRNA (EV-miRNA) is present in the hMSC-EVs using a next generation sequencing platform, Create differential EV-miRNA profiles using the described next-generation sequencing platform, and The therapeutic MSCs are engineered to overexpress one or more miRNAs identified in the EV-miRNA profile.

40. The method of claim 39, further comprising delivering the therapeutic MSCs to the subject.

41. The method of any one of claims 39 or 40, wherein the therapeutic MSCs overexpress hsa-miR-760, hsa-miR-3175, hsa-miR-885-3p, and hsa-miR-766-3p.

42. An oligonucleotide primer mixture comprising one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single target miRNA, and wherein the target miRNA is selected from the group consisting of hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-760 ... a-miR-6875-5p,hsa-miR-6799-5p,hsa-miR-6787-5p,miR-200b,miR-101,miR-145,miR-223,miR-494,miR-509-3p,miR-1246,miR-9,miR -138,miR-384,miR-600,miR-146a,miR-93,miR-17,miR-155,miR-199a-3p,miR-199a-5p,miR-126,miR-221,miR-31,miR-16 and / or miR-1343.

43. A kit for detecting the presence of at least one mesenchymal stromal cell-derived extracellular vesicle-related miRNA (MSC-EV-miRNA) in a biological sample, comprising: A primer mixture comprising one or more oligonucleotides, wherein each oligonucleotide is configured to hybridize to a single miRNA of interest, and wherein the miRNA of interest is selected from the group consisting of hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-mi R-6875-5p、hsa-miR-6799-5p、hsa-miR-6787-5p、miR-200b、miR-101、miR-145、miR-223、miR-494、miR-509-3p、miR-1246、miR-9、miR-1 38.miR-384,miR-600,miR-146a,miR-93,miR-17,miR-155,miR-199a-3p,miR-199a-5p,miR-126,miR-221,miR-31,miR-16 and / or miR-1343, Reagents for performing a nucleic acid assay using a nucleic acid pair to detect the at least one MSC-EV-associated miRNA, and Instructions for performing the assay to detect the at least one MSC-EV-associated miRNA.

44. The kit of claim 43, wherein the kit comprises at least four oligonucleotides for detecting at least four of the MSC-EV-associated miRNAs.

45. The kit of any one of claims 43 or 44, wherein the kit comprises at least eight oligonucleotides for detecting at least four of the MSC-EV-associated miRNAs.

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