Extracellular Vesicle MicroRNA and Its Use

Abstract

Compositions comprising miRNA (MSC-EV-miRNA) derived from mesenchymal stromal cell-derived extracellular vesicles and their use in subjects diagnosed with acute respiratory distress syndrome (ARDS) and other inflammatory diseases are disclosed herein. Also disclosed herein are methods for creating differential MSC-EV-miRNA profiles for any disease of interest (e.g., ARDS) and the use of miRNA profiles for creating miRNA-based therapies. Additional aspects of the disclosure relate to primer mixes for rapidly detecting specific miRNA profiles, and kits thereof.

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 63 / 349,551, filed on June 6, 2022, which is incorporated herein by reference in its entirety.

Background Art

[0002] Cell-based therapies utilizing mesenchymal stromal cells (MSCs) are increasingly being studied as potential therapeutics for various 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, the results of recent human clinical studies are still variable, suggesting that new approaches are needed to improve the therapeutic efficacy of MSC-based therapies (e.g., MSC-EVs).

Summary of the Invention

[0003] Each limitation of the present invention may encompass various embodiments of the present invention. Thus, it is expected that each limitation of the present invention involving any one element or combination of elements may be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components described in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways. 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 become apparent from the present specification and claims.

[0004] In some aspects, the present disclosure is a method for generating an EV-miRNA profile, comprising adding an activating factor to a culture of mesenchymal stromal cells (MSCs), using a miRNA sequencing platform to determine whether miRNA associated with at least one MSC extracellular vesicle (MSC-EV-miRNA) is present in the culture of MSCs, and creating an EV-miRNA profile of MSCs exposed to the activating factor based on the presence or absence of each miRNA, compared to a control. In some embodiments, the method further comprises using the MSC-EV-miRNA profile to identify a subject who receives MSCs for the 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.

[0005] Some aspects of the present disclosure relate to 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 culture of MSCs and the second biological sample in a second culture of MSCs, detecting whether at least one MSC-EV-miRNA is present in the first and / or second culture of MSCs using a next-generation sequencing platform, creating a differential EV-miRNA profile using a next-generation sequencing platform, and determining whether a subject suspected of having a disease is a candidate for MSC-based therapy based on the differential EV-miRNA profile.

[0006] Additional aspects of the present disclosure relate to a pharmaceutical composition comprising a miRNA associated with extracellular vesicles derived from at least one mesenchymal stromal cell (MSC-EV-miRNA) and a pharmaceutically acceptable excipient. Optionally, the pharmaceutical composition further comprises lipid nanoparticles (LNP) encapsulating at least one type of MSC-EV-miRNA. In certain embodiments, the pharmaceutical composition comprises LNP encapsulating at least 4 types of MSC-EV-miRNA. Further, in some additional embodiments, the pharmaceutical composition also comprises LNP encapsulating at least 14 types of MSC-EV-miRNA. Optionally, the LNP may comprise a targeting moiety. 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 cell therapy comprising a culture of engineered mesenchymal stromal cells configured to release a plurality of mesenchymal stromal cell - derived 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, hsa - miR - 6787 - 5p. In some embodiments, the cell therapy comprises MSC - EVs including hsa - miR - 760, hsa - miR - 3175, hsa - miR - 885 - 3p, and hsa - miR - 766 - 3p.

[0008] Certain aspects of the present disclosure further relate to a method of generating 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 culture of MSCs and the second biological sample in a second culture of MSCs, isolating human MSC - related extracellular vesicles (hMSC - EVs) from the cell cultures, using a next - generation sequencing platform to determine whether miRNA associated with at least one EV (EV - miRNA) is present in the hMSC - EVs, using a next - generation sequencing platform to generate a differential EV - miRNA profile, and engineering the therapeutic MSCs to overexpress one or more miRNAs identified by the EV - miRNA profile. In some embodiments, the method further comprises delivering the therapeutic MSCs to a subject. Optionally, the method may comprise therapeutic MSCs that overexpress hsa - miR - 760, hsa - miR - 3175, hsa - miR - 885 - 3p, and hsa - miR - 766 - 3p.

[0009] Aspects of the present disclosure also relate to an oligonucleotide primer mix comprising one or more oligonucleotides, each oligonucleotide being 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, hsa-miR-6787-5p.

[0010] Some aspects of the present disclosure also relate to a kit for detecting the presence of miRNA (MSC-EV-miRNA) associated with extracellular vesicles derived from at least one mesenchymal stromal cell in a biological sample, the kit comprising a primer mix comprising one or more oligonucleotides, each oligonucleotide being configured to hybridize to a single miRNA of interest, the miRNA of interest being 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, hsa-miR-6787-5p; reagents for performing a nucleic acid assay to detect miRNA associated with at least one MSC-EV using a nucleic acid pair; and instructions for use for performing an assay to detect miRNA associated with at least one MSC-EV. In some embodiments, it comprises at least four oligonucleotides for detecting at least four of the miRNAs associated with MSC-EV. In certain additional embodiments, the kit comprises at least eight oligonucleotides for detecting at least four of the miRNAs associated with MSC-EV.

[0011] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by like numerals. For clarity, not all components are shown in every drawing.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] Cell-based therapies using mesenchymal stromal cells (MSCs) are increasingly being studied as potential therapeutic agents for various diseases, including acute respiratory distress syndrome (ARDS) including ischemic stroke, myocardial infarction, and ARDS caused by SARS-CoV-2 infection. These approaches are based on the secondary anti-inflammatory effects of MSCs, which in the case of ARDS may suppress alveolar inflammation, promote endothelial repair, increase alveolar fluid clearance, and regulate the permeability of the lung epithelium and endothelium.

[0014] One mechanism by which MSCs regulate paracrine signaling is to release extracellular vesicles (EVs), including exosomes and microvesicles, for example during disease-mediated inflammatory responses. EVs are particles delimited by a lipid bilayer, unable to replicate, and encapsulating a cargo that includes multiple 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, generally, MSC-EVs are thought to exert their function through the transfer of cargo that acts as paracrine signaling agents for communication between adjacent and / or distal cells.

[0015] Several preclinical studies have consistently shown that systemic or intratracheal administration of MSCs and, more recently, systemic administration of MSC-EVs, result in improved clinical outcomes (e.g., improvement of lung injury in ARDS models). However, in various preclinical models (e.g., mouse models of ARDS), despite the success of MSC cell-based therapies for treating human diseases, the results of recent human clinical studies are still variable, suggesting that new approaches are needed to improve the therapeutic efficacy of MSC-based therapies (e.g., MSC-EVs).

[0016] As described herein, MSC-based paracrine responses, and thus potential therapeutic effects, can vary depending on the cellular microenvironment that the MSC encounters. For example, as shown in Example 1, human MSCs (hMSCs) cultured with bronchoalveolar lavage fluid (BALF) obtained from subjects diagnosed with ARDS (ARDS BALF) had different effects on hMSC-EV release, expression of characteristic EV cell surface tetraspanin protein markers, and miRNA content compared to hMSCs cultured with BALF obtained from healthy subjects (HV BALF). This discovery enables the generation of differential EV-miRNA profiles (e.g., using target prediction, functional analysis, and visual exploration) that highlight changes in miRNAs known to be associated with the therapeutic effects (e.g., paracrine effects) of hMSCs, such as those involved in the regulation of inflammation, cell cycle, proliferation, and apoptosis.

[0017] Thus, in some aspects, the present disclosure solves the foregoing problems by providing a method for determining a disease-specific MSC-EV-miRNA profile. The method can be used, in some embodiments, to identify miRNAs that function as regulators of the paracrine response of MSCs. For example, in some embodiments, the method can be used to identify miRNAs that regulate the MSC paracrine response in ARDS.

[0018] In other aspects, the present disclosure solves the foregoing problems by providing, for example, disease-specific MSC-EV-miRNA profiles that can be used to identify diseases for which the potential to benefit from MSC-based cell therapy is highest. For example, in some embodiments, the method includes obtaining biological samples from subjects with and without the disease, adding them to cultures of MSCs, profiling miRNA expression patterns, and creating differential MSC-EV-miRNA profiles. For example, a disease-specific differential MSC-EV-miRNA profile created by exposing MSC cultures to ARDS BALF or HV BALF contains multiple miRNAs involved in Wnt signaling and other cellular responses to stress, suggesting that MSC-based cell therapy may have therapeutic benefit in the setting of ARDS. On the other hand, an MSC-EV-miRNA profile (created for a particular disease of interest) that shows limited changes in miRNA expression patterns may suggest that MSC-based cell therapy may not have therapeutic benefit in the particular disease of interest.

[0019] In some aspects, the present disclosure further solves the foregoing problems by providing a method for modulating the content of MSC-EVs, for example, using genetic engineering. In some embodiments, the method includes creating differential MSC-EV-miRNA profiles and identifying miRNAs that counter pathophysiology such as pro-inflammatory responses associated with diseases such as ARDS. In some cases, the MSCs may be genetically modified, for example, to overexpress the identified miRNAs and package them into EVs, thereby improving the therapeutic efficacy of the MSCs after administration to a subject in need thereof.

[0020] In yet 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 can be particularly useful, for example, in situations where the EV-miRNA profile identifies a subset of miRNAs that regulate multiple paracrine responses. The subset of miRNAs can, in some cases, be encapsulated within microcarriers such as lipid nanoparticles, optionally contain targeting moieties, and can be administered by intravenous, intramedullary, intramuscular, intraperitoneal, or subcutaneous injection. In some cases, EVs can be used as drug delivery vehicles.

[0021] Method for creating an MSC-EV-miRNA profile Aspects of the present disclosure relate to methods for generating differential MSC-EV-miRNA profiles. In some embodiments, the method includes adding an activating factor to a culture of mesenchymal stromal cells (MSCs). Optionally, the activating factor can include various disease-related stimuli, such as chemical, mechanical, temperature, light stimuli, etc. For example, in some embodiments, the activating factor includes a biological sample obtained from a subject having a disease (i.e., a chemical stimulus), although 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., lymph), bile, pus, sputum, saliva, mucosal secretions, cerebrospinal fluid, plasma, menstrual blood, tears, gastric juice, amniotic fluid, aqueous humor, breast milk, earwax, bile, exudate, mucus, pericardial fluid, peritoneal fluid, pleural effusion, sebum, serous fluid, phlegm, sweat, vomit, bronchoalveolar lavage fluid (BALF), etc.

[0022] In other embodiments, the biological sample can include all human tissues (e.g., fresh, frozen, fixed, or processed) and / or human whole blood (e.g., peripheral blood and / or cord blood) and blood by-products (e.g., serum, plasma, buffy coat) and / or all human biological fluids (e.g., sputum, urine, bile) and / or human primary cells derived from human biological samples, and / or DNA from individual donors.

[0023] In other embodiments, the activating factor may comprise a solution containing one or more agents known to those skilled in the art to be associated with a disease (e.g., inflammation). In certain embodiments, the activating factor comprises one or more immune cells and / or inflammatory cells, such as Th1 cells, CD4+ cells, macrophages, dendritic cells, or any combination thereof. In other cases, the activating factor comprises one or more types of cytokines, such as, for example, lymphokines (cytokines made by lymphocytes), monokines (cytokines made by monocytes), chemokines (cytokines having chemotactic activity), and / or interleukins (cytokines made by one leukocyte 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-gamma, and TNF-alpha.

[0024] In some embodiments, the activating factor comprises mechanical stimulation. For example, many solid tumors (e.g., glioblastoma, prostate, breast) exhibit high solid stress driven by a hypoxic tumor microenvironment. In such cases, the activating factor may, for example, constitute the oxygen partial pressure of the MSC culture. For example, in some cases, the method comprises reducing the oxygen partial pressure of the MSC culture from about 20% oxygen to 1% - 5% oxygen (the percentages are volume percentages). In some embodiments, the method comprises reducing the oxygen partial pressure to 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, etc. of the total gas volume. In other embodiments, the method comprises reducing the oxygen partial pressure to 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, etc. of the total gas volume.

[0025] Other mechanical stresses, such as shear stress or cyclic mechanical strain, can also be used as activating factors. For example, in some embodiments, MSCs can be grown on microcarriers and expanded in a stirred batch bioreactor, and the stirring action induces fluid shear stress on the cultured MSCs. Other types of mechanical stimuli can include acoustic activation (e.g., subjecting a culture of MSCs to sonication) 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 enrich the MSC microenvironment and stimulate changes in the miRNA content of EVs. Combinations are also possible, for example, a culture of MSCs can be exposed to an activating factor solution and a low oxygen partial pressure.

[0026] After exposing the MSC cultures to the activating factor, mesenchymal stromal cell extracellular vesicles (MSC-EVs) can be recovered and the miRNA content within the MSC-EVs can be measured. MSC-EVs can be recovered from cell culture media using commercially available kits such as Exosome Isolation Kits (Miltenyli Biotec) and exoEasy Maxi Kit (Qiagen), or any other technique known to those skilled in the art, such as magnetic isolation, ultracentrifugation, differential ultracentrifugation, sequential centrifugation, size-based fractionation (e.g., tangential flow filtration and size exclusion chromatography), etc. Following the isolation of the EVs, the relevant miRNAs can be isolated (e.g., using standard laboratory practice). In most cases, commercially available kits can be purchased and used to extract the miRNAs (e.g., exoRNeasy Midi and Maxi kits from Qiagen). The miRNAs are then sequenced using nucleic acid-based detection assays (e.g., pyrosequencing on the 454 Life Sciences platform, polymerase-based sequencing by synthesis on the Illumina platform, sequencing by ligation on the ABI Solid Sequencing platform, or the HTG EdgeSeq miRNA Transcriptome Assay) to identify the miRNAs present in the EVs. The latter includes the identification of known miRNAs and the identification of novel miRNAs (e.g., by performing miRNA alignment analysis).

[0027] In some embodiments, the miRNA sequencing analysis measures the expression of 1000 to 5000 human miRNA transcripts. In some embodiments, the sequencing analysis measures human miRNA transcripts of 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, etc. In other embodiments, the sequencing analysis measures human miRNA transcripts of 5000 or less, 4000 or less, 3000 or less, 2000 or less, or 1000 or less.

[0028] After miRNA sequencing of MSC-EV-miRNA, differential expression analysis of miRNAs can be performed. Differential expression analysis is useful, for example, for comparing the effects of two different activators on MSC-EV-miRNA content (e.g., ARDS BALF vs. HV BALF), or the effect of a single activator over various time points. For example, in some embodiments, treatment of MSC cultures with ARDS BALF induces, inter alia, the 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, each relative to MSCs cultured with HV BALF.

[0029] Differential expression analysis (DEA) can be performed using any software known to those skilled in the art, such as DESeq2 of R (R package "DESeq2"). Although not wishing to be bound by theory, in some cases, miRNA sequence data may be prepared for differential expression analysis using variance stabilization transformation. In some embodiments, differential expression analysis includes performing significance analysis of microarrays (SAM) of normalized read counts using a one-class analysis approach to identify miRNAs that accounted for a large proportion in EVs from control samples. In other embodiments, differential expression analysis includes setting the delta value to 7 (i.e., ensuring the lowest false discovery rate [FDR]) using 1000 permutations. In some cases, miRNAs considered to be differentially expressed (DE) may exhibit an expression change of 1.2-fold or more, 1.5-fold or more, a 1.7-fold or more increase in expression, 2-fold or more, 2.5-fold or more, 3-fold or more, etc. In other embodiments, miRNAs considered to be differentially expressed may exhibit an expression change of 3-fold or less, 2.5-fold or less, 2-fold or less, 1.7-fold or less, 1.5-fold or less, 1.2-fold or less, etc. In some cases, miRNAs considered to be differentially expressed (DE) may be selected based on an expression change of 2-fold or more (i.e., average expression across samples exceeding 50 read counts) and an adjusted P-value (or FDR) of less than 0.05 after correction for multiple comparisons.

[0030] In some embodiments, the method further includes performing target prediction and enrichment analysis. Target prediction analysis identifies target mRNAs of miRNAs and helps to provide an understanding of the genes or gene networks they regulate. 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), and generally includes (1) determining miRNA:mRNA binding pairs. This can be done by identifying complementarity between miRNA sequences in the 3'-UTR of the mRNA sequence, (2) determining the degree of conservation of miRNA:mRNA binding pairs across species, and (3) observing evidence of miRNA targeting in mRNA-Seq or protein expression data, where high miRNA expression results in low protein expression of the gene and its target gene.

[0031] In some embodiments, after identifying the target mRNAs, gene set enrichment analysis can be performed. As would be understood by one of ordinary skill in the art, such analysis, for example, accounts for a large proportion in large gene sets or protein sets and can be used to identify the classification of genes or proteins that may be associated with disease phenotypes. Similar to other "omics platforms", there are several commercially available tools for performing gene enrichment analysis. Exemplary tools include, but are not limited to, NASQAR, PlantRegMap, Blas2Go, GREAT, MSigDB, and the like.

[0032] Additional aspects of the method include performing sparse partial least squares discriminant analysis (sPLS-DA). Without 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., a y variable, ARDS positive or negative patients) and independent variables (e.g., x variables, miRNA expression). This is achieved by using a latent variable approach, which allows for the analysis of a categorical response variable (e.g., ARDS positive or negative patient samples) as if it were a continuous variable. This enables the model to perform variable selection and classification in a one-step procedure. In other words, the implementation of sPLS-DA on sequencing data (e.g., miRNA sequencing data) can be used to create a mathematical formula that correlates x variables (e.g., EV-miRNA) with y variables (e.g., ARDS positive, ARDS negative, control). This formula can be used, for example, to classify a target disease as likely to respond or not respond to an MSC cell-based therapy. Additionally, this formula can be used to prescribe a cell-free drug therapy, for example, by providing the identification of therapeutic miRNAs and estimates of their relative concentrations (e.g., for encapsulation within lipid nanoparticles).

[0033] In some embodiments, performing the sPLS-DA analysis includes obtaining a miRNA sequence library. In some embodiments, the miRNA sequence library can include 1000 to 5000 human miRNA transcripts. In some embodiments, the library includes human miRNA transcripts that are 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, etc. In other embodiments, the library includes human miRNA transcripts that are 5000 or less, 4000 or less, 3000 or less, 2000 or less, or 1000 or less.

[0034] In some embodiments, performing sPLS-DA analysis includes randomly splitting miRNA sequencing data into training and test settings using a 0.7 / 0.3 split. In other embodiments, the training setting / test setting ratio may be 0.1 / 0.9 or greater, 0.2 / 0.8 or greater, 0.3 / 0.7 or greater, 0.4 / 0.6 or greater, 0.5 / 0.5 or greater, 0.6 / 0.4 or greater, 0.7 / 0.3 or greater, 0.8 / 0.2 or greater, 0.9 / 0.1 or greater. In some embodiments, the ratio of miRNAs in the training setting may be 0.9 / 0.1 or less, 0.8 / 0.2 or less, 0.7 / 0.3 or less, 0.6 / 0.4 or less, 0.5 / 0.5 or less, 0.4 / 0.6 or less, 0.3 / 0.7 or less, 0.2 / 0.8 or less, or 0.1 / 0.9 or less.

[0035] The method may include, in some embodiments, performing overlapping analysis and / or network analysis. Without wishing to be bound by theory, overlapping analysis may be useful, for example, when comparing differentially expressed genes between various experimental groups and / or analysis techniques (e.g., differential expression analysis vs. sPLS-DA). Network analysis may be used, for example, to visualize predicted "direct" interactions between differentially expressed miRNAs of interest and their target genes. Such an approach may enable the identification of one or more subsets of miRNAs that function as key regulators or "hubs" of MSC paracrine signaling (e.g., in response to a given activator, such as ARDS BALF).

[0036] ARDS-specific MSC-EV-miRNA profile Aspects of the present disclosure relate to, in some cases, using the methods described above to identify MSC-EV-miRNA profiles for a target disease such as, for example, ARDS. For example, Examples 1-8 highlight the use of the present method 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, which collectively regulate processes such as inflammation, cell cycle, proliferation, apoptosis, and Wnt signaling. In some embodiments, the differentially regulated miRNAs can be further screened for a reduced set of miRNAs that cooperate to form a putative in-silico regulatory network acting as putative hub regulators for the entire set of gene targets (about 1259 putative targets in the case of the ARDS profile). In the case of the MSC-EV-miRNA profile (generated from ARDS BALF and HV BALF), in some embodiments, the reduced set of miRNAs includes miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p.

[0037] Other diseases of interest may also be profiled using the methods described above. For example, MSC-EV-miRNA profiles can be generated for other lung diseases (e.g., chronic lung disease, chronic obstructive pulmonary disease, emphysema, asthma, etc.), neurological diseases (e.g., epilepsy, traumatic brain injury, brain injury in premature neonates, and stroke), ischemic diseases (e.g., myocardial infarction, chronic renal insufficiency respiratory failure), inflammatory joint diseases (e.g., osteoarthritis and rheumatoid arthritis), infectious diseases, various ophthalmic diseases, and cancer. In some embodiments, it is also possible to profile the content of MSC-EVs during regenerative processes (e.g., bone, liver, heart, muscle, blood cell regeneration).

[0038] Methods of using MSC-EV-miRNA profiles Aspects of the present disclosure relate to methods of identifying diseases that are likely to benefit from MSC therapies, such as, for example, cancer immunotherapy. For example, in some embodiments, the method includes obtaining a first biological sample from a healthy first subject and a second biological sample from a suspected subject 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 a first culture of MSCs and a second culture of MSCs, respectively, and detecting whether at least one MSC-EV-miRNA is present in the first and / or second cultures of MSCs, for example, using next-generation sequencing (e.g., microRNA-Seq). As described elsewhere herein, a series of analyses (e.g., functional prediction, overlap analysis, and discriminant analysis) can be performed after miRNA sequencing to generate a disease-specific differential EV-miRNA profile based on the presence or absence of each miRNA (relative to a control). In certain embodiments, a differential MSC-EV-miRNA profile showing overexpression of relevant miRNAs may suggest that a suspected subject having a disease may be a candidate for MSC-based therapy. Similarly, a miRNA profile showing limited changes in a differential miRNA expression profile may, in other embodiments, suggest that a suspected subject having a disease is not likely to be a candidate for MSC-based therapy.

[0039] Pharmaceutical composition Aspects of the present disclosure relate to pharmaceutical compositions for delivering one or more miRNAs from an MSC-EV-miRNA profile that do not contain intact MSCs or MSC-EVs. In some embodiments, the pharmaceutical composition comprises at least one naked miRNA (e.g., identified using an MSC-EV-miRNA profile). As used herein, "naked miRNA" refers to a miRNA that is not complexed with another compound (e.g., not encapsulated within a lipid nanoparticle). In some embodiments, the pharmaceutical composition comprises at least one miRNA (e.g., identified using an MSC-EV-miRNA profile) and a pharmaceutically acceptable excipient (e.g., a carrier). As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmacologically inert material used in conjunction with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include, but are not limited to, various materials known in the art, including saccharides (e.g., glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, coloring agents, physiological saline (e.g., phosphate buffered saline), and buffering agents.

[0040] In some embodiments, the pharmaceutical composition comprises at least one miRNA (identified using an MSC-EV-miRNA profile) and a delivery vehicle that encapsulates the at least one miRNA, such as a lipid nanoparticle. Optionally, the pharmaceutical composition comprises one or more miRNAs, two or more miRNAs, three or more miRNAs, five or more miRNAs, seven or more miRNAs, ten or more miRNAs, twelve or more miRNAs, fourteen or more miRNAs, sixteen or more miRNAs, or twenty or more miRNAs. In other embodiments, the pharmaceutical composition comprises twenty or fewer miRNAs, sixteen or fewer miRNAs, twelve or fewer miRNAs, ten or fewer miRNAs, seven or fewer miRNAs, five or fewer miRNAs, three or fewer miRNAs, or one or fewer miRNAs.

[0041] 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.

[0042] In some embodiments, the delivery vehicle may include lipid nanoparticles and / or liposomes. "Liposome" is a general term encompassing various single and multi-layer lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. These are formed spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure and take up water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicular structures in solution are also included. For example, the lipids may take on a micellar structure or may simply exist as a heterogeneous aggregate of lipid molecules.

[0043] At least one miRNA may be encapsulated within the aqueous interior of the liposome, may be interspersed within the lipid bilayer of the liposome, may be bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, may be trapped within the liposome, may be complexed with the liposome, may be dispersed in a lipid-containing solution, may be mixed with the lipid, may be combined with the lipid, may be contained as a suspension within the lipid, may be contained within or complexed with micelles, or may be associated with the lipid in other ways. The lipid, or lipid / nucleic acid composition, is not limited to any particular structure in solution. For example, they may be within a bilayer structure as micelles or may exist with a "collapsed" structure. They may also simply be dispersed in solution or may form aggregates that are not uniform in size or shape.

[0044] In another embodiment, the liposome comprises a transfection reagent (e.g., cationic and / or anionic lipids). In another embodiment, the liposome increases stability within cells, increases uptake efficiency, and improves biological activity. In another embodiment, the liposome is a hollow spherical vesicle composed of lipids arranged in a manner similar to the lipids that make up the cell membrane. In some embodiments, the liposome includes an internal aqueous space for trapping water-soluble compounds. In another embodiment, the liposome can deliver at least one MSC-EV-miRNA in an active form to cells.

[0045] In one embodiment, the composition comprises lipid nanoparticles (LNPs) and at least one miRNA.

[0046] The term "lipid nanoparticle" (LNP) refers to particles having at least one dimension on the nanometer order (e.g., 1 - 1000 nm) that contain one or more lipids. In some embodiments, the LNP is organized within an inverse lipid micelle, encapsulated within a lipid monolayer envelope, or contains at least one agent interposed between adjacent lipid bilayers (e.g., lipid bilayer - agent - lipid bilayer). In some embodiments, the morphology of the LNP differs from that of conventional liposomes characterized by a lipid bilayer surrounding an aqueous core, having a core with high electron density and being organized into inverse micelles around an agent encapsulated with a cationic / ionizable lipid.

[0047] In some embodiments, the lipid nanoparticles are substantially non - toxic. In certain embodiments, at least one agent, when present in the lipid nanoparticles, is resistant to degradation by intracellular or intercellular enzymes in an aqueous solution.

[0048] The LNP can include any lipid capable of forming particles to which at least one miRNA is attached, or in which at least one miRNA is encapsulated or complexed. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters), generally insoluble in water but soluble in many organic solvents. Exemplary lipids are shown elsewhere in this specification.

[0049] In one embodiment, the LNP includes one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids, anionic lipids, and pegylated (PEG - added) lipids.

[0050] In one embodiment, the LNP contains a cationic lipid. As used herein, the term "cationic or ionizable lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH is lower than the pKa of the ionizable group of the lipid and gradually becomes neutral as the pH value increases. At a pH value below the pKa, the lipid can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid includes a zwitterionic lipid that becomes positively charged as the pH decreases.

[0051] In various embodiments, the LNP contains a cationic or ionizable lipid, a stabilizing lipid, a sterol, and a polyethylene glycol to which the lipid is immobilized (i.e., a PEGylated lipid).

[0052] In certain embodiments, the LNP contains one or more stabilizing lipids (e.g., neutral or anionic lipids) that encapsulate the cargo and help stabilize particle formation during particle formation.

[0053] In various embodiments, the LNP further contains a steroid or a steroid analog.

[0054] In certain embodiments, the LNP contains one or more targeting sites that target the LNP to a cell or cell population. For example, in one embodiment, the targeting domain is a ligand that directs the 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-associated molecular pattern receptors.

[0055] In certain embodiments, the LNP is formed by co-injecting an aqueous solution of mRNA and an ethanol solution of lipid through a microfluidic device, resulting in the spontaneous formation of vesicles.

[0056] In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells to induce internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to receptors found on the cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding to a biomolecule in vivo, and the biomolecule bound to the LNP can then be recognized by a cell surface receptor and induce internalization. For example, in one embodiment, the LNP binds to systemic ApoE and causes uptake of the LNP and associated cargo.

[0057] Other exemplary LNPs and their manufacture are described in the art, for example, 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; BBasha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys 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 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): 1570-15788; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, which are incorporated herein by reference in their entirety.

[0058] Engineered MSCs for Cell Therapy Aspects of the present disclosure relate to cell therapies (cell therapeutics) that, in some cases, include genetically engineered MSC cells configured to overexpress one or more miRNAs identified using an MSC-EV-miRNA profile and package them into EVs. In certain embodiments, the cell therapy includes engineered MSCs (i.e., MSCs and EVs), but in some cases, the secreted EVs may be isolated and used to directly deliver the miRNA of interest to a subject (e.g., similar to LNPs).

[0059] In some embodiments, the MSCs may 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, the MSCs may 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.

[0060] In some embodiments, the cell therapy includes a culture of MSCs that overexpress a single miRNA of interest, e.g., a miRNA identified from an MSC-EV-miRNA profile. In certain embodiments, the cell therapy includes a mixture of subpopulations of MSCs, each subpopulation overexpressing a single miRNA of interest. The cell therapy may also include one or more subpopulations of MSCs engineered to overexpress multiple miRNAs (see below).

[0061] Aspects of the present disclosure relate, in some cases, to a method of generating therapeutic MSCs, wherein the therapeutic MSCs are genetically engineered MSC cells configured to overexpress one or more miRNAs identified from an MSC-EV-miRNA profile. In some embodiments, the method includes obtaining a first biological sample from a healthy first subject and 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 a first culture of MSCs and a second culture of MSCs, respectively, and detecting whether at least one MSC-EV-miRNA is present in the first and / or second culture of MSCs, for example, using next-generation sequencing (e.g., microRNA-Seq). As described elsewhere herein, a series of analyses (e.g., functional prediction, overlapping analysis, and discriminant analysis) can be performed after miRNA sequencing to create a differential EV-miRNA profile based on the presence or absence of each miRNA, compared to a first healthy subject (or other appropriate control). miRNAs overexpressed in the MSC-EV-miRNA profile can then be used as therapeutic targets for overexpression in clinically graded MSCs.

[0062] The MSC can be engineered to overexpress the miRNA of interest using any technique known to those skilled in the art, such as infection with a virus having the gene of interest (e.g., a recombinant gene) or direct transfer of plasmid DNA having the gene of interest (e.g., a recombinant gene). For example, any miRNA of interest, such as hsa-miR-760, may be cloned into a commercially available lentiviral vector system (e.g., XMIRXpress cloning lentiviral vector, System Biosciences) that includes an RNA sequence tag, such as XMotif, that targets the small RNA to exosomes for packaging. In some embodiments, the engineered MSC can express the recombinant gene of interest indefinitely (i.e., function like a cell line), while in some cases, the engineered MSC can express the recombinant gene of interest only transiently.

[0063] In some embodiments, the method includes engineering the MSC to overexpress two or more genes of interest. Any technique known to those skilled in the art can be used to engineer the MSC to overexpress multiple genes of interest and package them into EVs. For example, in some cases, different expression vectors each having a different miRNA gene of interest can be used. In some cases, a single vector containing multiple genes each having its own promoter may be constructed. Some additional options can include using a translational fusion approach where two genes of interest are genetically joined in-frame to ensure stoichiometric production of both miRNAs. Another strategy can include using an internal ribosome entry site (IRES), which facilitates ribosome binding to the second and subsequent transcription units. Other strategies and approaches are possible. The method according to certain embodiments further includes delivering the therapeutic MSC to a subject in need thereof (e.g., a subject diagnosed with ARDS).

[0064] Primers for rapid detection of miRNA profile Aspects of the present disclosure relate, in some cases, to oligonucleotide primer mixes. The primer mix can be used, for example, for quantification of miRNA expression in MSC-EVs (i.e., biomarkers for QC) to confirm that the correct combination of miRNAs is being made (e.g., the miRNAs identified in the MSC-EV-miRNA profile). In some cases, the primer mix includes a forward primer and / or a reverse primer. In some embodiments, the oligonucleotide primer mix includes a stem-loop reverse transcriptase (stem-loop RT) primer and / or a linear primer. In certain embodiments, the primer mix includes one or more oligonucleotides, each oligonucleotide being configured to hybridize to a single miRNA of interest, and 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, hsa-miR-6787-5p.

[0065] In some embodiments, an oligonucleotide primer mix that includes one or more oligonucleotide primers is used, and the presence (or absence) of a miRNA of interest in a sample can be detected (e.g., during quality control checks of MSC cell therapy). For example, in some embodiments, stem-loop hairpin RT is used to bind to the target miRNA at the 3' end and reverse transcription is performed using a reverse transcriptase (e.g., MultiScribe reverse transcriptase), and the RT product can be quantified using a conventional PCR (e.g., Taqman PCR) that includes a miRNA-specific forward primer, a reverse primer, and a dye-labeled probe (e.g., Taqman probe).

[0066] Quantitative RT-PCR using DNA primers is another example where a primer mix can be used to quantify the miRNA of interest in a sample. Although not wishing to be bound by theory, this method relies on poly(A) tailing of the miRNA followed by reverse transcription (RT) using tagged poly(T) primers. The RT product can then be quantified using standard PCR with a primer set (e.g., forward primer and reverse primer) specific for the target miRNA transcript (e.g., 5' tag and 3' tag).

[0067] In certain embodiments, the number of primers in the primer mix can be 1 or more primers, 2 or more primers, 4 or more primers, 6 or more primers, 8 or more primers, 10 or more primers, 12 or more primers, 14 or more primers, or 20 or more primers, or more. In other embodiments, the number of primers in the primer mix can be 20 or fewer primers, 14 or fewer primers, 12 or fewer primers, 10 or fewer primers, 8 or fewer primers, 4 or fewer primers, 2 or fewer primers, 2 or fewer primers, 1 or fewer primers, etc.

[0068] Primer kit Aspects of the present disclosure relate to kits for detecting the presence of at least one miRNA identified using an MSC-EV-miRNA profile in a biological sample, in some cases. In some embodiments, the kit includes a primer mix comprising one or more oligonucleotides, each oligonucleotide being configured to hybridize to a single miRNA of interest, and 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, hsa-miR-6787-5p. The kit may also include, in certain embodiments, reagents (e.g., fluorescent probes) for performing a nucleic acid assay (e.g., PCR) for detecting at least one MSC-EV-miRNA using nucleic acid pairs, and instructions for use for performing an assay for detecting at least one MSC-EV-miRNA. In some embodiments, the primer mix includes 1 to 14 primer pairs for detecting up to 14 different miRNAs. In other embodiments, the primer mix includes 1 to 28 primers for detecting 1 to 14 miRNAs of interest. In some embodiments, the kit can be used to screen a culture of MSCs (intended for cell therapy applications) for the presence of therapeutic miRNAs (identified via the MSC-EV-miRNA profile) for the treatment of ARDS and other inflammatory diseases (e.g., COPD, stroke, myocardial infarction).

[0069] Source of MSC cells The present disclosure, in some aspects, relates to obtaining mesenchymal stromal / stem cells (MSCs). In some embodiments, the MSCs are of human origin. In some embodiments, the MSCs are of animal origin (e.g., dog, cat, or monkey). Primary MSCs can be obtained from any suitable source (i.e., tissue) such as, for example, bone marrow (BM), adipose tissue (AD), and / or placental membrane / umbilical cord blood (UC), and can be cultured, for example, using a cell culture device. In some embodiments, the culture of MSCs may contain BM-MSCs, and in some cases, the culture of MSCs may contain AD-MSCs. In other embodiments, the culture of MSCs may contain UC-MSCs. MSCs may 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., the culture may contain 50% BM-MSCs and 50% AD-MSCs, where the percentages are relative to the total number of MSCs in the culture).

[0070] In some embodiments, the culture of MSCs contains BM-MSCs. In some cases, the BM-MSCs contain 10% or more, 25% or more, 50% or more, 75% or more, 100% or more of the total number of MSCs in the culture. In other embodiments, the BM-MSCs contain 100% or less, 75% or less, 50% or less, 25% or less, 10% or less of the total number of MSCs in the culture.

[0071] In some embodiments, the culture of MSCs contains AD-MSCs. In some cases, the AD-MSCs contain 10% or more, 25% or more, 50% or more, 75% or more, 100% or more of the total number of MSCs in the culture. In other embodiments, the AD-MSCs contain 100% or less, 75% or less, 50% or less, 25% or less, 10% or less of the total number of MSCs in the culture.

[0072] In some embodiments, the culture of MSCs contains UC-MSCs. In some cases, the UC-MSCs contain 10% or more, 25% or more, 50% or more, 75% or more, 100% or more of the total number of MSCs in the culture. In other embodiments, the UC-MSCs contain 100% or less, 75% or less, 50% or less, 25% or less, 10% or less of the total number of MSCs in the culture.

[0073] One skilled in the art would understand that the confluence of MSC cultures can have an adverse effect on EV production. Thus, in some embodiments, MSC cultures may be grown to a confluence of 25% or greater, 50% or greater, or 75% or greater. In certain embodiments, MSC cultures may be grown to a confluence of 75% or less, 50% or less, or 25% or less.

[0074] It is also generally understood that the number of passages of cells in MSC cultures (i.e., replicative senescence) can also have an adverse effect on MSC EV production. Thus, in some embodiments, MSC cultures can be passaged 3 to 8 times before showing morphological abnormalities. In some embodiments, MSC can be passaged 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more times. In other embodiments, MSC can be passaged 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less times.

[0075] In certain cases, the MSC cells may be immortalized MSC strains. Immortalized MSCs can be used, for example, to ensure batch reproducibility, avoid donor variability between individuals, and maintain biological activity during culture expansion. Any technique known to those skilled in the art can be used to immortalize MSCs. For example, in some embodiments, MSCs derived from embryonic stem cells can be immortalized by transfection with a lentivirus carrying the c-Myc oncogene.

[0076] In some aspects, the present disclosure relates to culturing mesenchymal stromal / stem cells (MSCs). MSCs can be cultured using any technique known to those skilled in the art, such as, for example, in a bioreactor, or other in vitro cell culture devices (e.g., culture dishes, multilayer cell culture flasks, hollow fiber bioreactors, stirred tank bioreactors, and spheroid aggregates of MSCs). In some embodiments, culturing MSCs using a 3D system (e.g., a bioreactor) results in a 40-fold to 100-fold increase in EV production compared to a 2D culture system (e.g., a culture dish). In some cases, culturing MSCs using a 3D system results in an increase in EV production that is 40-fold or more, 60-fold or more, 80-fold or more, or 100-fold or more compared to a 2D culture system. In other embodiments, culturing MSCs using a 3D system results in an increase in EV production that is 100-fold or less, 80-fold or less, 60-fold or less, or 40-fold or less compared to a 2D culture system.

[0077] In some embodiments, the MSC culture may be grown in a cell growth medium. In some cases, the cell growth medium comprises a defined cell growth medium. In certain embodiments, the defined growth medium is, for example, xeno-free and / or EV-free for use in clinical applications where variability in MSC source and animal contamination are of concern. In other embodiments, the cell growth medium comprises an undefined medium, for example, containing human serum or human platelet lysate. Human platelet lysate contains multiple growth factors, cytokines, hormones, proteins, carbohydrates, and lipids that stimulate cell proliferation and is often used as a substitute for fetal bovine serum.

[0078] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative and not limiting in any way the remainder of the present disclosure. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to herein.

[0079] [Examples] To more fully understand the invention described herein, the following examples are provided. The examples described in this application are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed as limiting the scope in any way.

[0080] [Example 1] Characteristics of Extracellular Vesicles (EVs) after Bronchoalveolar Lavage Fluid (BALF) Exposure Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) may be used as therapeutic agents for acute respiratory distress syndrome (ARDS). As described herein, the gene and protein expression of MSCs are modulated by the lung environment of ARDS. As described below, the effect of this environment on the characteristics and miRNA content of MSC-EVs was investigated.

[0081] BALF exposure does not significantly affect the number or size distribution of EVs, but decreases the CD63 expression of EVs secreted by hMSCs. An overall schematic of the study is shown in Figure 1, summarizing the experimental protocols and analytical approaches utilized. First, to determine whether the number and size of EVs change by exposing hMSCs to different lung environments, BALF was used as a clinical surrogate and hMSC-EVs were prepared from hMSCs exposed to BALF from ARDS patients or HVs, or hMSCs exposed to control medium. From NTA analysis, it was revealed that 90% of the EVs secreted by hMSCs were in the size range of 50 - 200 μm and there was no significant difference in the number of EVs regardless of whether the cells were exposed to control medium or BAEF (Figure 2A). Both HV and ARDS BALF exposures decreased CD63-positive EVs overall compared to the control, and HV BALF exposure decreased CD63 expression more than ARDS BALF exposure (Figure 2B, left). There was no difference in CD81 expression of EVs among the experimental conditions. The difference in CD63 expression between the HV condition and the ARDS condition led to a significant difference in the expression of double-positive (CD63 / CD81) EVs (Figure 2B, middle, right). CD9 expression was not detected (data not shown).

[0082] EVs directly derived from ARDS samples and EVs directly derived from HV BAEF samples differ in the number of EVs rather than the expression of tetraspanins. EVs isolated from hMSCs exposed to BALF showed a narrow size distribution and similar area under the curve (AUC) measurements, whereas the size distribution of EVs isolated from BALF samples alone encompassed a wider range, and approximately 20% of the EVs from both ARDS and HV were larger than 200 μm. Furthermore, ARDS BALF samples had significantly more particles than HV or control samples (Figure 2C). Flow imaging revealed two major findings that contrasted with the analysis of EVs isolated from hMSCs. Specifically, in both HV and ARDS BALF, the expression of CD63 and CD81 was increased compared to the control. Additionally, the expression of CD9 was detectable in EVs from both HV and ARDS BALF, whereas it was not expressed in EVs derived from hMSCs exposed to HV or ARDS BALF (Figure 2D).

[0083] [Example 2] EVs derived from quiescent control hMSCs contain miRNAs predicted to regulate inflammatory pathways. Before investigating the effect of the inflammatory lung environment on the miRNA cargo of EVs, miRNAs were profiled in EV preparations obtained from control hMSCs (exposed to serum-free medium only). A total of 770 miRNAs were found to be present, of which 48 accounted for a significantly large proportion, as defined by counts that significantly exceeded the average counts of all miRNAs across all biological replicates (observed / expected) (significant analysis of microarrays [SAM] score > 20, delta [δ] set to 7 (default), false discovery rate [FDR] = 0.0, Figures 3A and 3B). Enrichment analysis of these 48 miRNAs was performed with miRNet (DIANA miRTarbaSe v8.0) based on target prediction, and overexpression of miRNAs that regulate genes involved in, among other pathways, cell cycle, regulation of gene expression, immune system regulation, immune system cytokine signaling, and in particular, 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 miRNAs that accounted for a large proportion and the experimentally identified gene targets. The top miRNAs and gene targets predicted in the network were involved in the regulation of the cell cycle (Figures 3D and 3E).

[0084] [Example 3] The miRNAs in hMSC-EVs are not randomly packaged and can be changed by environmental factors In this experiment, we investigated whether the miRNA cargo of EVs changes by exposing hMSCs to BALF from different environments. EVs were isolated from conditioned media from hMSCs exposed to either serum-free medium (control, N = 16), BALF from ARDS patients (ARDS, N = 12), or BALF from healthy volunteers (HV, N = 14). The expression levels of 2,083 miRNAs contained in the EVs isolated from these 42 samples were profiled using miRNA whole transcriptome sequencing. Principal component analysis (PCA) of the non-filtered, normalized miRNA counts revealed clustering of samples by treatment assignment, suggesting that the miRNA content may change by exposing hMSCs to different environments rather than being randomly packaged.

[0085] [Example 4] hMSCs differentially package miRNAs into EVs in response to different inflammatory environments To determine the effect of different inflammatory environments on the miRNA cargo of EVs, three pairwise comparisons were performed: HV vs. control (DE = 153 miRNAs), ARDS vs. control (DE = 97 DE miRNAs), and ARDS vs. HV (DE = 126 miRNAs). miRNAs were considered differentially expressed if they met a statistical cut-off of more than a two-fold change in expression (Log2 fold change [FC]) and FDR < 0.05. The volcano plots in Figures 4A - 4C show miRNAs that accounted for a large proportion for each comparison and miRNAs that were insufficient as samples. The top 10 predicted pathways that were consistently regulated in each comparison are shown in Figures 4A - 4C. EVs from hMSCs exposed to HV BALF had more miRNAs associated with decreased expression of genes involved in the cell cycle and the signaling of TGFβ, VEGF, and EGFR compared to the control. (Figure 4A). In contrast, EVs from hMSCs exposed to HV BALF had fewer miRNAs involved in the cellular response to stress, including the organization of the extracellular matrix and the activation of hypoxia-inducible factor (HIF), compared to the control. For some groups, such as genes involved in platelet activation, signaling, and aggregation, individual genes accounted for a proportion without excess or deficiency. Compared to the control, EVs from hMSCs exposed to ARDS BALF had a large proportion of miRNAs predicted to inhibit genes involved in the cellular response to stress and interferon signaling, and miRNAs predicted to promote the expression of genes involved in the selective induction of canonical mitochondrial apoptosis of BH3 (e.g., Bax and Bad) in response to developmental cues or stress signals such as DNA damage were insufficient as samples (Figure 4B). Comparing the miRNAs differentially expressed in EVs of ARDS vs. HV, it was surprising that there were more miRNAs that were insufficient as samples than in each of the previous comparisons of control vs. HV or ARDS. Genes affected included those involved in the cell cycle, DNA synthesis, TLR4, and NF-κB activation after exposure to ARDS BALF.In contrast, miRNAs that accounted for a large proportion in hMSCs exposed to ARDS as compared to HV were enriched to regulate genes involved in interferon, activin (related to TGF-β), and FGFR signaling (Figure 4C).

[0086] Repeated analysis demonstrated consistent differences in the expression of EV miRNAs among experimental groups Venn diagrams were used to visualize the overlap between lists of miRNAs differentially regulated in each comparison (Figures 5A - 5D). Consistent differential expression in more than two comparisons was used to filter miRNAs that were likely to vary in each of the three conditions (i.e., control vs HV or exposure to BALF of ARDS patients, Figures 4B - 4C). The miRNA expression patterns were visualized by plotting the Log2 FC of miRNA expression for each miRNA that overlapped across each comparison (Figures 5E - 5G). The enrichment analysis of miRNAs in each repeated analysis is shown in Figures 5E - 5G. Functional analysis showed that miRNAs predicted to be involved in regulation of gene expression, cellular stress, RNA processing, alternative splicing, and TGF-β receptor complex signaling were differentially expressed in all comparisons with control hMSC-EV (N = 23) (the top 10 pathways enriched in the analysis are shown in Figure 5E). miRNAs regulated in all comparisons with HV BALF exposure samples (N = 57) were of particular interest because they were regulated in the opposite direction in EVs from hMSCs exposed to ARDS BALF. Depending on the direction of miRNA change, these miRNAs were predicted to increase or decrease pathways involved in, among other things, cell cycle and pathways involved in infection, similar to signal transduction by TGF-β regulation (Figure 5F). miRNAs responding to ARDS BAEF were predicted to be involved particularly in cytokine signaling, interferon-β and signaling, and programmed cell death, etc. (Figure 5G).

[0087] [Example 5] Identification of 52 miRNAs strongly related to the biological and therapeutic effects of hMSCs Since the miRNAs that were differentially present in all comparisons seemed to be packaged within hMSC-derived EVs under all experimental conditions, they were considered to have biological and / or therapeutic significance. Thus, if hMSCs confer beneficial effects, these miRNAs are not specifically present within hMSC-derived EVs but are likely involved in the mechanism that confers such effects. From the overlap of the three pairwise comparisons, it was demonstrated that 13 miRNAs were differentially regulated in all hMSC-EV preparations (Figure 6A). To highlight the effect of ARDS-BALF on differential miRNA packaging, separate pairwise comparisons combining the effects of the control and HV (control + HV) were performed. A total of 52 miRNAs were found to be robustly differentially expressed in all three comparisons, which also included 13 overlapping miRNAs (Figure 6B; Table 1).

[0088] The pathways predicted to be regulated by the 13 co-expressed miRNAs in Overlap 1 (Figure 6A) were found to affect genes particularly involved in Wnt signaling and mRNA processing and stability (Figure 6C). The miRNAs co-regulated in Overlap 2 (52 miRNAs, Figure 6B) interacted with genes known to be involved in interferon and cytokine signaling, Wnt, Toll-like receptor (TLR3 / 4), and nerve growth factor (NGF) signaling, as well as programmed cell death (Figure 6D). The similar patterns of miRNA expression changes in each comparison were visualized as horizontal bar graphs. Heatmaps summarizing the consistency of miRNA expression changes across all 42 biological samples are shown in Figures 6E and 6F.

[0089] To identify miRNAs potentially associated with the biological and therapeutic effects of hMSCs, miRNAs present in EVs were isolated from control hMSCs and differentially expressed across all treatment conditions (including demonstration of maintained differential expression when combining the effects of unstimulated and healthy volunteers (control + HV vs ARDS)). Network analysis of two overlapping miRNAs (N = 52, Figure 7A) showed that co-regulated miRNAs and their gene targets formed a functionally related interaction network. Two of these networks, particularly notable in acute lung injury, were involved in inflammasome activation and cellular stress (Figures 7B and 7C). Sample-level data are shown as box-and-whisker plots of Log2-normalized counts of the top 20 miRNAs from the two networks (ranked by FDR, Figure 8A), demonstrating the trend of differential expression of EV-derived miRNAs.

[0090]

Table 1

[0091] [Example 6] Differential expression of the top 20 miRNAs contained in BALF-EV strongly suggests a biological effect of exposure to miRNAs in hMSC EVs rather than cross-contamination from BALF samples. We evaluated the miRNAs contained in EVs directly isolated from BALF of HV (N = 4), ARDS patients (N = 4), and serum-free medium (N = 5) used to incubate hMSCs during the BALF exposure experiment, and determined whether the hMSC EV-miRNA content reflected the carry-over of EVs in BALF that adhered to hMSCs without being washed away before collecting BALF-exposed hMSC-EVs. Isolation of EVs and miRNAs, and sequencing of miRNAs were performed as described for hMSC-EV studies. Differential expression analysis was performed in two separate ways. The first differential expression was determined using DESeq2, and the list of miRNAs differentially expressed in BALF was compared with the list of miRNAs differentially expressed in EVs. Separately, due to the small sample size, the Mann-Whitney test was used to compare the group medians.

[0092] Of the top 20 miRNAs of interest differentially expressed in EVs derived from hMSCs exposed to ARDS BALF (Figure 8A), 13 miRNAs were not differentially expressed in EVs directly obtained from ARDS BALF compared to HV BALF. Among the 7 miRNAs that were differentially expressed in BALF and differentially present in hMSC-derived EVs, 3 had opposite directions of change (Figures 8B - 8C), suggesting different biological effects of exposure to both HV and ARDS BALF rather than cross-contamination from EVs seen in either HV or ARDS BALF samples.

[0093] [Example 7] Signature of overlapping discriminative miRNAs results from supervised classification analysis Discriminant analysis was used to determine whether EVs exposed to BALF of HV or ARDS and control hMSC-derived EVs could be classified using a reduced miRNA signature. Samples were split into training and test settings (70% and 30% respectively), and Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) was used to develop a classification model with the training data. A two-component model that selected 15 and 5 miRNAs in the first two components respectively was found to have the lowest balanced discriminant error rate (0.0883). The 20-miRNA signature was found to identify samples into appropriate treatment groups (ARDS BALF exposure, HV BALF exposure, control), with the first component separating ARDS from other classes and the second component resolving HV and control (Figure 9A). The ability of the 20 miRNAs to accurately classify samples into treatment groups was verified using leave-one-out cross-validation (LOOCV) in the test cohort. The area under the receiver operating characteristic curve for ARDS vs HV+control was based on the predicted distance and averaged 0.9643 across all cross-validations (Figure 9B). The accuracy of the signature in predicting each classification (class) in the test setting was 0.92, 0.85, and 0.92 in the ARDS group, HV group, and control group respectively.

[0094] Of the 20 classifiers identified using sPLS-DA, a total of 14 were found to be differentially expressed among treatment groups. Functional analysis and visual exploration of miRNA-target interactions in miRNet revealed that four of the 14 differentially expressed classifiers (miRNA-760, miRNA-3175, miRNA-855-3p, and miRNA-766-3p) form a putative in-Silico regulatory network acting as putative "hub" regulators for 1259 putative targets, 75 of which are involved in cellular responses to stress and Wnt signaling (enrichment scores = 9.7E-9 and 3.2E-9, Figures 10B and 10C).

[0095] To understand how the pathways predicted to be regulated by gene targets might be related to acute lung injury, a more detailed analysis of these 14 miRNAs was performed (Table 2). miRNA-766-3p had a direct association with genes involved in the cellular stress response, while miRNA-885-3p, miRNA-3175, and miRNA-760 each contributed to the enrichment of genes involved in Wnt signaling (Figures 10A and 10B). miRNA-885-3p directly interacted with genes involved in pro-inflammatory cytokine regulation, apoptosis, chemotherapy resistance, proliferation, and metastasis. miRNA-766-3p regulated genes that inhibit inflammation by acting through the NF-kB signaling pathway. miRNA-664b-3p, miRNA-4644, miRNA-6803-5p, miRNA-6869-5p, miRNA-3940-5p, and miRNA-766-3p were involved in growth regulation. miRNA-3175 promoted epithelial-mesenchymal transition by targeting Smad 7. miRNA-760 was considered a potential tumor suppressor as it negatively regulated oncogenic proteins and reduced proliferation, cell cycle progression, migration, and differentiation. Table 2 shows an overview of the miRNA-level results and functional enrichment that are likely related to acute lung injury for the top 14 differentially expressed miRNA classifiers and overlapping miRNAs of interest.

[0096]

Table 2

[0097] [Example 8] Discussion Regarding the fate and action of exogenously administered hMSCs and their EV products in the inflammatory environment of clinical lung diseases, basic knowledge still remains insufficient. It has previously been shown that the ARDS inflammatory environment using clinical BALF as a substitute has a significant impact on hMSC gene and protein expression, recognition by the host immune system, and downstream effects on related immune effector cells. In some embodiments, it has been shown that the ARDS inflammatory environment also affects hMSC-EV tetraspanin expression and related miRNA content. A data-driven approach was used to identify the most abundant and consistently expressed miRNAs in hMSC-EVs. As a result of analyzing differences in expression patterns, EVs isolated from hMSCs exposed to ARDS 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 TGF-β, VEGF, and EGFR signaling. Finally, an EV miRNA signature was identified and used to classify samples into treatment groups. In the future, these signatures may provide information on the biological activity of hMSCs or serve as efficacy markers. Of the 14 miRNAs that passed both the differential analysis and discriminant analysis filters, 10 were mostly novel and there was little literature data to elucidate their potential functions. In contrast to empirical determination, computational prediction suggests important roles in the regulation of intercellular, cell matrix, oxidative stress, fatty acid metabolism, programmed cell death, and endothelial cell activation (Table 1). Based on what is known about the remaining four (miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p), these miRNAs were predicted to form interaction networks with known target genes and regulate important processes in the acute lung. For example, there are cellular responses to stress and Wnt signaling, strongly suggesting the biological role of miRNAs contained in hMSC-derived EVs.

[0098] EVs are increasingly recognized as mediating the anti-inflammatory and other effects of their parent hMSCs. This includes preclinical models of acute lung injury and other lung diseases, where EVs exert effects equivalent to, and sometimes greater than, those of the parent 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 mechanism by which hMSC-EVs affect the inflammatory lung environment remains unclear. So far, the initial focus has been on miRNAs associated with EVs. For example, miRNA-27 and its target gene VAV3 have been shown to be involved in cell infiltration and cell adhesion during acute lung injury.

[0099] In this study, substantial and significant differences were observed in the miRNA profiles associated with hMSC-EVs obtained under different BALF exposure conditions. Interestingly, in ARDS-exposed hMSC-EVs, the number of isolated miRNAs was reduced compared to HV-exposed hMSC-EVs. One possible explanation is that variability is lost due to the disease. Variability determines plasticity, i.e., the degree to which genes change their expression in response to environmental fluctuations. Under pathological stress, this plasticity can be lost. hMSCs exposed to BALF samples from both ARDS and HV generated EVs containing miRNAs with general anti-inflammatory activities, such as inhibition of the cellular response to stress, IFN, TGF-β, VEGF, and EGFR signaling. In contrast, EVs isolated from control hMSCs exposed to serum-free medium were predicted to be less affected by these pathways. Additionally, EVs isolated from hMSCs exposed to a healthy non-inflammatory environment contained miRNAs that led to proliferation, platelet activation, HIF signaling, and activation of the extracellular matrix. These are all pathways involved in wound healing and tissue remodeling.

[0100] As an attempt to identify the major miRNAs, 14 notable miRNAs were identified by two different bioinformatics strategies, DEA and sPLS-DA. This list was further narrowed down by using network analysis and visual exploration of miRNA-target interactions in miRNet to search for miRNAs that work together. Using this strategy, four "hub" regulatory factor miRNAs: miRNA-760, miRNA-3175, miRNA-885-3p, and miRNA-766-3p were identified, and these are known to be involved in the cellular response to stress and Wnt signaling. Interestingly, Wnt is an evolutionarily conserved pathway that regulates important aspects of cell fate determination, cell migration, cell polarity, neural patterning, and organogenesis during embryonic development. The Wnt / β-catenin pathway is involved in the induction, promotion, and abnormal repair of acute lung injury. One strength of these observations is that functional predictions are limited to miRNA-mRNA interactions that have been experimentally demonstrated to occur, based on large-scale experimental profiles of miRNA-mRNA interaction data.

[0101] Despite the fact that the hMSCs used in this study were carefully washed after BALF exposure, it was possible that some of the BALF-derived EVs were internalized or remained "stuck" to the cells. There is little literature on this point, and to address this, equivalent analyses were performed on EVs isolated from the same BALF samples and EVs isolated from the serum-free medium used to incubate control hMSCs. There were overlapping miRNAs in the raw BALF samples and control medium, but a significant number of miRNAs that were differentially expressed were identified. For example, miRNA-885-3p, miRNA3652, and miRNA-4763-3p were not found to be differentially expressed between the untreated BALF samples and the control medium, but significant differences were observed in the EVs isolated from the exposed hMSCs. These observations strongly suggest the biological effects of BALF exposure rather than cross-contamination from EVs found in the BALF samples.

[0102] The research described here is evidence of the plasticity of hMSCs and their EVs, and importantly, provides many mechanistic hypotheses to be evaluated regarding the differences in miRNAs associated with hMSC-EVs. From these observations, our understanding of the complex interplay of inflammatory and other pathways involved in the actions of hMSCs in the lung has increased, providing important information for the development of more effective hMSC-based cell therapies for ARDS and other lung diseases.

[0103] [Example 9] Materials and Methods Human BALF Samples Collection and processing of BALF samples from healthy volunteers (HV) and ARDS patients were performed as described previously. Briefly, HVs underwent standard fiberoptic bronchoscopy of the right middle lobe at the University of Dartmouth (exclusion criteria for HVs were a history of cardiorespiratory disease, smoking or regular tobacco use, use of immunomodulatory drugs). BALF samples from ARDS patients without sepsis were collected prospectively as part of an unrelated clinical study. For HV lavage, 20 cc of sterile saline was used, and the samples were centrifuged and the supernatants stored at -70°C. For lavage of ARDS patients, standard 40-ml mini-BALF with sterile saline was used in intubated ARDS patients, and the BALF samples were likewise centrifuged and stored.

[0104] Exposure of hMSCs to BALF in vitro hMSCs were 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 pre-characterized according to the criteria of the International Society for Cell and Gene Therapy. hMSCs were used at passage 3-5 and were the same as those used in recent studies of hMSC administration to non-COVID ARDS patients and previous studies of the effect of BALF on the actions of hMSCs.

[0105] miRNA and NTA Analysis To prepare EVs for miRNA and nanoparticle tracking analysis (NTA), hMSCs were seeded in 6-well plates (2 x 10 5 cells / well, 2 wells / BALF sample or control) under the cell culture conditions described above 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 20% (v / v) concentration of ARDS or HV BALF sample. Control hMSCs were exposed to serum-free medium only. After culturing 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 per well. After culturing for 48 hours (37°C), the conditioned medium was collected, passed through a 0.8 μm syringe filter, and processed for EV preparation, NTA, and miRNA evaluation as described below.

[0106] Expression of tetraspanin To evaluate the expression of characteristic cell surface tetraspanins (CD9, CD63, CD81) by imaging flow cytometry, 2x10 6 cells (each condition) were exposed to ARDS or HV BALF samples or serum-free medium (control) as described above. Since the BALF volume required for exposure was large (20% v / v) and the amount of available BALF was limited, pools rather than individual samples of HV or ARDS were used for these studies.

[0107] Preparation of extracellular vesicles (EVs) and isolation of miRNA Isolation of EVs from the conditioned medium of BALF-exposed hMSCs or direct isolation of EVs from the BALF samples themselves was prepared according to the recent recommendations of the International Society for Extracellular Vesicles. EVs and EV-derived miRNAs were isolated from the conditioned medium 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, EVs were isolated using ExoQuick-TC® (catalog number EXOTC50A; System Biosciences, Palo Alto, CA) according to the manufacturer's protocol to avoid interference with the elution column in the NTA protocol. This was because background contamination from the kit elution column was observed when performing preliminary NTA on samples separated with the exoRNeasy kit. This contamination hindered the accurate assessment of the particle number and particle size distribution using NTA. Subsequently, a precipitation-based separation method (ExoQuick-TC®) that does not rely on an elution column was utilized to prepare EVs for characterization by NTA and imaging flow cytometry. This approach resulted in contamination-free and highly reproducible results.

[0108] Nanoparticle tracking analysis (NTA) The particle size and concentration of EVs were measured using NTA (ZetaView (registered trademark), Particle Metrix Inc, Germany, 488 nm laser). All samples were analyzed at 25°C after daily calibration of the instrument according to the manufacturer's recommendations. Samples were diluted to an appropriate concentration with ultrapure water before analysis. Video recording was performed with fixed settings for all samples (scattering mode: sensitivity 85, shutter 75, fluorescence mode: sensitivity 95, shutter 32, both: minimum brightness 20, minimum size 5, maximum size 200). Videos of all 11 positions for each sample were recorded for 5 cycles (1 cycle corresponds to 1 second) at each position and analyzed with ZetaView (registered trademark) analysis software (version 8.03.08.02).

[0109] Imaging of flow cytometry Imaging flow cytometry was performed using an AMNIS ImageStreamX (registered trademark) Mark II flow cytometer (AMNIS / LumiNex, Seattle, WA, USA). Briefly, antibodies were added to the samples and incubated at room temperature for 1 hour. Analyses were performed using unstained EV samples (uEV), NaCl-HEPES buffer with antibodies added but no EV samples, and stained samples with 1% NP40 (Calbiochem, San Diego, CA, USA) added as controls according to the recommended MIFlowCyt-EV guidelines. After staining without washing, the samples were diluted with PBS and analyzed using the built-in autosampler for 96-well round-bottom plates. The acquisition time was set at 5 minutes per well. Data were acquired at a magnification of 60×, low flow rate, and with the bead removal option disabled. Data analysis was performed as described above using IDEAS software (version 6.2). Fluorescent events were plotted against side scatter (SSC). To improve the detection of fluorescent images, the composite mask function (MC and NMC) was used. Images were analyzed by the spot counting function to determine whether they matched (cluster detection). All data points containing multiple objects were excluded from the analysis. Events with low side scatter values (less than 500) and high fluorescence intensity (higher than 300) were considered uEV. The average concentration was calculated according to the acquisition volume and time.

[0110] miRNA Sequencing and Differential Expression Analysis EVs were directly isolated as described above from hMSCs (control, N = 16) exposed to serum-free medium, or individual BALF samples obtained from ARDS patients (N = 16), HVs (N = 16), or pure BALF samples obtained from HVs (N = 4) and ARDS patients (N = 4). RNA was isolated from each EV preparation as described above, and only samples with an A260 / A280 of 1.80 or higher that passed quality control were used for RNA sequencing (control = 16; ARDS = 12; HV = 14). A total of 35 ng of eRNA (EV-derived RNA) preparation was used for miRNA sequencing performed using the HTG EdgeSeq miRNA Whole Transcriptome Assay (miRNA WTA, according to the manufacturer's instructions) to measure the expression of 2,083 human miRNA transcripts using a next-generation sequencer (NGS). The raw read counts of each of the 42 samples were input into DESeq2 for differential expression analysis. Analysis data were created using variance-stabilizing transformation. Significance analysis of microarrays (SAM) of the normalized read counts was performed using a one-class analysis approach to identify miRNAs overexpressed in EVs derived from control hMSCs. The δ value was set to 7 (the best delta with the lowest false discovery rate [FDR] selected by the software) using 1,000 permutations, and the FDR cutoff was 0 (%). Differential expression analysis (DEA) was performed using DESeq2 in R (R package "DESeq2"). Four comparisons were made: (1) control vs. HV, (2) ARDS vs. control, (3) ARDS vs. HV, (4) ARDS vs. combined control + HV. Strict selection criteria were used to reduce the FDR rate. miRNAs considered to be differentially expressed (DE) were selected based on a change in expression of 2-fold or more (average expression across samples with 50 read counts or more) and an adjusted P value (or FDR) < 0.05 after multiple comparison correction. miRNAs that were consistently overexpressed were identified by the sum of pairwise comparisons and visualized using a Venn diagram.

[0111] Target prediction and functional analysis were performed using miRNet (mirnet.ca). Enriched pathways were selected based on a hypergeometric test of miRNAs with FDR > 0.05 from Reactome. Putative relationships were obtained using DIANA-TarBase v8 (a collection of experimentally supported miRNA-gene interactions).

[0112] ARDS, HV, supervised classification analysis of control hMSC EV miRNAs Sparse partial least squares (PLS) discriminant analysis (sPLS-DA) (R package "mixOmics") was performed to determine the most discriminative features from -2000 miRNAs using the normalized expression profiles from ARDS or HV BALF exposure or control hMSC-derived EVs to classify samples into these categories. Briefly, samples were split into a training set and a test set at a 0.7 / 0.3 split. Leave-one-out cross-validation (LOOCV) was used to select the optimal parameters of the model. The number of components required to discriminate the classes (usually the number of classes (K-l) works best, so up to 4 components were considered) and the number of features (discriminable miRNAs) per component (5 - 20 features per component were tested). The combination of components and features with the lowest balanced error rate (BER) was selected for the final model (2 components with 15 and 5 features respectively). The prediction distance used to measure the classification error was calculated using the maximum distance. The performance of the model for the training set and the prediction of the test set results were evaluated using the accuracy, sensitivity, and specificity of each class assignment. The area under the receiver operating characteristic curve (AUROC) of the all-pairwise comparisons of PLS-DA was based on the predicted maximum distance averaged over all cross-validations and was intended to complement the analysis rather than evaluate the performance of the model.

[0113] Statistical analysis Data from NTA and image flow analysis The particle size and count obtained with ZetaView (registered trademark) were plotted on the X-axis from 2.5 to 903 μm in 100-μm bins, and the area under the curve (AUC) of each sample was calculated. The data were analyzed using one-way analysis of variance (one-way ANOVA) with group (control, HV, ARDS) as a factor, and a Bonferroni post hoc test was performed to identify differences between group means. The number of positively stained particles and the number of double-positive particles (CD63 / CD81) for each tetraspanin antibody (CD9, CD63, CD81) were also analyzed within each tetraspanin type. All analyses and graphs were performed using Prism (version 9.3, GraphPad Software).

[0114] miRNA data To determine the differential expression of miRNAs found in EVs derived from BALF and EVs derived from hMSCs, for those that passed the normality test (Kolmogorov-Smirnov), an unpaired T-test with Welch's correction (not assuming equal standard deviations) was performed. For those that were not normally distributed, a two-sided Mann-Whitney test was performed for each miRNA (p = 0.05). Statistical analysis was performed using GraphPad Prism software. The Mann-Whitney test was used to evaluate the difference between two groups. A P-value less than 0.05 was considered significant, but for RNA sequencing data analyzed by DESeq, a multiple hypothesis correction FDR less than 0.05 was considered significant. Spearman correlation was calculated in base R using the t-distribution to calculate the P-value when there were ties in the ranks.

[0115]

Table 3

[0116] References: TIFF2025519331000009.tif229156TIFF2025519331000010.tif229156TIFF2025519331000011.tif243155TIFF2025519331000012.tif235158TIFF2025519331000013.tif177156

[0117] Other embodiments In the claims, articles such as "a", "an", and "the" may mean one or more unless the contrary is indicated or is not apparent from the context. A claim or specification that includes "or" between one or more members of a group is considered satisfied if, unless the contrary is indicated or is otherwise apparent from the context, one, two or more, or all of the members of the group are present in, employed in, or otherwise related to a given product or process. The present invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise related to a given product or process. The present invention includes embodiments in which two or more, or all, of the members of the group are present in, employed in, or otherwise related to a given product or process.

[0118] Furthermore, the present invention encompasses all modifications, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same base claim. When elements are presented as a list, for example in Markush group format, each sub-group of the elements is also disclosed and any element can be removed from the group. Generally, when the present invention or an aspect of the present invention is referred to as including a particular element and / or feature, it is to be understood that a particular embodiment of the present invention or an aspect thereof consists of or consists essentially of such element and / or feature. For the sake of brevity, those embodiments are not specifically described herein in such words. Also, note that the terms "comprising" and "containing" are intended to be open-ended and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Further, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges can be assumed to be within the described range to one-tenth of the unit of the lower limit of the range, for any particular value or sub-range within the described range, in different embodiments of the present invention, unless the context clearly dictates otherwise.

[0119] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are hereby incorporated by reference into this specification. In case of any conflict between the incorporated references and this specification, this specification shall prevail. Further, any particular embodiment of the present invention that pertains to the prior art can be explicitly excluded from any one or more of the claims. Such embodiments are considered to be known to one of ordinary skill in the art and thus can be excluded even if no explicit exclusion is described herein. Any particular embodiment of the present invention can be excluded from any claim for any reason, regardless of whether it is related to the existence of the prior art.

[0120] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. One of ordinary skill in the art will understand that various changes and modifications may be made to this specification without departing from the spirit or scope of the invention as defined in the following claims.

Claims

**Claim 1** A method for generating an EV - miRNA profile, comprising: adding an activating factor to a culture of mesenchymal stromal cells (MSCs); using a miRNA sequencing platform to determine whether miRNA associated with at least one MSC extracellular vesicle (MSC - EV - miRNA) is present in the culture of MSCs; and creating an EV - miRNA profile of MSCs exposed to the activating factor based on the presence or absence of each miRNA, compared to a control. **Claim 2** The method of claim 1, further comprising identifying a subject receiving MSCs for treatment of a disease using the MSC - EV - miRNA profile. **Claim 3** The method according to any one of claims 1 or 2, wherein the miRNA comprises hsa - miR - 7107 - 5p. **Claim 4** The method according to any one of claims 1 to 3, wherein the miRNA comprises hsa - miR - 6803 - 5p. **Claim 5** The method according to any one of claims 1 to 4, wherein the miRNA comprises hsa - miR - 6798 - 5p. **Claim 6** The method according to any one of claims 1 to 5, wherein the miRNA comprises hsa - miR - 760. **Claim 7** The method according to any one of claims 1 to 6, wherein the miRNA comprises hsa - miR - 6727 - 5p. **Claim 8** The method according to any one of claims 1 to 7, wherein the miRNA comprises hsa - miR - 4763 - 3p. **Claim 9** The method according to any one of claims 1 to 8, wherein the miRNA comprises hsa - miR - 3652. **Claim 10** The method according to any one of claims 1 to 9, wherein the miRNA comprises hsa - miR - 885 - 3p. **Claim 11** The method according to any one of claims 1 to 10, wherein the miRNA comprises hsa - miR - 766 - 3p. **Claim 12** The method according to any one of claims 1 to 11, wherein the miRNA comprises hsa - miR - 3175. **Claim 13** The method according to any one of claims 1 to 12, wherein the miRNA comprises hsa - miR - 6893 - 5p. **Claim 14** The method according to any one of claims 1 to 13, wherein the miRNA comprises hsa - miR - 6875 - 5. **Claim 15** The method according to any one of claims 1 to 14, wherein the miRNA comprises hsa - miR - 6799 - 5p. **Claim 16** The method according to any one of claims 1 to 15, wherein the miRNA comprises an 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. 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 culture of MSCs and the second biological sample in a second culture of MSCs, Detecting, using a next-generation sequencing platform, whether at least one MSC-EV-miRNA is present in the first and / or second culture of MSCs, Creating a differential EV-miRNA profile using a next-generation sequencing platform, and Determining, based on the differential EV-miRNA profile, whether a subject suspected of having a disease is a candidate for MSC-based therapy A method comprising.

18. A pharmaceutical composition comprising at least one microRNA associated with extracellular vesicles derived from mesenchymal stromal cells (MSC-EV-miRNA), and A pharmaceutically acceptable excipient.

19. The pharmaceutical composition according to claim 18, further comprising lipid nanoparticles (LNP) encapsulating at least one MSC-EV-miRNA.

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

21. The pharmaceutical composition according to any one of claims 19 or 20, wherein the LNP encapsulates at least 14 MSC-EV-miRNAs.

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein the LNP comprises a targeting moiety.

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

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

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

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

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

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

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

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

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

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

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

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

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

36. The pharmaceutical composition according to any one of claims 18 to 35, wherein the miRNA comprises 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 comprising a culture of engineered mesenchymal stromal cells configured to release a plurality of mesenchymal stromal cell extracellular vesicles (MSC-EV), wherein the plurality of MSC-EV comprise 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, 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 therapy according to claim 37, wherein the MSC-EV comprise hsa-miR-760, hsa-miR-3175, hsa-miR-885-3p, and hsa-miR-766-3p.

39. A method of generating therapeutic mesenchymal stromal cells (MSC), 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 culture of MSC and the second biological sample in a second culture of MSC; isolating human MSC-related extracellular vesicles (hMSC-EV) from the cell cultures; determining, using a next-generation sequencing platform, whether miRNA associated with at least one EV (EV-miRNA) is present in the hMSC-EV; creating a differential EV-miRNA profile using a next-generation sequencing platform; and engineering the therapeutic MSC to overexpress one or more miRNAs identified by the EV-miRNA profile. A method comprising the steps above.

40. The method according to claim 39, further comprising delivering the therapeutic MSC to a subject.

41. The method according to claim 39 or 40, wherein the therapeutic MSC overexpresses hsa-miR-760, hsa-miR-3175, hsa-miR-885-3p, and hsa-miR-766-3p.

42. An oligonucleotide primer mix comprising one or more oligonucleotides, each oligonucleotide being configured to hybridize to a single miRNA of interest, the miRNA of interest being 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, 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 miRNA associated with extracellular vesicles derived from at least one mesenchymal stromal cell (MSC-EV-miRNA) in a biological sample, A primer mix comprising one or more oligonucleotides, each oligonucleotide being 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, 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, primer mix, Reagents for performing a nucleic acid assay for detecting miRNAs associated with at least one MSC-EV using nucleic acid pairs, and A kit comprising instructions for performing an assay for detecting miRNAs associated with at least one MSC-EV.

44. The kit according to claim 43, comprising at least four oligonucleotides for detecting at least four of the miRNAs associated with MSC-EV.

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

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