MicroRNA-based particles for the treatment of immune response dysregulation

Abstract

Disclosed herein are therapeutic particles based on miRNA mimetics. The particles include synthetic miRNA or miR-187-3p mimetics encapsulated in lipid nanoparticle (LNP) carriers, or synthetic miR-193b-5p inhibitors encapsulated in lipid carriers, or a combination thereof. The lipid nanoparticle carriers are composed of at least four types of lipids, including: a) ionizable cationic lipids selected to bind and protect negatively charged miRNA, facilitate endosomal escape, and be positively charged in the formulation buffer (pH 4), which are neutral in the storage buffer; b) sterols in the structure of the lipid nanoparticles (LNPs); c) structural helper lipids selected to contribute to lipid nanoparticle stability and / or enhance endosomal release; and d) PEGylated lipids selected to stabilize the therapeutic particles and protect them from opsonization.

Description

[Technical Field]

[0001] The present disclosure relates to the delivery of microRNA inhibitors and microRNA mimetics for the treatment of dysregulated immune responses, including myocardial dysfunction in acute respiratory distress syndrome and sepsis. [Background technology]

[0002] Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI, preclinical inference) are disorders of acute inflammation that disrupt the pulmonary endothelial and epithelial barriers. 1 Triggering events, such as microbial or viral infection or other injurious stimuli, can result in cellular damage to either side of the alveolar-capillary membrane (epithelial and endothelial), loss of membrane permeability with influx of edema fluid, and dysregulated activation of the innate immune response, resulting in gap formation. Multiple therapeutic agents are important to consider in a holistic strategy to protect against lung injury: modulation of the cytokine storm, reduction of edema, and repair of tight junction damage. A major barrier to successful treatment may be the induction of an unintended immune response.

[0003] A recent approach to treating lung injury is mesenchymal stromal cell therapy, which has unfortunate limitations due to treatment delays. 2 This drawback of MSC therapy highlights the need for a pre-fabricated acellular product that can be deployed quickly and reliably at the point of need, which represents a benefit derived from the use of synthetic carriers.

[0004] Recently, dos Santos et al. demonstrated that microRNAs (miRNAs) are involved in regulating the host response to injury, inflammation, and infection. 1~6miRNAs are endogenous, non-coding, single-stranded RNAs that are 18–24 nucleotides long. Functionally, miRNAs interfere with protein synthesis by directly degrading or inhibiting the translation of target genes at the post-transcriptional level. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded ribonucleic acids (RNAs) that are 18–24 nucleotides long. They function by base pairing with complementary sequences within messenger RNA (mRNA) molecules. miRNAs inhibit protein synthesis by directly degrading or inhibiting the translation of target genes. miRNAs typically affect the expression of more than one protein and affect multiple pathways. They target specific proteins, unlike, for example, longer small interfering RNAs (siRNAs). MicroRNAs are several thousand nucleotides long and also differ from messenger RNAs (mRNAs), which provide templates for protein synthesis (protein translation). The inhibitory function of microRNAs occurs inside the cytoplasm but outside the nucleus.

[0005] Nucleic acids are susceptible to degradation by endo- and exonucleases. Therefore, delivery of nucleic acids in therapeutic agents has been achieved through carriers such as viral shells, polymers, and lipid nanoparticles. For example, lipid nanoparticles (LNPs) have been demonstrated in the first LNP gene therapy for liver amyloidosis (Onpattro™) and COVID vaccines from Moderna and Pfizer. LNPs contain several lipid types. Cationic ionizable lipids are used to condense anionic nucleic acid cargo. Additionally, structural lipids, by way of non-limiting example, can create the outer shell of the carrier, increasing its circulation time, rigidity, and aiding in cargo release, if desired. Specific targeting moieties can also be included on the LNP surface or conjugated to the nucleic acid.

[0006] Acute lung injury resulting in acute respiratory distress syndrome can be caused by a variety of factors, including infectious and noninfectious triggers. The most common cause is a dysregulated response to infection (sepsis). Noninfectious causes, such as pancreatitis, aspiration of gastric contents, smoke inhalation, and inhalation of toxic gases, severe trauma associated with shock, and transfusion of blood products (i.e., transfusion-associated acute lung injury), can also result in ARDS. More recently, new causes of ARDS have emerged, including vapor inhalation-associated lung injury and drug-induced ARDS caused by various agents, such as immunotherapies including checkpoint inhibitors. Of relevance is the increasing recognition of the role of viral infections as important causes of ARDS: SARS-CoV (2003), H1N1 influenza (2009), MERS-CoV (2012), and SARS-CoV-2 (2019), which led to the COVID-19 pandemic. Furthermore, exposure to an injurious mechanical ventilator can further exacerbate acute lung injury. Repetitive cyclic stretch, hyperextension, alveolar collapse and deformation injury contribute significantly to increased lung injury and the resulting morbidity and mortality associated with ARDS.

[0007] The pathophysiology of ARDS is not fully understood. Diffuse alveolar damage is a characteristic histological finding associated with ARDS. It is characterized by neutrophilic alveolitis and hyaline membrane deposition. Alveolar-capillary membrane damage is associated with alveolar-capillary membrane desquamation (cell detachment from the membrane due to injury or death), which is associated with loss of membrane permeability, diffuse alveolar hemorrhage, proteinaceous exudative edema in the alveoli, and the formation of fibrin-rich hyaline membrane deposits along the exposed alveolar basement membrane in areas of parenchymal epithelial and endothelial lung injury.

[0008] Damage to the robust pulmonary epithelial barrier impairs fluid transport by the alveolar epithelium, a normal mechanism for facilitating alveolar flooding and maintaining dry airspaces. Injury to type II cells can impair surfactant production. Surfactant can also be inactivated by alveolar flooding. Concomitant damage and shedding of the pulmonary epithelial glycocalyx, a layer of glycosaminoglycans and proteoglycans that covers the alveolar surface, is also proinflammatory. Activation and injury of the alveolar epithelium also leads to the shedding of anticoagulant molecules and the release of tissue factor from the pulmonary epithelium into the alveolar space. These changes favor intraalveolar fibrin formation, which drives hyaline membrane formation. The alveolar epithelium is an important barrier against pathogens and can secrete antibacterial proteins, such as surfactant proteins A and D; therefore, epithelial injury can also increase susceptibility to secondary infections. The capillary endothelium forms a barrier between circulating blood cells and plasma and the pulmonary interstitium and airspaces. Injury to the pulmonary endothelium is a key feature of ARDS and is characterized by the formation of gaps between endothelial cells and the upregulation of adhesion molecules such as P-selectin and E-selectin and endothelial injury mediators such as angiopoietin-2. Various stimuli, including circulating pathogens or their products, endogenous disease-associated molecular patterns, inflammatory cytokines, and cell-free hemoglobin, can induce endothelial injury. Severe injury to the pulmonary epithelium can also induce pulmonary endothelial injury. Although the mechanism is not well understood, direct intercellular communication between pulmonary epithelial cells and endothelial cells and the transfer of reactive oxygen species contribute to the injury. Like the pulmonary epithelium, the endothelium is covered with a glycocalyx that is easily damaged and sloughed off, exposing adhesion molecules and favoring edema formation. Endothelial injury leads to the shedding of anticoagulant molecules on the endothelial surface, such as thrombomodulin and endothelial protein C receptor, and the upregulation of procoagulant molecules that favor microvascular thrombus formation.

[0009] Damage to the alveolar-capillary membrane contributes to the clinical features associated with ARDS: exudative pulmonary edema (alveolar flooding—radiographically associated with bilateral diffuse airspace disease), impaired gas exchange (manifesting as severe hypoxia), and increased work of breathing (manifesting as respiratory distress). Alveolar flooding is further exacerbated by disruption of the lung's normal fluid transport mechanism, which normally pumps alveolar edema into the interstitium for clearance by lymphatic fluid. In addition to contributing to ventilation-perfusion mismatch, edema fluid contributes to surfactant inactivation, resulting in miliary atelectasis and end-expiratory alveolar collapse, as well as reduced lung compliance and increased work of breathing, requiring higher inspiratory pressures. Activation of procoagulant pathways on the pulmonary endothelium can lead to pulmonary microvascular thrombosis, which increases dead space (when ventilation occurs in areas of the alveoli / lung that are not perfused); increased dead space ventilation contributes to severe gas exchange impairment and is associated with higher mortality in ARDS. Unlike blood vessels in the systemic circulation, the response of the pulmonary circulation to hypoxia is to cause vasoconstriction, leading to further hypoxia and increased pulmonary vascular resistance. Microvascular thrombosis and severe damage to the microvascular bed can further contribute to pulmonary arterial hypertension and acute right ventricular dysfunction, both of which contribute to poor clinical outcomes.

[0010] Local and systemic acute inflammation is a hallmark of ARDS and contributes to pulmonary epithelial and endothelial injury. Early in the course of ARDS, cellular activation by pattern and damage-recognition receptors leads to increased expression of adhesion molecules on epithelial and endothelial surfaces and secretion of cytokines and chemokines. Neutrophils are not normally found in the alveolar space. Increased secretion of neutrophil chemoattractants leads to the infiltration of activated neutrophils, which, in their activated state, can release a variety of damaging mediators, including reactive oxygen species, proteases, and inflammatory lipid-derived mediators such as prostaglandins and leukotrienes. Neutrophil extracellular traps, composed of DNA, histones, and proteases, are also released into the airspace during these pathophysiological processes and can increase inflammation by activating the NRLP3 inflammasome, which initiates the local release of interleukin-1-β and interleukin-18. Neutrophil recruitment is primarily driven by tissue-resident and recruited macrophages (circulating monocytes that enter the lung and become activated macrophages), but interstitial fibroblasts may also contribute. Macrophage pattern recognition receptors bind disease- or pathogen-associated molecular patterns, activating macrophages to a pro-inflammatory phenotype and resulting in the release of inflammatory cytokines and neutrophil chemoattractants such as interleukin-8. Pulmonary epithelial and endothelial cells can also release neutrophil chemoattractants.

[0011] Exposure to repetitive cyclic stretch in this pro-inflammatory environment, where cells are more susceptible to mechanical injury, is associated with alterations in mechanosensation, mechanotransduction, and lung compartmentalization (ultrastructural damage to the alveolar-capillary membrane), leading to leakage of the "inflammatory soup" into the circulation and consequent distal organ damage—multiple organ dysfunction syndromes, including acute kidney injury, acute encephalopathy, cardiomyopathy, hemodynamic instability, liver dysfunction, intestinal dysfunction, and possibly worsening muscle dysfunction, contributing to increased morbidity, mortality, and post-ARDS and post-ICU syndrome.

[0012] Resolution of inflammation is a coordinated process requiring the downregulation of pro-inflammatory pathways and the upregulation of anti-inflammatory pathways. Regulatory T cells play a crucial role in orchestrating this process. Neutrophils are cleared from airspaces by apoptosis and phagocytic clearance by alveolar macrophages. Pro-resolution mediators, including lipoxins and resolvins, are a family of bioactive lipid mediators that also play a role in the resolution of lung injury and inflammation. Restoration of alveolar epithelial fluid transport requires regeneration of the alveolar epithelium, which may be necrotic in ARDS. Several cells can serve as progenitors for regenerating the epithelium, and their relative roles may depend on the severity of epithelial injury. Once a robust epithelial barrier is restored, various endogenous factors, including catecholamines and corticosteroids, can upregulate alveolar fluid clearance. The role of interstitial cells, such as fibroblasts, in the acute and resolving phases of ARDS is poorly understood. Pro-fibrotic pathways can be triggered as early as the first day of ARDS, leading to pulmonary fibrosis. Fibrosis may impede weaning from mechanical ventilation and, together with physiological restrictions and reduced diffusing capacity, may cause long-term deterioration of lung function.

[0013] Although no formal definition of septic cardiomyopathy exists, accepted diagnostic criteria include acute global decreased biventricular contractility, left ventricular dilation, decreased response to fluids and catecholamines, and the absence of coronary syndrome. 8、7 The presence of myocardial dysfunction is associated with a mortality rate of 70–90%, in contrast to a mortality rate of 20% in patients without myocardial involvement. 10、9、10 A reduced left ventricular ejection fraction (LVEF <45%) occurs in 60% of patients with septic shock during the first 3 days. 7 In patients who die, myocarditis is found in 27% of autopsy specimens, as well as degenerative changes. 10、11 Its pathogenesis is complex and incompletely understood, involving a combination of immune dysregulation, oxidative stress, calcium dysregulation, autonomic nervous system dysregulation, endothelial dysfunction, and catastrophic mitochondrial injury. 11、12、13Myocardial dysfunction in sepsis is a treatable hallmark, and miRNAs may contribute to the characteristics of sepsis-induced cardiomyopathy. 14、15、16 . Summary of the Invention

[0014] The present disclosure provides: A synthetic microRNA or mimetic thereof encapsulated in a lipid nanoparticle (LNP) carrier; and Lipid nanoparticle carriers containing at least four types of lipids that are independent of each other Including, a) an ionizable cationic lipid selected to be positively charged in the formulation buffer; b) sterols, c) structural helper lipids, and d) PEGylated lipids The present invention provides a microRNA-based particle comprising:

[0015] The synthetic microRNA may be any one or combination of miR-187-3p or miR-193b-5p inhibitors or mimetics thereof.

[0016] The synthetic microRNA may be any one of or a combination of miR-193b-5pinh, miR-187-5p, hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p and hsa-miR-6787-5p.

[0017] The ionizable cationic lipid may be positively charged in a formulation buffer having a pH ranging from about pH 3 to about pH 5.5.

[0018] The ionizable cationic lipid is neutral in a storage buffer having a pH ranging from about pH 7 to about pH 8. The storage buffer may be phosphate buffered saline (PBS) having a pH of about 7.4.

[0019] The ionizable cationic lipids may be any one or combination of saturated lipids, unsaturated lipids, single-tail lipids, multi-tail lipids, polymeric lipids, biodegradable lipids, or branched-tail lipids.

[0020] The ionizable cationic lipids may include neutral or true fats, waxes, cutin, suberin, phospholipids, sphingolipids, lipoproteins, terpenes, prostaglandins, or sterols.

[0021] The sterol is selected to complex with apolipoprotein E, thereby promoting endocytosis through the low density lipid receptor.

[0022] Sterols can be selected based on improved intracellular delivery.

[0023] Structural helper lipids can be selected to contribute to the stability of the lipid nanoparticles and / or to enhance endosomal release.

[0024] The structural helper lipid may be a cylindrical lipid such as phosphatidylcholine.

[0025] The structural helper lipid may be cone-shaped to favor the formation of a hexagonal II phase and facilitate endosomal release of the oligonucleotide.

[0026] The structural helper lipid can be a sterol, which can be cholesterol. The PEGylated lipid can be selected so that it stabilizes the particle and protects it from opsonization before it reaches its intended target.

[0027] The PEGylated lipid may be a polyethylene glycol (PEG) derivative attached to a lipid moiety.

[0028] The PEGylated lipids may be DMG-PEG2000 or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 ALC-0159 or (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, DSPE-PEG, DPPE-PEG, DOPE-PEG, DMPE-PEG with PEG lengths varying from 0.2 to 5 kDa.

[0029] The ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid may be in the mol % ratio range of about 40-70: 30-45: 3-16: 0.5-1.5.

[0030] The ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid may be in the mol % ratio range of about 45-55: 37-40: 8-12: 1-1.7.

[0031] The ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid may be in the mol % ratio range of about 50:38.5:10:1.5.

[0032] The lipid carrier may further comprise any one or combination of prodrug lipids or lipid carriers non-covalently functionalized with peptides, proteins, glycoproteins, polysaccharides, or combinations thereof to aid in tissue-specific targeting. The prodrug lipids may include, by way of non-limiting example, any one or combination of anti-inflammatory drugs, inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs), chemokine receptor antagonists, immune response modifiers, immunomodulatory drugs, mast cell stabilizers, T cell activation inhibitors, and TNF-binding proteins.

[0033] The at least four component lipid carrier composition may include the prodrug lipid in a molar fraction ranging from about 1 to about 15%, offset by the removal of sterols and structural helper lipids to offset the addition of the prodrug lipid, wherein the fraction of the prodrug lipid ranges from about 5 to about 10%.

[0034] The present disclosure provides a composition comprising a microbubble and a plurality of the above-described microRNA-based particles.

[0035] Multiple miRNA-based particles can be used to form a coating on the outer or inner surface of a microbubble.

[0036] The composition may further comprise one or more prodrugs.

[0037] The present disclosure provides a medicament for the treatment of immune response dysregulation, comprising a miRNA-based particle or composition disclosed above.

[0038] The dysregulated immune response may be acute respiratory distress syndrome.

[0039] Dysregulation of the immune response may be the myocardial dysfunction in sepsis.

[0040] Dysregulation of the immune response may result in any one or a combination of acute kidney injury, acute encephalopathy, hemodynamic instability, liver dysfunction, intestinal dysfunction, and muscle dysfunction, all of which may contribute to increased morbidity, mortality, and post-ARDS, post-cardiomyopathy, and post-ICU syndrome.

[0041] The present disclosure provides an RNA collector consisting of a synthetic or naturally occurring RNA molecule, which has multiple binding sites for target miRNAs and captures the target miRNAs. [Brief explanation of the drawings]

[0042] Embodiments will now be described, by way of example only, with reference to the drawings. [Figure 1] FIG. 1 is a schematic diagram illustrating a lipid nanoparticle formulation disclosed herein. [Figure 2-1] Figure 2A shows the RNA encapsulation efficiency determined by the RiboGreen assay for exemplary LNPs described herein. Figure 2B shows the hydrodynamic size (bars) and polydispersity index (dots) of exemplary LNPs described herein. Figure 2C shows the zeta potential of exemplary LNPs described herein. Figures 2A, 2B, and 2C therefore demonstrate that standardized characterization methods confirm the quality of LNPs. There are no significant differences between LNPs based on size, polydispersity, or zeta potential. [Figure 2-2] Figure 2A shows the RNA encapsulation efficiency determined by the RiboGreen assay for exemplary LNPs described herein. Figure 2B shows the hydrodynamic size (bars) and polydispersity index (dots) of exemplary LNPs described herein. Figure 2C shows the zeta potential of exemplary LNPs described herein. Figures 2A, 2B, and 2C therefore demonstrate that standardized characterization methods confirm the quality of LNPs. There are no significant differences between LNPs based on size, polydispersity, or zeta potential. [Figure 3] Figure 3 shows the LNP composition derived from mass spectrometry analysis of the LNPs after preparation compared to the lipid film. In the absence of RNA, cholesterol increases upon loading and decreases upon formation. The ionizable lipid MC3 shows the opposite trend. [Figure 4A] In two different LNP formulations (designated KC2 and MC3 based on their ionizable lipids), the addition of 2 mM CaCl2 to the formulation buffer increased GFP mRNA translation to a significant extent at different levels of treatment. [Figure 4B] In two different LNP formulations (designated KC2 and MC3 based on their ionizable lipids), the addition of 2 mM CaCl2 to the formulation buffer increased GFP mRNA translation to a significant extent at different levels of treatment. [Figure 5-1]Figures 5A, 5B, 5C, and 5D show the effect of LNP miR-193b-5p INH on viral replication in vitro. Figure 5A shows staining of occludin and E-cadherin along the periphery of the cells and an overlay of tight junctions and adherens junction proteins. Figure 5B shows 10 mM FAM-tagged siRNA delivered in BEAS2b cells for 24 hours, demonstrating 100% transfection efficiency. Figure 5C shows the miR-193b-p5 copies / µL contained in digital droplets compared to uninfected controls. Figure 5D shows that PR8 viral hemagglutinin (HA), RNA polymerase subunit 1 (PB1), and neuraminidase (NA) expression was significantly reduced 24 hours after infection in cells treated with LNP miR-193b-5p INH compared to the LNP with negative control (NC). [Figure 5-2] Figures 5A, 5B, 5C, and 5D show the effect of LNP miR-193b-5p INH on viral replication in vitro. Figure 5A shows staining of occludin and E-cadherin along the periphery of the cells and an overlay of tight junctions and adherens junction proteins. Figure 5B shows 10 mM FAM-tagged siRNA delivered in BEAS2b cells for 24 hours, demonstrating 100% transfection efficiency. Figure 5C shows the miR-193b-p5 copies / µL contained in digital droplets compared to uninfected controls. Figure 5D shows that PR8 viral hemagglutinin (HA), RNA polymerase subunit 1 (PB1), and neuraminidase (NA) expression was significantly reduced 24 hours after infection in cells treated with LNP miR-193b-5p INH compared to the LNP with negative control (NC). [Figure 6-1]Figures 6A, 6B, and 6C show a non-limiting example of the biodistribution of LNP miR-193b-5p INH in PR8-infected mice. Figure 6A shows a schematic diagram of the experimental design. Mice were infected at time 0. Therapeutic agents or control empty particles were delivered on day 4 post-infection. Mice were humanely sacrificed and then echocardiograms were performed on day 6. Figure 6B shows fluorescence images of organs collected on day 6. Images were taken at 6, 12, 24, 48, 72, 96, and 120 hours after intravenous injection. Ex vivo fluorescence images and corresponding optical densities of major organs dissected 24 hours post-injection are shown. Figure 6C shows digital quantification of fluorescence in mock mice. Figure 6D shows digital quantification of fluorescence in PR8 mice, demonstrating significant and pronounced recruitment of LNP to the spleen, liver, and lungs (N=3). [Figure 6-2] Figures 6A, 6B, and 6C show a non-limiting example of the biodistribution of LNP miR-193b-5p INH in PR8-infected mice. Figure 6A shows a schematic diagram of the experimental design. Mice were infected at time 0. Therapeutic agents or control empty particles were delivered on day 4 post-infection. Mice were humanely sacrificed and then echocardiograms were performed on day 6. Figure 6B shows fluorescence images of organs collected on day 6. Images were taken at 6, 12, 24, 48, 72, 96, and 120 hours after intravenous injection. Ex vivo fluorescence images and corresponding optical densities of major organs dissected 24 hours post-injection are shown. Figure 6C shows digital quantification of fluorescence in mock mice. Figure 6D shows digital quantification of fluorescence in PR8 mice, demonstrating significant and pronounced recruitment of LNP to the spleen, liver, and lungs (N=3). [Figure 6-3]Figures 6A, 6B, and 6C show a non-limiting example of the biodistribution of LNP miR-193b-5p INH in PR8-infected mice. Figure 6A shows a schematic diagram of the experimental design. Mice were infected at time 0. Therapeutic agents or control empty particles were delivered on day 4 post-infection. Mice were humanely sacrificed and then echocardiograms were performed on day 6. Figure 6B shows fluorescence images of organs collected on day 6. Images were taken at 6, 12, 24, 48, 72, 96, and 120 hours after intravenous injection. Ex vivo fluorescence images and corresponding optical densities of major organs dissected 24 hours post-injection are shown. Figure 6C shows digital quantification of fluorescence in mock mice. Figure 6D shows digital quantification of fluorescence in PR8 mice, demonstrating significant and pronounced recruitment of LNP to the spleen, liver, and lungs (N=3). [Figure 7-1] Figures 7A, 7B, and 7C show a non-limiting example of systemic administration of LNP-miR-193b-5p INH on day 4 after H1N1 / PR8 infection, which attenuates injury in WT mice without apparent harm to miR-193bKO. Figure 7A shows lung histology: the large square shows hematoxylin and eosin staining (40x) and immunohistochemistry for occludin staining (small square, bar = 100 μm). LNP miR-193b-5p INH attenuates occludin loss in PR8-infected WT mice. Arrows indicate areas of occludin staining, peripheral bronchioles (Br.), and alveoli (Alv.). Figure 7B shows bronchoalveolar lavage fluid (BALf), total cell, and polymorphonuclear cell (PMN) counts. Figure 7C shows the fold change (FC) in expression demonstrating increased expression of the miR-193b-5p target gene occludin and decreased expression of the pro-inflammatory gene interleukin 6 (IL-6) compared to 36B4 (housekeeping gene) in mice that received LNP-miR-193b-5p INH compared to scrambled. Circles represent values ​​for individual mice (N = 3–5). [Figure 7-2]Figures 7A, 7B, and 7C show a non-limiting example of systemic administration of LNP-miR-193b-5p INH on day 4 after H1N1 / PR8 infection, which attenuates injury in WT mice without apparent harm to miR-193bKO. Figure 7A shows lung histology: the large square shows hematoxylin and eosin staining (40x) and immunohistochemistry for occludin staining (small square, bar = 100 μm). LNP miR-193b-5p INH attenuates occludin loss in PR8-infected WT mice. Arrows indicate areas of occludin staining, peripheral bronchioles (Br.), and alveoli (Alv.). Figure 7B shows bronchoalveolar lavage fluid (BALf), total cell, and polymorphonuclear cell (PMN) counts. Figure 7C shows the fold change (FC) in expression demonstrating increased expression of the miR-193b-5p target gene occludin and decreased expression of the pro-inflammatory gene interleukin 6 (IL-6) compared to 36B4 (housekeeping gene) in mice that received LNP-miR-193b-5p INH compared to scrambled. Circles represent values ​​for individual mice (N = 3–5). [Figure 8-1] Figure 8A shows a non-exhaustive predicted role of miR-193b in acute lung injury based on known gene targets, Tarbase v8.0. Figure 8B further shows a non-exhaustive predicted role of miR-193b in acute lung injury in REACTOME. Figure 8C further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on KEGG pathways. Figure 8D further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on GO biological pathways. Figure 8E shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, TarBase v8.0. Figure 8F shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, miRTarbase v8.0. [Figure 8-2]Figure 8A shows a non-exhaustive predicted role of miR-193b in acute lung injury based on known gene targets, Tarbase v8.0. Figure 8B further shows a non-exhaustive predicted role of miR-193b in acute lung injury in REACTOME. Figure 8C further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on KEGG pathways. Figure 8D further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on GO biological pathways. Figure 8E shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, TarBase v8.0. Figure 8F shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, miRTarbase v8.0. [Figure 8-3] Figure 8A shows a non-exhaustive predicted role of miR-193b in acute lung injury based on known gene targets, Tarbase v8.0. Figure 8B further shows a non-exhaustive predicted role of miR-193b in acute lung injury in REACTOME. Figure 8C further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on KEGG pathways. Figure 8D further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on GO biological pathways. Figure 8E shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, TarBase v8.0. Figure 8F shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, miRTarbase v8.0. [Figure 8-4]Figure 8A shows a non-exhaustive predicted role of miR-193b in acute lung injury based on known gene targets, Tarbase v8.0. Figure 8B further shows a non-exhaustive predicted role of miR-193b in acute lung injury in REACTOME. Figure 8C further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on KEGG pathways. Figure 8D further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on GO biological pathways. Figure 8E shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, TarBase v8.0. Figure 8F shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, miRTarbase v8.0. [Figure 8-5] Figure 8A shows a non-exhaustive predicted role of miR-193b in acute lung injury based on known gene targets, Tarbase v8.0. Figure 8B further shows a non-exhaustive predicted role of miR-193b in acute lung injury in REACTOME. Figure 8C further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on KEGG pathways. Figure 8D further shows a non-exhaustive predicted role of miR-193b in acute lung injury based on GO biological pathways. Figure 8E shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, TarBase v8.0. Figure 8F shows a non-exhaustive predicted role of miR-187-3p in acute lung injury based on known gene targets, miRTarbase v8.0. [Figure 9-1]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-2]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-3]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-4]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-5]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-6]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-7]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-8]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 9-9]Figure 9A shows exemplary data and a schematic diagram of the MoA for miR-187-3p: Schematic of an in vivo experiment. Figure 9B further shows the mean ± SEM of the percent change in echo-derived ejection fraction. Figure 9C further shows TNFα associated with 36-b4. Figure 9D shows the survival probability at 72 hours after CLP. Figure 9E shows the results of neonatal cardiomyocytes exposed to LPS with ciMSCs versus platelet EVs with or without Dynasore™ (DYN, an inhibitor of endocytosis). Bars represent the mean ± SEM of IL-6 / 18S. Figure 9F shows a Volcano plot to visualize the 66 miRs found to be enriched in ciMSC-EVs compared to platelet EVs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis versus those who died without sepsis. Figure 9I shows the copy number of miR-187-3p in whole blood from nine patients enrolled in the CISSI clinical trial (Cellular Immunotherapy for Septic Shock). Figure 9J shows a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we postulate IL-6 and S100AI. [Figure 10A]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10B]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10C]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10D]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10E]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10F]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10G]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10H]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 10I]Figure 10A shows that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro, A) immunofluorescence images show LNP uptake by primary mouse neonatal cardiomyocytes loaded with 10 mM miR-187-3p mimics. Figure 10B further shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery. Figure 10C further shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours. Figure 10D further shows representative immunofluorescence images demonstrating LNP uptake in the heart, lungs, and spleen. Figure 10E further shows the mean ± SEM of left ventricular (LV) ejection fraction (EF) and left ventricular fractional shortening (FS) versus CLP. Figure 10F further shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p compared to saline and empty LNP. Figure 10G further shows the mean ± SEM of TNFW18S in healthy tissue 48 hours after CLP. Figure 10H shows a schematic diagram of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either a miR-187-3p inhibitor or a miR-187-3p mimic. Figure 10I shows the survival probability compared to saline-treated CLP. [Figure 11A]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11B]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11C]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11D]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11E]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11F]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11G]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11H]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 11I]Figure 11A shows that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Primary mouse neonatal cardiomyocytes were transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS, for 24 hours (A-B). Figure 11B further shows the mean ± SEM of the fold change (FC) in the gene / 18S. Figure 11C further shows a Western blot of S100A1 protein levels in cardiomyocytes. Figure 11D further shows the results for S100A1KO-derived neonatal cardiomyocytes treated with LPS for 24 hours. Bars represent mean ± SEM fold changes (FC) in gene / 18S relative to control and LPS. Figure 11E shows mean ± SEM relative to vehicle / empty vector showing increased expression of TNFα compared to LPS, vs. vehicle, vs. LPS. Figure 11F shows survival probability relative to WT-CLP. Figure 11G shows mean ± SEM ejection fraction % (EF) measured 48 hours after CLP, WT-Sham. Figure 11H shows mean ± SEM plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours relative to pre-CLP. Figure 11I shows a Western blot of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p. [Figure 12-1]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 12-2]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 12-3]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 12-4]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 12-5]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 12-6]Figure 12A shows novel miRs and targets: a schematic diagram for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows results for neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS. Mean + SEM relative LUX activity compared to vehicle and LPS. Figure 12C shows box plots of miR-574-3p / U6 from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died of sepsis. Figure 12D shows results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours. Bars represent mean ± SEM for miR574-3p / U6. Figure 12E shows additional results for neonatal cardiomyocytes treated with LPS or miR574-3p mimics for 24 hours, mean ± SEM for S IO0AI / 18S. Figure 12F shows further results for neonatal cardiomyocytes treated with LPS or miR574 / 3p mimics for 24 hours, showing the mean ± SEM of IL-6 / 18S. Figure 12G shows the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. [Figure 13] FIG. 13 shows the enhanced cardiac delivery of miRNA using LNPs combined with microbubbles and released using ultrasound. [Figure 14] FIG. 14 shows the benefit of ultrasound-induced LNP release of miRNA as reflected in improved ventricular ejection fraction in rat hearts. DETAILED DESCRIPTION OF THE INVENTION

[0043] Various embodiments and aspects of the present disclosure are described with reference to the details discussed below. The following description and drawings are illustrative of the present disclosure and should not be construed as limiting the disclosure. The drawings are not necessarily to scale. Some specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described to provide a concise discussion of embodiments of the present disclosure.

[0044] definition As used herein, the terms "comprises" and "comprising" should be interpreted as inclusive and open-ended, rather than exclusive. Specifically, when used herein, including the claims, the terms "comprises" and "comprising" and variations thereof mean that the specified features, steps, or components are included. These terms should not be interpreted to exclude the presence of other features, steps, or components.

[0045] As used herein, the word "exemplary" means "serving as an example, instance, or illustration" and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0046] As used herein, the terms "about" and "approximately," when used in conjunction with a particle size range, a mixture composition, or other physical property or characteristic, are meant to cover slight variations that may exist at the upper and lower limits of the size range, so as to not exclude embodiments in which, on average, many of the dimensions are met but which may statistically fall outside of this range. Unless otherwise specified, the terms "about" and "approximately" mean plus or minus 25% or less. There is no intention to exclude embodiments such as these from the present disclosure.

[0047] Unless otherwise specified, any specified range or group should be understood to refer to each and every member of the range or group individually, as well as to each and every possible subrange or subgroup contained therein, and to any subrange or subgroup therein, as a similar shorthand method. Unless otherwise specified, the present disclosure expressly includes each and every specific member and combination of subranges or subgroups.

[0048] As used herein, the term "on the order of," when used in conjunction with an amount or parameter, refers to a range ranging from approximately one-tenth to ten times the stated amount or parameter.

[0049] As used herein, microRNAs (miRNAs) refer to endogenous, non-coding, single-stranded small RNAs, approximately 18-24 nucleotides in length. They may be longer, for example, up to 30 nucleotides in length. Functionally, miRNAs interfere with protein synthesis by directly degrading or inhibiting the translation of target genes at the post-transcriptional level. Two major types of miRNAs exist: intracellular miRNAs and circulating miRNAs, which are differentially expressed under different pathological and physiological conditions. As expression regulators, miRNAs target approximately 60% of protein-coding genes and play important roles in biological processes, including, but not limited to, cell proliferation, apoptosis, differentiation, immunity, and inflammation. miRNAs are typically characterized by their stability, specificity, and selectivity. Compared to traditional therapeutic agents that target specific receptors or proteins, strategies using miRNAs offer additional novelty because miRNAs typically affect the expression of more than one protein and thus multiple pathways. At their targets, miRNAs can produce potent, sustained, and global biological effects through endogenous gene silencing at nanomolar concentrations.

[0050] As used herein, the phrase "synthetic microRNA (miRNA)" or "miRNA mimic" refers to a synthetic mimic of a naturally occurring miRNA. Synthetic miRNAs have some or all of the functions of naturally occurring miRNAs, and may also have additional functions. For example, synthetic miRNAs may have improved resistance to exo- and endonucleases compared to natural miRNAs. Synthetic microRNAs in delivery systems may have enhanced endosomal release and intracellular targeting, which may provide improved therapeutic outcomes compared to naturally occurring microRNAs. Other advantages of synthetic microRNAs compared to natural miRNAs may be enhanced features such as improved carrier loading, improved therapeutic efficacy, improved cell-specific targeting, and improved safety. Non-limiting examples of synthetic microRNAs include, but are not limited to, those with the following features:

[0051] 1.1 Synthetic miRNA of 20-24 bp in length, where the miRNA is double-stranded and has a passive passenger strand.

[0052] 1.2 Terminal modifications: phosphorothioate to reduce nuclease activity; PS linkers at the 3' and 5' ends to reduce nuclease activity.

[0053] 1.3 and were produced using engineered polymerases with different amounts of PS at the termini to incorporate a C3 spacer.

[0054] 1.3.1 2'-O-methyl bases (increases thermostability; more resistant to endonucleases; delivered in vivo).

[0055] 1.3.2 2'-F-purine (to increase Tm');

[0056] 1.3.3 N1-methyl-Ψ (can only use its Watson-Crick face to base pair with another nucleoside, thus preventing it from forming wobble base pairs with other nucleotides (G, U, and C)). The RNA-modifying enzyme, Nep1, can methylate the N1 position.

[0057] 1.3.4 2'-O-Methoxy-ethyl bases (2'MOE). 1.3.5 Locked nucleic acids (LNA). 1.3.6 Modifications including, for example, mismatched or wobble nucleic acids, glycans and peptides or combinations thereof.

[0058] Both synthetic and naturally occurring miRNAs are referred to herein as miRNAs.

[0059] Another group of synthetic miRNAs are miRNA inhibitors, which can be used to suppress miRNAs. A common approach to correcting aberrant miRNA expression is based on the synthesis of antisense oligonucleotides with complementary sequences. 17 miRNA inhibitors are often synthetic single-stranded nucleic acids, 18–22 monomers in length, designed to specifically bind to endogenous miRNAs that would otherwise bind to their target mRNA molecules and prevent their translation. "Sponge" inhibitors can bind to two or more copies of miRNA. miRNA inhibitors are generally synthetic, and they may contain non-natural nucleic acid moieties to enhance their function. Non-limiting examples include sugar modifications -20-OMe: 20-O-methyl, 20-F: 20-fluoro-RNA, LNA: locked nucleic acid, UNA: unlocked nucleic acid, and 20-MOE: 20-O-methoxyethyl; backbone modifications -PO: phosphodiester, PS: phosphorothioate, PACE: phosphonoacetate, PMO: phosphorodiamidate morpholino oligomer, and PNA: peptide nucleic acid. miRNA inhibitors may not have a passenger strand. The miRNA inhibitor may be composed of multiple passenger and / or multiple guide strands simultaneously.

[0060] We provide several specific, non-limiting examples of LNPs that can be prepared using microfluidics. As shown in Figure 1, a typical LNP formulation contains four lipids: an ionizable lipid, cholesterol, a structural helper lipid, and a PEGylated lipid. The ionizable cationic lipid is positively charged in the formulation buffer (pH range of 3.5-5.5, preferably at a pH of about 4) and binds to and protects the negatively charged miRNA, facilitating endosomal escape, but is neutral in the storage buffer (PBS, pH 7.4). 7 Cholesterol is essential in the structure of LNPs and is important for LDL receptor-mediated endocytosis, for example, by complexing with apolipoprotein E or its variants. PEGylated lipids stabilize the particle and protect it from opsonization before it reaches its intended target.

[0061] As used herein, the term "ionizable lipid" refers to a lipid containing an ionizable group. This ionizable group can favorably interact with either the microRNA or the microRNA inhibitor. In the event of cationization, its role may include facilitating the loading of the combination into the carrier by binding to the negatively charged microRNA. Once the carrier enters the cell's endosome, the ionizable lipid may have a pKa that adjusts to the changing pH within the endosome as it evolves over time or with maturation, allowing for enhanced release of the carrier and endosome. This enhancement may result in a change in the affinity of the ionizable lipid for the microRNA or a change in the interaction between the ionizable lipid and the carrier or the endosomal wall. The ionizable lipid may contain one or more ionizable groups. The pKa of the ionizable group may be in the range of pKa = 4 to pKa = 6.9.

[0062] When the ionizable group is an amine, the lipid head group may be characterized by a primary, secondary, or tertiary amine. Ionizable lipids may take the form of branched molecules. Ionizable lipids must be amphiphilic, with aliphatic or hydrophobic tails characterized by varying degrees of length, branching, or both. The aliphatic segments may contain carbon or sulfur, and the bonds connecting them may be saturated or unsaturated, exhibiting different isomers. Lipid selection can be tailored to obtain either a monolayer or multilayer structure around the microRNA. It can be selected to interact with additional lipids to enhance microRNA loading.

[0063] Non-limiting examples of structural classes of ionizable lipids include unsaturated lipids (containing unsaturated bonds), multi-tail lipids (having two or more tails), polymeric lipids (containing polymers or dendrimers), biodegradable lipids (characterized by biodegradable bonds), and branched-tail lipids. Combinations of these structural features within a particular ionizable lipid are possible. As used herein, the term "lipid" refers to neutral or true fats, waxes, cutin, suberin, phospholipids, sphingolipids, lipoproteins, terpenes, prostaglandins, and sterols.

[0064] As used herein, the term "lipid" encompasses neutral or true fats, waxes, cutin, suberin, phospholipids, sphingolipids, lipoproteins, terpenes, prostaglandins, and sterols.

[0065] Furthermore, the term "phospholipid" is defined as a lipid molecule containing a hydrophilic "head" consisting of one or more phosphate groups and two hydrophobic fatty acid "tails." Typically, these components are connected by a glycerol molecule, creating a phospholipid, preferably a glycerol-phospholipid. Furthermore, the phosphate groups are often modified with simple organic molecules such as choline (to give phosphocholine) or ethanolamine (to give phosphoethanolamine).

[0066] Exemplary phospholipids relevant to the present invention include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0) PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0067] Other non-limiting examples of phospholipids include: Phospholipids: Examples include TIFF2026501811000002.tif234147 and TIFF2026501811000003.tif121147.

[0068] * The notation "carbon chain length:modification" is used, where modifications are represented by "0" for the default saturated configuration or a specific number of modifications. "C" and "t" indicate cis and trans configurations of double bonds, "y" represents a triple bond, and "me" indicates a methyliso branch, all followed by the carbon atom position of the modification.

[0069] ** Head groups refer to phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS).

[0070] As used herein, a "helper lipid" refers to a lipid that contributes to the stability of lipid nanoparticles or enhances their delivery. An example of a helper lipid that provides greater stability is a cylindrical lipid such as phosphatidylcholine. Conversely, a helper lipid with a conical shape can favor the formation of a hexagonal II phase and promote the endosomal release of oligonucleotides. Dioleoylphosphatidylethanolamine (DOPE) is an example of a helper lipid that promotes endosomal release.

[0071] Another helper lipid that promotes endosomal release is sterol, such as cholesterol.Cholesterol can be classified into three regions: head, body, and tail.Examples include vitamin D3, vitamin D2, calcipotriol (group 1); stigmasterol, beta-sitosterol (group 2); and buterin, lupeol, ursolic acid, oleanolic acid (group 3).A preferred embodiment is beta-sitosterol.

[0072] The term "PEGylated lipid" or PEG lipid refers to a polyethylene glycol (PEG) derivative attached to a lipid moiety (e.g., DMG or DSPE). PEG lipids serve various functions, including increasing circulation time, resistance to aggregation, and reducing nonspecific uptake of liposome-encapsulated (LNP) drugs. PEG lipids affect lipid nanoparticle properties by influencing particle size, stability, blood circulation time, and targeted delivery. The extent of these effects depends on the proportion and properties of the PEG lipid, such as PEG molar mass and lipid length. Differences in circulation time between lipid nanoparticle-siRNA formulations may be due to faster degradation of certain PEG lipids, enhancing cellular uptake and endosomal escape.

[0073] Non-limiting examples of PEG lipids include DMG-PEG2000 or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 ALC-0159 or (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, DSPE-PEG, DPPE-PEG, DOPE-PEG, DMPE-PEG (with PEG lengths varying from 0.2 to 5 kDa). Further non-limiting examples of PEG lipids are provided in Table 1.

[0074] [Table 1]

[0075] The notation used is "carbon chain length:modification", where modifications are represented by "0" for the default saturated configuration or a specific number of modifications. "C" and "t" indicate the cis and trans configuration of the double bond, followed by the carbon atom position of the modification.

[0076] Prodrug lipids: The term "prodrug lipid" refers to a lipid prodrug or drug-lipid conjugate in which the drug is covalently bound to a lipid moiety such as a diglyceride, phosphoglyceride, or fatty acid. The rationale for using prodrugs with lipid nanoparticles (LNPs) is based on the notion that nucleic acid delivery requires a vehicle such as LNPs, which can potentially enhance immune stimulation. To mitigate this, potent immunosuppressants such as dexamethasone have been co-administered with LNP formulations of siRNA. For example, anti-inflammatory prodrugs can effectively suppress cytokine production after intravenous administration of immunostimulatory oligodeoxynucleotides loaded into LNPs. LNPs tend to accumulate in phagocytes of the immune system after systemic administration, and direct association of the prodrug with LNPs allows for more specific delivery to immune cells, potentially reducing the required dose.

[0077] Compared with higher doses of free drug, incorporation of low levels of prodrug in LNPs containing unmodified mRNA, miRNA, or plasmid DNA can significantly reduce inflammatory cytokine levels after intravenous administration. 19 This can be achieved, in a non-limiting example, by chemically conjugating a lipophilic acyl / alkyl moiety to the drug or prodrug via a biodegradable linker, allowing for its incorporation into the nanoparticle.

[0078] A simple test could demonstrate the benefits of incorporating a prodrug into lipid nanoparticles compared to injecting the drug separately. For example, injection of LNPs containing modified miRNA into the interscapular space of the skin can result in skin lesions due to an immune response. Administration of the drug before LNP-miRNA injection can improve this response, demonstrating the potential benefits of incorporating a prodrug. The efficacy of the miRNA in the presence of the prodrug lipid can be demonstrated to ensure its efficacy is maintained.

[0079] This can be compared to any improvement observed when mice are injected with LNP-miRNA containing, for example, 10 mol% prodrug (preferably, the fraction of lipids other than ionizable lipids can be reduced to maintain nucleic acid loading).

[0080] It may be advantageous to demonstrate that prodrug lipids (e.g., 10 mol%) do not reduce the efficacy of miRNA compared to LNPs that do not carry the prodrug lipid. The immunomodulatory benefits of adding prodrug lipids may not be limited to LNPs with nucleic acid cargo, but may also be advantageous for the incorporation of other species, such as proteins or complex sugars, carried with the LNP.

[0081] Other components that may be advantageously included in lipid nanoparticles include, but are not limited to, proteins, peptides, glycans, small molecule (less than 300 daltons) drugs, aggregates of these, whether preformed or not, metal nanoparticles, nucleic acids, triggerable release of drug or prodrug cargo, or molecules that may serve to provide other functions such as enhanced lipid nanoparticle uptake by cells, enhanced release of drugs from LNPs after entry into cells, or enhanced targeting to different tissues, cell types, organelles containing cells, or macromolecular complexes within the cytoplasm or nucleus.

[0082] Other advantageous additions to lipid nanoparticles may include increasing the cholesterol fraction to enhance endosomal release, divalent ions, changes in the N / P ratio, changes in the anionic / cationic lipid ratio to optimize tissue targeting, alternative ionizable lipids, variations in alkyl chains, including changes in chain length, position and double bond isomers, and S to C substitutions, etc. These can be characterized, for example, by diameter, zeta potential, lipid composition using LC-MSMS, loading, and transfection efficiency (GFP mRNA), unloaded, and loaded.

[0083] Methods for making nanoparticles A non-limiting summary of the method for producing lipid nanoparticles is as follows: DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 are mixed in ethanol at a molar ratio of 50:10:38.5:1.5, and miRNA is suspended in acetate buffer. The miRNA and lipid solution are then mixed in a herringbone microfluidic mixer to form LNPs. The resulting mixture is then purified by centrifugal filtration before storage in PBS.

[0084] A more detailed and non-limiting explanation for creating LNPs by incorporating a lipid film into the preparation step follows. A lipid film containing DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5 can be prepared by mixing the separate lipid components dissolved in chloroform. For LNP formulation, an aliquot can then be dried with inert gas (N or Ar) and placed in a vacuum chamber overnight to remove residual solvent. The film can then be stored at -20°C. On the day of formulation, the lipid film can be dissolved in ethanol at a concentration of 6.8 mM (a total of 3.6 mg of lipid in 1 mL of ethanol). A miRNA solution can be prepared by suspending 5 nmol of miRNA mimic or inhibitor in 1200 μL of 0.1 M acetate buffer (pH 4). The miRNA and lipid solutions can then be mixed by microfluidic mixing using a flow ratio of 3:1, respectively, and a total flow rate of 9 mL / min. After mixing, the LNPs can be diluted to 12 mL with PBS (pH 7.4) and purified by filtration using a 30 kDa cutoff centrifugal filter, then resuspended in PBS and sterile filtered through a 0.45 μm membrane syringe filter. For characterization, a 50 μL aliquot can be collected and diluted 20-fold in PBS, after which the size, polydispersity index (PDI), and zeta potential can be measured. The concentration and median size of the LNPs can be measured, for example, at 2 x 10 per milliliter (ml). 5 The concentration of LNPs can be determined by nanoparticle tracking analysis at a particle dilution factor of 1. The LNPs can then be stored in PBS at 4°C before use.

[0085] After filtering the LNP, the encapsulation efficiency can be measured.The loading of the LNP can be quantified using a fluorescence-based assay.To ensure accurate quantification, a standard curve can be generated using the same RNA loaded into the LNP.The encapsulation efficiency can be calculated using the following formula:

number

[0086] Nanovesicles as alternative miRNA / miRNA-INH carriers Another class of carriers for nucleic acid therapeutics (such as siRNA, mRNA, miRNA, and miRNA inhibitors) are cell membrane-derived nanovesicles. 20 These nanovesicles have several beneficial characteristics: because their composition is that of cell membranes, they exhibit long circulation lifetimes and high biocompatibility. Furthermore, because these vesicles contain nearly the same composition (in terms of lipids, proteins, glycans, etc.) as the cell type from which they are derived, they can endogenously target tissues similar to that of the cell line from which they are derived. It is envisioned that these nanovesicles will be produced from isolated human cells of the following types or cell lines and all possible sublineages listed below:

[0087] bone marrow stromal cells, non-limiting examples of which may include HS-5 and HS-27A; myeloblasts, non-limiting examples of which may include HL-60, CMK, HEL, K-562, KASUMI-1, KG-1, LAMA-84, M-07e, MONO-MAC-1, MV4-11, NB-4, OCI-AML2, OCI-AML5, SIG-M5, THP-1, AR230, KCL22, U-937; isolated primary human neutrophils; eosinophils, non-limiting examples of which may include EoL-1, AML14, AM14.3D10 mast cells, non-limiting examples of which may include HMC-1, LAD-1, LAD-2, LUVA lymphoblasts, non-limiting examples of which may include SR4;11, MOLT-4, CCRF-CEM, BDCM; pulmonary epithelial, non-limiting examples of which may include primary small airway epithelial cells; normal human (HSAEC), Beas2B, A549; and endothelial cell lines, non-limiting examples of which may include 293; primary umbilical vein endothelial cells; normal human (HUVEC), and primary aortic endothelial cells; normal human (HAEC).

[0088] These nanovesicles have the added benefit of being readily loadable with hydrophilic cargo, such as small molecules, proteins, and nucleic acids, either passively during the cavitation process or actively by pH gradient loading after nanoparticle cavitation and isolation. Hydrophobic drugs can be incorporated into the nanovesicles during the cavitation process or by adding them to the solution with the purified nanovesicles, where they spontaneously incorporate with the lipid bilayer.

[0089] Non-limiting examples of potential cargoes to be loaded into the nanovesicles are small molecules such as cisplatin, TPCA-1, piceatannol, resolvin D2, ceftazidime, and resolvin D1; and nucleic acids such as DNA plasmids, siRNA, mRNA, miRNA, and miRNA inhibitors.

[0090] Microbubbles as miRNA delivery agents when combined with miRNA-carrying LNPs The microbubbles are typically 0.5 to 10 μm in size. The gas inside the microbubbles is typically a perfluorogas, such as perfluoropropane (C3F8), perfluorobutane (C4F 10 ), and sulfur hexafluoride (SF6) are approved for biomedical use. The gas inside the microbubble is surrounded or contained by a membrane that may be composed of polymers, biocompatible biopolymers, surfactants, proteins, lipids, or a combination of these. The membrane may be a molecular liquid with its molecular components intermixed to various degrees, or it may be made from a membrane composed of surface-packed nanoparticles. Various methods of microbubble preparation include sonication, cross-linking polymerization, nebulization, reconstitution, and evaporation of a solvent emulsion. Sonication is often preferred for microbubble preparation, for example, by an ultrasonic needle in a syringe that contacts the gas with the surfactant. The resulting microbubbles in the syringe can then be injected into a patient.

[0091] One use for microbubbles is to use them as drug carriers, where drugs can be loaded into the microbubbles in a syringe and later released in the patient by the application of additional ultrasound. Thus, the microbubbles can prevent the release of substances before they reach the area of ​​therapeutic and / or diagnostic interest.

[0092] Different compositions of the microbubble shell, for example, varying neutral, anionic phospholipids, and polyethylene glycol-conjugated phospholipids in appropriate ratios, can affect the relative stability of the microbubbles for ultrasound-based imaging or drug delivery.

[0093] Nanoparticle carriers of substances can be combined with microbubbles for localized delivery using ultrasound. Microbubbles and lipid nanoparticles can be mixed before administration (e.g., by intravenous injection), or they can be administered at different times. Ultrasound-excited microbubbles can be used to transiently permeabilize tissues or tissue components through which the nanoparticles can then pass, for example, enhancing delivery of the nanoparticle contents to a desired location, for example, at the ultrasound focus. 21 .

[0094] Formulations for use with the production of coated microbubbles Depending on their composition, nanoparticles may merge (fully or partially mix) with the surfactant that stabilizes the microbubbles, or they may act independently as a surfactant to stabilize the microbubbles. In the second case, the nanoparticles may have their composition altered by their function as a surfactant that stabilizes the microbubbles, or the nanoparticle composition may not be altered by their function as a surfactant that stabilizes the microbubbles. In this last case, the nanoparticles can in some cases be viewed as nanoparticle coatings. The composition of the nanoparticles may be such that they have individual components that interact separately with the gas inside and the liquid outside. Alternatively, the nanoparticles may be intermediate between gas and liquid, with a surface energy that lies between them and thus stabilize the microbubbles. A combination of these two properties may also be advantageous.

[0095] Identity of the miRNA mimic used to create the microbubbles In the case of solid lipid nanoparticles containing microRNAs used as therapeutic agents in conjunction with microbubble delivery techniques, the lipid nanoparticles may retain their identity as intact particles, or their components may be distributed within or create a membrane.The miRNA mimic composition can be expected to affect the degree to which nanoparticle integrity or dispersion occurs.This will depend on the stability of the lipid nanoparticles.Because miRNA mimic aggregates of a certain size preferentially cross cell membranes, transfection events after ultrasound-induced local release of miRNA mimics can be enhanced if the majority of the lipid nanoparticles are intact.

[0096] The release profile of the miRNA mimic can be predicted depending on the stability of the miRNA mimic in its environment and the intensity of the externally applied ultrasound that causes its release.

[0097] MicroRNA inhibitors can also be administered with the microbubbles as described above.

[0098] usefulness Non-limiting examples of utility include: Microparticles can be used to deliver drugs that act as therapeutic agents; Microparticles may have combined uses as drug delivery agents and diagnostic agents; Microparticles can be combined with other therapeutic agents, such as small molecule drugs, to perform combination therapies that can address different therapeutic targets or enhance the effect on a single target; By varying the composition of the microparticle, or more specifically, the amount of miRNA and / or additional drug it contains, the dosing of the therapeutic agent can be varied; this can improve the efficacy and / or safety of the therapy; Microparticles can be designed to release the loaded microRNA at different times, for example, in a delayed-release formulation to control the administration time or reduce the potential toxicity of the loaded miRNA; Microparticles can be designed to target a specific organ or set of organs, enhance function as needed, and reduce the possibility of adverse side effects; MicroRNA particles can be designed to release their cargo when an external force is induced, such as by an electromagnetic field or ultrasound, for example. An external trigger then allows the cargo to be released at the desired time and location. Microparticles can be designed to release their cargo depending on the physiological environment they are found in, non-limiting examples of which are local temperature, pH, or ionic strength. Microparticles can also be used to screen for existing or unknown physiological or diagnostic effects, either by themselves or in combination with other agents.

[0099] The present disclosure provides a pharmaceutical composition for treating immune response dysregulation, comprising the miRNA-based particle or the composition disclosed above. The immune response dysregulation may be acute respiratory distress syndrome, or it may be myocardial dysfunction in sepsis, or a combination of both.

[0100] Dysregulation of the immune response may result in any one or a combination of acute kidney injury, acute encephalopathy, hemodynamic instability, liver dysfunction, intestinal dysfunction, and muscle dysfunction, all of which may contribute to increased morbidity, mortality, and post-ARDS, post-cardiomyopathy, and post-ICU syndrome.

[0101] This treatment is equally applicable to human and animal subjects. (Example)

[0102] Non-limiting exemplary data relates to natural or synthetic miR-193b-5p INH mimetics and natural or synthetic miR-187-5p in four lipid vehicles that are effective in treating cells and mice for two models of lung injury resulting from viral and bacterial infection. Possible product description for miR-193b-5p INH: Formulation for increasing pulmonary surfactant in patients with injured lungs. Possible product description for miR-187-5p mimetics: Formulation for treating cardiomyopathy.

[0103] 1.miRNA Example data: Lipid nanoparticles: Referring to Figure 1, an exemplary lipid nanoparticle (LNP) containing four lipids: an ionizable lipid (DLin-MC3-DMA), cholesterol, a structural helper lipid (DSPC), and a PEGylated lipid (DMG-PEG2000) in a mol% ratio of 50:38.5:10:1.5, respectively, used to encapsulate microRNA (miRNA). Microfluidics was used to control the mixing of the lipid-containing solution and the miRNA-containing solution. The formulation contains an ionizable cationic lipid that is positively charged in the formulation buffer (pH range of approximately 3.5-5.5, preferably at a pH of approximately 4) to bind and protect the negatively charged miRNA, but is neutral in the storage buffer (PBS, pH 7.4). When endocytosed by acidic endosomes, it reionizes, facilitating endosomal escape. 12Cholesterol is important in the structure of LNPs and for endosomal release. Cholesterol is a non-limiting example of a sterol. PEGylated lipids stabilize the final LNP structure and are gradually replaced by a protein corona (including opsonins that lead to opsonization). In the formulations tested by the inventors, the corona is derived from Apo-E, and endocytosis is generally believed to be mediated by the LDL receptor.

[0104] Modified ionizable lipids can be achieved by simple aliphatic epoxide reaction with amines. The fraction of loaded cationic lipids can govern which organs are targeted. LNPs can be characterized, for example, by diameter, zeta potential, lipid composition by LC-MSMS, loading, and transfection efficiency (GFP mRNA).

[0105] Lipid films containing DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5 were prepared by mixing the separate lipid components dissolved in chloroform. For LNP formulation, aliquots were then dried with inert gas (N2 or Ar) and then placed in a vacuum chamber overnight to remove residual solvent. The lipid film was dissolved in ethanol at a concentration of 6.8 mM (3.6 mg total lipid per mL of ethanol). The miRNA solution was prepared by suspending 5 nmol of miRNA mimic or inhibitor in 1200 μL of 0.1 M acetate buffer (pH 4). The miRNA and lipid solutions were then mixed by microfluidic mixing (NanoAssemblr Benchtop™) using a flow ratio of 3:1 and a total flow rate of 9 mL / min, respectively. After mixing, the LNPs were diluted to 12 mL with PBS (pH 7.4) and purified by filtration using a 30 kDa cutoff centrifugal filter, then resuspended in PBS and stored in a refrigerator at 4 °C. Standardized characterization methods confirm the quality of our LNPs, as shown in Figure 2. There are some differences between LNPs in terms of size, polydispersity, and zeta potential; encapsulation efficiency would benefit from optimization.

[0106] There may be benefits to increasing the cholesterol fraction to enhance endosomal release. Typically, only 1-2% of LNPs exit endosomes and release their cargo. Increasing cholesterol and / or changing the sterol type (e.g., to β-sitosterol) can dramatically increase LNP cargo release and, therefore, transfection efficiency. It was previously assumed that LNP component fractions would not change significantly upon miRNA loading. However, we have now developed a reliable mass spectrometry method to measure the actual cholesterol (and lipid) components, as shown by the results in Figure 3. We find that compositional changes can be significant. Therefore, accurate preparation and post-loading characterization are crucial.

[0107] One way to test the value of increasing the cholesterol fraction is to increase the mol% of cholesterol (and other sterols) and compare LNP uptake with GFP fluorescence derived from RNA-loaded LNP mRNA. We have found that adding divalent ions to lipid formulations improves transfection. Figure 4 illustrates the value of this approach.

[0108] The anionic / cationic lipid ratio can be varied to optimize lung targeting. This approach differs from the work of Cheng and coworkers. 14 microRNA mimetics and inhibitors have various constraints on their structure and characteristic regulatory parameters in nucleic acid-loaded LNPs that were tested and reported in Cheng's study.

[0109] Exemplary data on in vitro testing of LNPmiR-193b-5pINH formulations are presented below. miR-193b-5p is expressed in endothelial, epithelial, and bone marrow-derived cells in response to TNFα and INFβ. Ocln is expressed in endothelial, epithelial, and myeloid cells in mouse and human lungs. We demonstrated the uptake of fluorescently labeled miRNA mimics and inhibitor-loaded LNPs in primary human distal bronchial airway epithelial cells (BEAS2b), primary human microvascular endothelial cells (HPMEC), and a non-small cell lung cancer cell line (CALU-3). All express Ocln (Figure 5A, example shown for CALU3). Briefly, influenza A / PR / 8 / 34 (H1N1 / PR8) virus stocks were prepared in MDCK (Madin-Darby canine kidney) cells (viral titers were determined by plaque assay). Cells were inoculated with virus (multiplicity of infection MOI = 1) ± empty LNPs (2.7 × 10 9LNP / ml; LNP containing a scrambled / negative RNA control (NC, 10 nM); or LNP miR193b-5p INH (10 nM). Figures 5B and 5C show that LNP was able to deliver miR-193b-5p to cells (expression levels were reduced according to digital droplet PCR (ddPCR), indicating that the inhibitor had an effect) and reduced viral replication as measured by semi-quantitative PCR (qRT-PCR) for the PR8 viral protein, as shown in Figure 5C (n = 2). Concurrently, Jurkat E6.1 (an immortalized line of human T lymphocytes) and BEAS2b cells were used to determine cytotoxicity, as measured by standard Annexin V / PI and PrestoBlue cell viability assays.

[0110] A non-limiting example of an in vitro H1N1 screening design is as follows: In vitro H1N1 screening design: CALU-3 human lung cells (which form tight junctions in culture) and BEAS2b (expressing a tight junction protein) are cultured in DMEM-F12 (1:1) medium, and MDCK cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. A virus stock of influenza A / PR / 8 / 34 (H1N1) (ATCC: VR-1469) is prepared in MDCK cells, and the virus titer is determined by plaque assay. The virus stock is diluted in serum-free medium for adsorption. An initial dose-response study is performed to establish doses of selected drugs and / or metals that do not affect CALU-3 cell viability (see cell viability assay).

[0111] Subsequently, monolayers of CALU-3 cells in 6-well plates were adsorbed with H1N1 at a multiplicity of infection (MOI) of 0.05, 0.5, or 5 for 60 minutes at 37°C, washed three times with PBS, and incubated in medium containing 2% FBS and the indicated concentrations of the compounds of interest. Samples (100 μL / well) at 1, 4, 8, 24, 48, and 72 hours were supplemented with the same volume of medium containing the corresponding compound concentrations. The collected medium was centrifuged at 20,000 x g for 1 minute, and virus production in the culture supernatant was determined by plaque assay. Plaque-forming units (PFU) were determined by plaque assay using 6-well plates. Briefly, MDCK cell monolayers were adsorbed with 10-fold serial dilutions of the supernatant and overlaid with a 1:1 mixture of 1.8% low-melting-point agarose and 2X virus growth medium (2X DMEM containing 2X VGM, 6 mg / ml bovine serum albumin, and 2 mg / ml TPCK-trypsin). After 72 hours of incubation, cells were fixed with 10% formaldehyde solution, and plaques were detected by staining with 0.1% crystal violet in 20% methanol.

[0112] Cell viability assay CALU-3 monolayer cells were seeded in 96-well plates and adsorbed with H1N1 at an MOI of 5 or the same volume of 1X VGM for 60 minutes, washed three times with 1X PBS, and incubated in medium containing 2% fetal bovine serum and the components of interest. To determine the effect of drug exposure on antiviral activity, cells were incubated with the treatment for 1, 4, 8, 24, and 48 hours. At the indicated time points, the treatment was removed and the wells were replenished with medium supplemented with 2% FBS. At 72 hours postinfection, cell viability was measured based on membrane integrity, where all cells were stained with Hoechst 3334241 and nonviable cells were stained with ethidium homodimer-I. Cells were incubated for 20 minutes at 37°C and imaged using an INCell Analyzer 2200 platform (GE Healthcare). Cell counts were performed using Developer Toolbox 1.9 software (GE). EC50 values ​​were calculated using the number of viable cells. A non-infected control should be included to determine CC50. We found that treatment with LNP improved the viability of virus-infected cells.

[0113] Therefore, the preferred route of administration is IV, even though efficient delivery to the lungs may be limited by biodistribution or / and removal of the active drug by other organs.Ultrasound can be seen as an alternative means to guide the delivery of effective therapeutic agents across the edema-filled alveoli, and as a non-limiting example, decafluorobutane microbubbles, which exhibit a net positive surface potential when negatively charged lipids are included in the outer layer of the lipid nanoparticles, can be integrated with the lipid nanoparticles envisioned above.

[0114] Exemplary data on miRNA delivery using microfluidic-loaded lipid nanoparticles We present non-limiting exemplary data on the in vivo delivery of LNP-miR-193b-5p INH. We delivered empty fluorescent LNP or LNP loaded with labeled miRNA via nasopharyngeal, intratracheal instillation, aerosolization, or intravenous administration at 6, 12, 24, 48, 72, 96, or 120 hours. Intranasal delivery resulted in increased fluorescence in the nasopharynx and oropharynx (with a slight increase in the lungs, not shown). When correlated with tissue, both intratracheal and aerosolized LNPs reached the lungs but were preferentially found in non-injured areas. Intravenous administration revealed LNPs in severely injured areas of the lung. We cut frozen sections and were able to visualize labeled miRs in the severely injured alveolar space (up to 96 hours) using miRNAscope in situ hybridization (data not shown). Labeled LNP and miR-INH were detected in extrapulmonary organs (particularly the spleen and liver, Figures 6B-6D), associated with preliminary evidence of reduced inflammatory gene expression in these organs (data not shown), suggesting additional benefit to extrapulmonary organs from systemic delivery. Fluorescent LNP or labeled miRNA were not detectable beyond 96 hours postinfusion.

[0115] miR-193b-5p expression was reduced as demonstrated by digital droplet PCR (ddPCR) in samples harvested 48 hours after LNPmiR-193b-5INH administration (i.e., day 6 post-infection). miR-193bKO and wild-type (WT) littermates (8-12 weeks old) were infected intranasally (IN) with H1N1 (PR8 strain A / Puerto Rico / 8 / 1934). 7Infection with a median tissue culture infectious dose [TCID] of 100 mg / mL results in 90-100% mortality by day 7. Mice develop progressive weight loss, hypothermia, and arterial hypoxemia. Four days after infection, mice were randomized to receive equal volumes (100 μl) of intravenous (IV): (i) empty LNP (6.3E11 / mouse), (ii) LNP scrambled negative control (NC 0.4 nmol / mouse), or (iii) LNP miR-193b-5p inhibitor ([INH], 0.4 nmol / mouse). Mice were sacrificed on day 6 for evaluation of effects on lung injury (histology), inflammation (BALf total cell count, PMN extravasation, and IL-6 expression), and target gene Ocln expression (Figures 7A-7C). These data strongly suggest that inhibition of miR-193b reduces virus-induced lung injury compared to scrambled controls; empty LNPs were not associated with an overt increase in lung inflammation in WT or miR-193bKO mice. We tested an exemplary test formulation of LNPmiR-193b-5pINH in an H1N1 mouse model of influenza infection.

[0116] In vivo outcome measures to help determine efficacy include survival. Mice are monitored for morbidity, as indicated by clinical signs of disease, including weight loss, lethargy, wrinkled fur, hunched posture, labored breathing, and hypoxemia (pulse oximetry). Detailed assessment of lung injury development includes, but is not limited to, histology, lung injury score (LIS), BALf (total cell count, neutrophilia rate, and myeloperoxidase (MPO) activity assay), and mediators in BALf and lung tissue lysates using prefabricated mouse-specific cytometric bead arrays. Assaying Th1 and Th2 responses is useful. Membrane permeability can be determined by measuring total protein, Evans blue dye, and IgM leakage in BALf. Viral load can be quantified by plaque-forming units (PFU) / ml, HAU, immunohistochemical analysis for PR8 antigen in the lung, Western blot for viral NP, and qRT-PCR for viral transcripts: hemagglutinin (HA), neuraminidase (NA), and polymerase (PB1). Those skilled in the art can determine cell-specific regulation of miR-193b-5p by in situ hybridization in lung tissue using a Basescope with immunostaining for cell-specific markers (CK18 (epithelial cell marker) or / and CD31 (endothelial cell marker)) by immunofluorescence or brightfield immunohistochemical analysis to detect pre-miR-193b, -3p, -5p, and occludin (probes for mmu-pre-miR-193b, -5p, and -3p). The immune response can be analyzed by determining the integrated optical density (IOD) per μm2 of stained area using Image-Pro Plus software. If desired, in addition to measuring miR-193b -5p and -3p by qRT-PCR, one skilled in the art can determine function by assessing target mRNA expression levels and off-target effects by RNA sequencing analysis, as previously published.Total RNA from WT and KO mice infected with PR8 and treated with miR-193b-5p INH, mimetics (MIMs), or respective control oligonucleotides can be hybridized to RNAseq arrays.

[0117] Exemplary in vitro testing of formulations of LNPmiR-193b-5p-INH miR-193b-5p is expressed in endothelial, epithelial, and bone marrow-derived cells in response to TNFα and INFβ ( 15 and 16 Ocln is expressed in endothelial, epithelial, and myeloid cells in mouse and human lungs. We demonstrated the uptake of fluorescently labeled miRNA mimics and inhibitor-loaded LNPs in primary human distal bronchial airway epithelial cells (BEAS2b), primary human microvascular endothelial cells (HPMEC), and a non-small cell lung cancer cell line (CALU-3). All of these cells express Ocln (Figure 5A, example shown for CALU3). Briefly, influenza A / PR / 8 / 34 (H1N1 / PR8) virus stocks were prepared in MDCK (Madin-Darby canine kidney) cells (virus titers were determined by plaque assay). Cells were infected with virus (multiplicity of infection = 1) ± empty LNPs (2.7x10 9 LNP / ml); LNP containing scrambled / negative RNA control (NC, 10 nM) 22、23 or exposed to LNP miR193b-5p INH (10 nM) as published.

[0118] Figures 5B and 5C show that LNPs can deliver miR-193b-5p into cells (expression levels were reduced according to digital droplet PCR (ddPCR) and inhibitors had an effect), and that the LNPs were able to deliver miR-193b-5p into cells (see references ( 24 and 25(N=2) shows that viral replication was reduced as measured by semi-quantitative PCR (qRT-PCR) for PR8 viral proteins as described in (). Concurrently, cytotoxicity was determined using Jurkat E6.1 (an immortalized line of human T lymphocytes) and BEAS2b cells, as measured by standard Annexin V / PI and PrestoBlue cell viability assays.

[0119] Exemplary in vivo delivery of LNP-miR-193b-5p-INH We delivered empty fluorescent LNPs or LNPs loaded with labeled miRNAs via nasopharyngeal, intratracheal instillation, aerosolization, or intravenous administration at 6, 12, 24, 48, 72, 96, or 120 hours. Intranasal delivery resulted in increased fluorescence in the nasopharynx and oropharynx (with a slight increase in the lungs, not shown). When correlated with tissue, both intratracheal and aerosolized LNPs were found in the lungs but were preferentially found in non-injured areas. Intravenous administration revealed LNPs in severely injured areas of the lungs. We cut frozen sections and were able to visualize labeled miRs in the severely injured alveolar space (up to 96 hours) using miRNAscope in situ hybridization (data not shown). Labeled LNP and miR-INH were detected in extrapulmonary organs (particularly the spleen and liver, Figures 6B, 6C, and 6D), associated with preliminary evidence of reduced inflammatory gene expression in these organs (data not shown), suggesting additional benefit to extrapulmonary organs from systemic delivery.

[0120] Fluorescent LNP or labeled miRNA was not detected beyond 96 hours post-injection. miR-193b-5p expression was reduced as demonstrated by digital droplet PCR (ddPCR) in samples harvested 48 hours after LNPmiR-193b-5INH administration (i.e., day 6 post-infection). miR-193bKO and wild-type (WT) littermates (8-12 weeks old) were infected intranasally (IN) with H1N1 (PR8 strain A / Puerto Rico / 8 / 1934). 26 ). 10 7Infection with a median tissue culture infectious dose [TCID] of 100 mg / mL results in 90-100% mortality by day 7. Mice experience progressive weight loss, hypothermia, and arterial hypoxemia. 20 Four days after infection, mice were randomized to receive equal volumes (100 μl) of intravenous (IV): (i) empty LNP (6.3E11 / mouse), (ii) LNP scrambled negative control (NC 0.4 nmol / mouse), or (iii) LNP miR-193b-5p inhibitor ([INH], 0.4 nmol / mouse). (a) Mice were sacrificed on day 6 for evaluation of lung injury (histology), inflammation (BALf total cell count, PMN extravasation, and IL-6 expression), and target gene Ocln expression (Figures 7A-7C). Our data strongly suggest that inhibition of miR-193b reduces virus-induced lung injury compared to the scrambled control; empty LNP was not associated with an apparent increase in lung inflammation in WT or miR-193bKO mice.

[0121] Figures 7A-7C show that systemic administration of LNP-miR-193b-5pINH on day 4 after H1N1 / PR8 infection attenuates injury in WT mice without overtly harming miRbKO. We performed approximate dose-finding and administration timing experiments by IV delivery of LNP-miR-193b-5pINH 6 hours after cecal ligation and puncture (CLP) (data not shown), which resulted in a trend toward reduced inflammatory cell infiltration, edema formation, and histological lung injury (data not shown). The data also show the preservation of ejection fraction, fractional shortening, and alpha (α)-myosin heavy chain (Myh6) expression compared with LNP alone or LNP-NC. 27、28 Together, they show a trend towards reduced bacterial counts and sepsis-induced cardiomyopathy, as well as reduced TNF and S100A8 (important in the injured heart) expression (data not shown).

[0122] The exploration and proven role of miR-193b-5p and miR-187-3p in ARDS / acute lung injury i) A primer on ARDS / acute lung injury is available in the references 29 Available at.

[0123] We found that miR-193b-5p binds to occludin (a major component of tight junctions) and surfactant protein C (a key protein involved in reducing surface tension), resulting in post-transcriptional degradation and reduced protein expression. Inhibition of miR-193b-5p is beneficial in three models of acute lung injury: a model of aseptic sepsis (endotoxemia caused by infusion of lipopolysaccharide into the lungs, a model of direct lung injury); cecal ligation and puncture (sepsis-induced ARDS, a model of secondary extrapulmonary ARDS); and a model of influenza A virus-induced lung injury (reducing lung injury). Preventing occludin degradation in our mouse model of ARDS resulted in reduced alveolar-capillary barrier function, neutrophil infiltration, inflammatory cytokine and chemokine expression, reduced bacterial and viral loads, and improved survival.

[0124] We further found that the absence of miR-193b-5p protected mouse lungs from the loss of surfactant C during viral ARDS. Surfactant retention was associated with reduced picrosirius red staining (a stain for collagen and amyloid), suggesting reduced ARDS-associated fibrosis in mice lacking miR-193b-5p.

[0125] (i) Prediction (based on known gene targets Tarbase v8.0) - Role of miR-193b in acute lung injury: Known targets of miR-193b-5p and putative pathways that may be affected by miR-193b-5p inhibition are shown in Figures 8A and 8B. Antiviral (red); fibrosis (TGFb / Wnt pink). HUGO gene symbols are shown for each node related to known gene targets of miR-193b-5p. Inhibition of miR-193b-5p is predicted to (i) reduce viral load by decreasing the expression of viral replication-related genes and (ii) reduce lung fibrosis by decreasing activation of the TGFb / Wnt / beta-catenin-related pathway.

[0126] (ii) Discovery / Proof-of-Concept miR-187-3p Mimetics We demonstrated in vivo and in vitro that miR-187a-3p replacement therapy using miR-187a-3p mimetics transfected into primary neonatal cardiomyocytes further demonstrated the regulation of miR-187a-3p and target genes (Itpkc, Lrrc59, and Tbl1xr1) involved in sepsis-induced cardiac dysfunction. Tbl1xr1 (transducin beta-like 1 X-linked receptor 1) is essential for Wnt-β-catenin activation, and inhibition of canonical Wnt signaling prevents heart failure. Furthermore, TNFα, IL-6, and IL-12p40 (key pro-inflammatory mediators in sepsis) are published targets of miR-187a-3p. TarBase, a database of experimentally supported miR targets, has shown that intracellular adhesion molecule-1 (ICAM-1), Jumonji and AT-Rich interacting domain-containing 2 (Jarid2), and S100 calcium-binding protein A1 (S100A1) and A4 (S100A4) are involved in myocardial dysfunction. We have discovered previously unknown roles for microRNA-187-3p (miR-187a-3p) and its target genes S100 calcium-binding protein A1 (S100A1) and Jumonji and AT-Rich interacting domain-containing 2 (Jarid2) in sepsis and sepsis-induced myocardial dysfunction.

[0127] (iii) Prediction (based on known gene targets miRTarbase v8.0) - Role of miR-187-3p in acute lung injury: miR-187-3p is less well studied and has fewer known targets (Tarbase and miRTarbase v8.0), as shown in Figures 8C and 8D. The inventors identified miR-187a-3p and determined its therapeutic mechanism of action, which involves post-transcriptional inhibition of the pro-inflammatory mediators TNFα, interleukin-6 (IL6), and cardiac alarmin S100A1. Novel promising miRs for further testing were also identified. Exemplary data are provided as support for the central claims.

[0128] MicroRNA-based therapeutics for sepsis-induced myocardial dysfunction: exemplary data To identify miR biotargets for the treatment of myocardial dysfunction in sepsis 36、37、38 , clonally expanded and immortalized human mesenchymal stromal cell line (ciMSCs) 39 )—preparing a therapeutically relevant, homogeneous, and renewable source of extracellular vesicles (EVs) 40、41、42、43 Preclinical studies have shown a significant reduction in inflammation, organ injury, and mortality as well as increased bacterial killing after intravenous MSC administration. 44、45、46 The contents of EVs—mRNA, microRNA, lipids, and proteins—may confer paracrine bioactivity to MSCs. 47、48、49、50、68、69 Administration of ciMSC-derived EVs has been shown to be a promising model for brain injury. 51 and models of graft-versus-host disease 39 Given the immunomodulatory properties of ciMSC-EVs, we tested whether these same EVs could also prevent left ventricular dysfunction in a preclinical model of sepsis contribution of S100A1 to sepsis-induced cardiomyopathy.

[0129] Figures 9A-9I show exemplary data and a schematic diagram of the mechanism of action (MoA) of miR-187-3p. Figure 9A shows a schematic diagram of the in vivo experiment. Male and female C57BL / 6 mice (12-14 weeks) randomized to CLP or sham surgery (sham) received antibiotics (imipenem) and fluid resuscitation (saline). Mice were further randomized to EV preparations derived from ci-MSCs or platelets (5x10E4 cell equivalents / g BW) delivered IV 6 hours after CLP, and evaluation was performed 72 hours after CLP. Figure 9B shows the mean ± SEM of the percent change in echo-derived ejection fraction, and Figure 9C shows the TNFα-associated effect of 36-b4. * p ≤ 0.05; ** p≦0.01. Figure 9D shows the survival probability at 72 hours after CLP, and as shown in Figure 9E, * p ≤ 0.05, **p≦0.01, showing the effect on neonatal cardiomyocytes (2×10E5 / cell) exposed to LPS (10 mg / mL x 24 h) with ciMSCs vs. platelet EVs (2.5x or 5x dose) with or without Dynasore (DYN, an inhibitor of endocytosis). Bars are mean ± SEM of IL-6 / 18S vs. vehicle. * *p<0.05, N=3. Figure 9F shows results for two independent replicates of ci-MSC and platelet EVs sent for miRNA sequencing (HTG Molecular Diagnostics). Using a Volcano plot, 66 miRs were visualized and found to be enriched in ciMSC-EVs compared with platelet EVs (false discovery rate <0.01; fold change >2). miRs 187-3p and 574-3p were identified as the most enriched miRs in EVs derived from ciMSCs. Figure 9G shows that miR-187-3p expression was increased in hearts from septic mice receiving ciMSC-EVs. Figure 9H shows a box plot of miR-187-3p / U6 levels from formalin-fixed, paraffin-embedded postmortem human hearts from patients who died with sepsis versus non-sepsis. Data are median ± IQR and represent the mean ± standard deviation (IQR) for non-sepsis. * p<0.05, N=6.

[0130] Figure 9I shows miR-187-3p copy numbers in whole blood from nine patients enrolled in a cellular immunotherapy for septic shock trial; symbols represent values ​​from individual patients (N = 3 / group) and healthy controls (N = 6). RNA was isolated from whole blood collected at baseline, 24, and 72 hours after MSC infusion. MSC dose (0.3 million, 1 million, or 3 million cells / kg) was used. Figure 9J provides a schematic diagram of the MoA for miR-187-3p. miR-187-3p is decreased during sepsis. miR-187-3p reduces the expression of TNFα, and we hypothesize that it binds IL-6 and S100A1 post-transcriptionally (to the 3'UTR, leading to mRNA degradation) to attenuate both pathogen and damage-associated molecular pattern receptor stimulation, reducing inflammation and myocardial dysfunction.

[0131] Figures 10A-10I show that delivery of LNPs carrying miR-187-3p attenuates inflammatory gene expression, organ dysfunction, and death. In vitro: Figure 10A, immunofluorescence images show the uptake of LNPs (green fluorescence) by primary mouse neonatal cardiomyocytes (red fluorescence) loaded with 10 mM miR-187-3p mimics. Figure 10B shows the mean ± SEM of miR187-3p / U6 24 hours after LNP-miR-187-3p delivery, n=3. Figure 10C shows the mean ± SEM of TNFW18S in response to LPS (mg / mL) ± LNP-miR-187-3p over 24 hours, n=4. In vivo: Delivery of exogenous miR-1873p to septic mice was examined. Cy5-tagged LNPs were injected intravenously (IV) into C57BL / 6 mice. Figure 10D provides representative immunofluorescence images showing LNP uptake in the heart, lungs, and spleen. Figure 10E provides the mean + SEM of left ventricular (LV) ejection fraction (EF) and fractional shortening (FS) for CLP (relative to CLP + empty LNP). ** p≦0.001); Figure 10F shows the survival probability 48 hours after CLP in mice receiving LNP-miR-187-3p (n-11) compared to saline (n-13) and empty LNP (n-12); *p≦0.05. Figure 10G shows the mean±SEM of TNFW18S in healthy tissues 48 hours after CLP. * p ≤ 0.05, ** p≦0.01 (n-3-10). Figure 10H provides a schematic of the reconstitution experiment. Mice were randomized to receive ciMSC-EVs 6 hours after CLP with either miR-187-3p inhibitors or miR-187-3p mimetics. Figure 10I shows survival probability compared to saline-treated CLP, log-rank test. * p ≤ 0.05, ** p ≤ 0.01, *** p≦0.001 is indicated.

[0132] Figures 11A-11I show that S100A1 is a target of miR-187-3p and that S100A1-deficient mice are resistant to sepsis. Figure 11A shows primary mouse neonatal cardiomyocytes transfected with the full-length S100A1 3'UTR fused to a luciferase (LUX) expression vector and treated with either miR-187 scramble (SCR), miR-187-3p mimic (MIM), or inhibitor (INH), alone or together with LPS (1 μg / mL), for 24 hours (Figures 11A and 11B). Figure 11C shows a Western blot of S100A1 protein levels in cardiomyocytes (N=1). Figure 11D shows S100A1KO-derived neonatal cardiomyocytes treated with LPS (1 μg / mL) for 24 hours. Bars are mean ± SEM of fold change (FC) in genes / 18S for control and LPS, relative to control * *p<0.05, *p≦0.05 vs. LPS (n=3-8). Neonatal cardiomyocytes were transfected with S100A1 expression vector or empty vector and then treated with LPS (1 μg / mL) for 24 hours. Figure 11E shows the mean ± SEM for vehicle / empty vector treatments demonstrating increased expression of TNFα compared to LPS. *p<0.05 vs. vehicle. * p<0.05. #p≦0.05 vs. LPS (n=3). S100A1KO and WT mice (10-12 weeks) were randomized to CLP or sham surgery. Figure 11F shows the survival probability; WT-CLP vs.* p<0.001. Figure 11G provides the mean ± SEM of ejection fraction (EF) (%) measured 48 hours after CLP. WT-relative to Sham * *p<0.05, n=5-10. Figure 11H provides plasma levels of S100A1 (ng / ml) measured at 24 and 48 hours. Bars are mean ± SEM, relative to pre-CLP. * p<0.05, ** *p<0.01, n=5-10. Figure 11I shows a Western blot (N=1) of changes in S100A1 protein levels in hearts from CLP mice treated with LNP-miR-187-3p.

[0133] Figures 12A-12G show novel miRs and targets: Figure 12A provides a schematic for testing the effects of the top 10 miRs identified in ci-EVs in human cardiomyocytes. Figure 12B shows neonatal cardiomyocytes transfected with an IL-6 3' UTR LUX expression plasmid or an empty plasmid and treated with LPS (10 μg / mL for 24 h). Bars are the mean + SEM of relative LUX activity (relative to vehicle). * p<0.05; relative to LPS * *p<0.05, n=8). Figure 2C provides box plots of miR-574-3p / U6 derived from formalin-fixed, paraffin-embedded, postmortem human hearts from patients who died with sepsis (N=6) versus patients who died without sepsis (N=6). Neonatal cardiomyocytes were treated with LPS (10 μg / mL) or miR574-3p mimics (10 nM) for 24 h. Bars are mean ± SEM for miR574-3p / U6 (Figure 12D), S100A1 / 18S (Figure 12E), and IL-6 / 18S (Figure 12F); relative to vehicle / scrambled. * *p<0.05, N=3. Figure 12G provides the mean ± SEM of miR574-3p (copies / ng) in WT mice 48 hours after CLP treated with saline or miRI87 LNPs. * p<0.05 (N=5–7).

[0134] Figure 13 shows ultrasound-targeted microbubble destruction (UTMD)-mediated delivery of miR187a-3p-enhanced miR187a-3p expression in the myocardium. Fisher rats (8-10 weeks old) received miR187a-3p mimetic LNP or scrambled LNP delivery, alone or with microbubbles (MB), after ischemia / reperfusion (I / R) or sham surgery. Data presented as box plots show the median and interquartile range of miR187a-3p / U6 expression in infarcted (left), peri-infarct (center), and remote (right) myocardium compared to sham. Relative to sham * *p<0.05. n=2-6. Note that the data are not normally distributed and are therefore presented as box plots. LNPs were diluted to 1x10 in 3 mL of saline solution. 9 The LNP-cationic microbubble complex was mixed with 10 microbubbles and injected into rats via the jugular vein. For ultrasound-targeted microbubble destruction (UTMD), high-power ultrasound with a pulse interval of 10 cardiac cycles at end-systole was transmitted to the left ventricle via a transducer with a frequency of 5 MHz, a depth of 2 cm, and an output of 120 V during intravenous infusion of the LNP-cationic microbubble complex via the jugular vein over a 5-minute period. The probe was positioned to cut the LV in the transverse plane (short axis) at the midpapillary level, and the transducer was slowly moved from base to apex to allow maximal myocardial delivery. Ultrasound transmission was continued for an additional 25 minutes after LNP-microbubble infusion to ensure maximal delivery.

[0135] Figure 14 shows that post-I / R delivery of miR187a-3p improves echo-derived parameters of LV function. Bars represent mean + SEM of echo-derived ejection fraction (left) and fractional shortening (right) 3 days after I / R treatment with miR187a-3p (IDT formulation) LNPs, scrambled LNPs alone, or scrambled LNPs combined with microbubbles (MB) or sham surgery. Relative to sham **p<0.05. n=2-3 / group. Quite surprisingly, the dosage required to achieve the observed effect was approximately 100-fold lower than that required when the therapeutic agent was delivered systemically without localized triggered release using ultrasound. This greatly improves the therapeutic index.

[0136] Significance and Impact Our innovations anticipate new approaches to RNA-based therapeutics to help unravel the disconnect between mortality and inflammation and uncover unknown disease processes. Our approach can be used to develop personalized (determine miR levels in humans and use replacement or inhibition therapies, if necessary) and precise (target specific treatable traits) therapeutics. 96 ) will yield libraries of LNPs / RNAs that can be used alone or in combination to advance the care of patients with sepsis. The strength of this innovation lies in the potential for rapid clinical application of innovative therapeutic agents.

[0137] References 1. Butt Y., Kurdowska A., Allen TC Acute Lung Injury: A Clinical and Molecular Review. Arch Pathol Lab Med. 140, 345-350 (2016). 2. Sweeney RM, Griffiths M., McAuley D. Treatment of acute lung injury: current and emerging pharmacological therapies. Semin Respir Crit Care Med. 34, 487-498 (2013). 3. Bruscella, P. et al. Viruses and miRNAs: More Friends than Foes. Front Microbiol 8, 824 (2017). 4. Dos Santos, C. C. et al. Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury. Eur Respir J 59, 2004216 (2022). 5. Lee, L. K., Medzikovic, L., Eghbali, M., Eltzschig, H. K. & Yuan, X. The Role of MiRs in Acute Respiratory Distress Syndrome and Sepsis, From Targets to Therapies: A Narrative Review. Anesth Analg 131, 1471-1484 (2020). 6. Younes, N. et al. Mesenchymal stromal / stem cells modulate response to experimental sepsis-induced lung injury via regulation of miR-27a-5p in recipient mice. Thorax 75, 556-567 (2020). 7. Kulkarni, J.A., Witzigmann, D., Thomson, S.B. et al. The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 16, 630-643 (2021). 8. Sam Chen, Josh Zaifman, Jayesh A. Kulkarni, Igor V. Zhigaltsev, Ying K. Tam, Marco A. Ciufolini, Yuen Yi C. Tam, Pieter R. Cullis, Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids, Journal of Controlled Release, 286, 2018, 46-54. 9. W. Chatham, R.P. Kimberly Treatment of lupus with corticosteroids Lupus, 10 (2001), pp. 140-147. 10. M.T. Abrams, M.L. Koser, J. Seitzer, S.C. Williams, M.A. Dipietro, W. Wang, A.W. Shaw, X. Mao, V. Jadhav, J.P. Davide, P.A. Burke, A.B. Sachs, S.M. Stirdivant, L. Sepp- Lorenzino Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: effect of dexamethasone co-treatment Mol. Ther., 18 (2010), pp. 171-180 11. D.C. Litzinger, J.M. Brown, I. Wala, S.A. Kaufman, G.Y. Van, C.L. Farrell, D. Collins Fate of cationic liposomes and their complex with oligonucleotide in vivo Biochim. Biophys. Acta, 1281 (1996), pp. 139-149 12. Kulkarni, J.A., Witzigmann, D., Thomson, S.B. et al. The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 16, 630-643 (2021). 13. Patel, S. et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA Nature communications 11, 1-13 (2020). 14. Cheng Q., Wei T., Farbiak L., Johnson L. T., Dilliard S. A., Siegwart D. J. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 15, 313-320 (2020). 15. Dos Santos, C. C. et al. Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury. Eur Respir J 59, 2004216 (2022). 16. Dos Santos, C. C. et al. Preventing Occludin Tight-Junction Disruption via Inhibition of miR-193b-5p Attenuates Viral Load and Influenza-induced Lung Injury, in preparation. 17. Lima, J.F., et al. Anti-miRNA oligonucleotides: A comprehensive guide for design, RNA Biology, 15, 338-352 (2018). 18. Tenchov, R. et al, Lipid Nanoparticles─From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement, ACS Nano 15, 16982-17015 (2021). 19. Chen S, et al Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids. J Control Release. 286, 46-54 (2018). 20. Wang J, et al. MiR-101a loaded extracellular nanovesicles as bioactive carriers for cardiac repair. Nanomedicine. 27, 102201 (2020). 21. Ogawa, K. et al. Focused ultrasound / microbubbles-assisted BBB opening enhances LNP-mediated mRNA delivery to brain, J Control Release, 348, 34-41 (2022). 22. Younes, N. et al. Mesenchymal stromal / stem cells modulate response to experimental sepsis-induced lung injury via regulation of miR-27a-5p in recipient mice. Thorax 75, 556-567 (2020). 23. Dos Santos, C. C. et al. Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury. Eur Respir J 59, 2004216 (2022). 24. Dos Santos, C. C. et al. Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury. Eur Respir J 59, 2004216 (2022). 25. Dos Santos, C. C. et al. Preventing Occludin Tight-Junction Disruption via Inhibition of miR-193b-5p Attenuates Viral Load and Influenza-induced Lung Injury, in preparation. 26. Guttman, J. A. & Finlay, B. B. Tight junctions as targets of infectious agents. Biochimica et Biophysica Acta (BBA) - Biomembranes 1788, 832-841 (2009). 27. Smeding, L. et al. Salutary effect of resveratrol on sepsis-induced myocardial depression. Crit Care Med 40, 1896-1907 (2012). 28. dos Santos, C. C. et al. Sepsis-induced myocardial depression is associated with transcriptional changes in energy metabolism and contractile related genes: a physiological and gene expression-based approach. Crit Care Med 38, 894-902 (2010). 29. Matthay, M.A., Zemans, R.L., Zimmerman, G.A. et al. Acute respiratory distress syndrome. Nat Rev Dis Primers 5, 18 (2019). PATENT CITATIONS 1. WO2020219941A1 2019-04-26 2020-10-29 Genevant Sciences Gmbh Lipid nanoparticles. 2. US11141378B2 2022-09-20 2030-06-30 Arbutus Biopharma Lipid formulations for delivery of therapeutic agents. 3. CN105555279B 2019-12-06 2034-06-25 London Health Sciences Centre Research Inc MicroRNA inhibition for treatment of sepsis

Claims

1. A synthetic miRNA or mimetic thereof encapsulated in a lipid nanoparticle (LNP) carrier; and Lipid nanoparticle carriers containing at least four types of lipids that are independent of each other Including, a) an ionizable cationic lipid selected to be positively charged in the formulation buffer; b) sterols, c) structural helper lipids, and d) PEGylated lipids miRNA-based particles, including

2. 10. The miRNA-based particle of claim 1, wherein the ionizable cationic lipid is positively charged in a formulation buffer having a pH ranging from about pH 3 to about pH 5.

5.

3. The miRNA-based particle of claim 1 or 2, wherein the ionizable cationic lipid is neutral in a storage buffer having a pH in the range of about pH 7 to about pH 8.

4. The miRNA-based particle of claim 3 , wherein the storage buffer is phosphate buffered saline (PBS) having a pH of about 7.

4.

5. 5. The miRNA-based particle of any one of claims 1 to 4, wherein the ionizable cationic lipid is any one or combination of a saturated lipid, an unsaturated lipid, a single-tail lipid, a multi-tail lipid, a polymeric lipid, a biodegradable lipid, or a branched-tail lipid.

6. The miRNA-based particle of any one of claims 1 to 4, wherein the ionizable cationic lipid comprises a neutral or true fat, a wax, a cutin, a suberin, a phospholipid, a sphingolipid, a lipoprotein, a terpene, a prostaglandin, or a sterol.

7. The miRNA-based particle of any one of claims 1 to 6, wherein the sterol is selected to facilitate endocytosis by low density lipid receptors by complexing with apolipoprotein E.

8. The miRNA-based particle of any one of claims 1 to 7, wherein the sterol is selected on the basis of improving intracellular delivery.

9. The miRNA-based particle of any one of claims 1 to 8, wherein the structural helper lipid is selected to contribute to the stability of the lipid nanoparticle and / or to enhance endosomal release.

10. The miRNA-based particle of any one of claims 1 to 9, wherein the structural helper lipid is a cylindrical lipid such as phosphatidylcholine.

11. The miRNA-based particle of any one of claims 1 to 9, wherein the structural helper lipid is cone-shaped, favoring the formation of a hexagonal II phase and facilitating endosomal release of the oligonucleotide.

12. The miRNA-based particle of any one of claims 1 to 11, wherein the structural helper lipid is a sterol.

13. The miRNA-based particle of claim 12 , wherein the sterol is cholesterol.

14. 14. The miRNA-based particle of any one of claims 1 to 13, wherein the PEGylated lipid is selected to stabilize the particle and protect it from opsonization before it reaches its intended target.

15. The miRNA-based particle of any one of claims 1 to 14, wherein the PEGylated lipid is a polyethylene glycol (PEG) derivative attached to a lipid moiety.

16. 16. The miRNA-based particle of any one of claims 1 to 15, wherein the PEGylated lipid is DMG-PEG2000 or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 ALC-0159 or (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (Pfizer vax), DSPE-PEG, DPPE-PEG, DOPE-PEG, DMPE-PEG with PEG lengths varying from 0.2 to 5 kDa.

17. 17. The miRNA-based particle of claim 1, wherein the ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid is in the mol% ratio range of about 40-70: 30-45: 3-16: 0.5-1.

5.

18. 17. The miRNA-based particle of any one of claims 1 to 16, wherein the ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid is in the mol% ratio range of about 45-55: 37-40: 8-12: 1-1.

7.

19. 17. The miRNA-based particle of any one of claims 1 to 16, wherein the ratio of ionizable lipid, sterol, structural helper lipid, and PEGylated lipid is in the mol% ratio range of approximately 50: 38.5: 10: 1.

5.

20. 20. The miRNA-based particle of any one of claims 1 to 19, wherein the lipid carrier further comprises any one or combination of a prodrug lipid, or a lipid carrier non-covalently functionalized with a peptide, protein, glycoprotein, polysaccharide, or combination thereof to assist in tissue-specific targeting.

21. The miRNA-based particle of claim 20, wherein the prodrug lipid includes, by way of non-limiting example, any one or combination of anti-inflammatory drugs, inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs), chemokine receptor antagonists, immune response modifiers, immunomodulators, mast cell stabilizers, T cell activation inhibitors, and TNF binding proteins.

22. 21. The miRNA-based particle of claim 20, wherein the composition of the at least four component lipid carrier comprises a prodrug lipid in a molar fraction ranging from about 1 to about 15%, offset by removing sterols and structural helper lipids to offset the addition of the prodrug lipid, wherein the fraction of the prodrug lipid ranges from about 5 to about 10%.

23. 23. The miRNA-based particle of any one of claims 1 to 22, wherein the synthetic miRNA is a miR-187-3p or miR-193b-5p inhibitor or a mimetic thereof, either alone or in combination.

24. 23. The miRNA-based particle of any one of claims 1 to 22, wherein the synthetic miRNA is any one or combination of miR-193b-5pinh, miR-187-5p, hsa-miR-7107-5p, hsa-miR-6803-5p, hsa-miR-6798-5p, hsa-miR-760, hsa-miR-6727-5p, hsa-miR-4763-3p, hsa-miR-3652, hsa-miR-885-3p, hsa-miR-766-3p, hsa-miR-3175, hsa-miR-6893-5p, hsa-miR-6875-5p, hsa-miR-6799-5p and hsa-miR-6787-5p.

25. miR-187-3p, miR-193b-5p, miR-187-5p, 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 miR-885-3p, miR-766-3p, miR-3175, miR-6893-5p, miR-6875-5p, miR-6799-5p and miR-6787-5p inhibitors or mimetics thereof, and wherein the carrier is a cell membrane-derived nanovesicle.

26. The miRNA-based particle of claim 25 , wherein the nanovesicle is derived from an isolated human cell.

27. 27. The miRNA-based particle of claim 26, wherein the isolated human cell is a bone marrow stromal cell, a myeloblast, an isolated primary human neutrophil, an eosinophil, a mast cell, a lymphoblast, a kidney endothelial cell, or an alveolar basal epithelial cell.

28. 28. The miRNA-based particle of any one of claims 23 to 27, wherein the microRNA inhibitor is a synthetic single-stranded nucleic acid of 18 to 24 monomers in length designed to specifically bind to an endogenous miRNA that would otherwise bind to its target mRNA molecule and prevent its translation.

29. The miRNA-based particle of claim 28, wherein the nucleic acid backbone comprises one or more phosphorothioates (PS) replacing natural phosphodiester (PO) bonds in naturally occurring nucleotides.

30. The miRNA-based particle of claim 28, wherein one or more ribose sugars of the nucleic acid contain a modification at the 2'-O position, the modification being 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'MOE), or 2'-fluoro (2'-F).

31. The miRNA-based particle of claim 30, wherein the conformationally constrained analog to RNA comprises a constrained 2'-O-ethyl (cEt), locked nucleic acid (LNA), or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) prepared by placing a methyl bridge from the 2'-O to 4'-C position of the ribose sugar, replacing one or more naturally occurring RNA bases.

32. 29. The miRNA-based particle of claim 28, wherein the microRNA inhibitor comprises a peptide nucleic acid and a phosphorodiamidate morpholino oligomer (PMO) as charge-neutral nucleotides.

33. 29. The miRNA-based particle of claim 28, wherein the microRNA inhibitor is conjugated at the 3' end to a lipid, peptide, or sugar moiety.

34. The miRNA-based particle of claim 28, wherein the microRNA inhibitor has a three-carbon chain spacer (C3 spacer) at the 3' or 5' end or internally.

35. 29. The miRNA-based particle of claim 28, wherein the microRNA inhibitor has 3' or 5' terminal phosphorylation.

36. 28. The miRNA-based particle of any one of claims 23 to 27, wherein the microRNA mimetic is a synthetic single- or multi-stranded nucleic acid having a length of 18 to 24 monomers that binds to the miRNA binding region of the target gene.

37. The miRNA-based particle of claim 36, wherein the nucleic acid backbone comprises one or more phosphorothioates (PS) replacing natural phosphodiester (PO) bonds in naturally occurring nucleotides.

38. The miRNA-based particle of claim 36, wherein one or more ribose sugars of the nucleic acid contain a modification at the 2'-O position, the modification being 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'MOE), or 2'-fluoro (2'-F).

39. 37. The miRNA-based particle of claim 36, wherein the nucleic acid comprises a conformationally constrained analogue to RNA.

40. The miRNA-based particle of claim 39, wherein the conformationally constrained analog to RNA comprises a constrained 2'-O-ethyl (cEt), locked nucleic acid (LNA), or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) prepared by placing a methyl bridge from the 2'-O to 4'-C position of the ribose sugar, replacing one or more naturally occurring RNA bases.

41. 37. The miRNA-based particle of claim 36, wherein the microRNA mimetic comprises peptide nucleic acids and phosphorodiamidate morpholino oligomers (PMOs) as charge-neutral nucleotides.

42. 37. The miRNA-based particle of claim 36, wherein the microRNA mimetic is conjugated at the 3' end to a lipid, peptide, or sugar moiety.

43. 37. The miRNA-based particle of claim 36, wherein the microRNA mimetic comprises two strands, a longer guide strand integrated with two or more passenger strands.

44. 37. The miRNA-based particle of claim 36, wherein the microRNA mimetic has a three-carbon chain spacer (C3 spacer) located at the 3' or 5' end, or internally on the guide or passenger strand.

45. 45. The miRNA-based particle of claim 44, wherein the guide or passenger strand has a 3' or 5' terminal phosphorylation.

46. 46. ​​A composition comprising microbubbles and a plurality of miRNA-based particles, each miRNA-based particle being as defined in any one of claims 1 to 45.

47. 47. The composition of claim 46, wherein the plurality of miRNA-based particles form a coating on the outer or inner surface of the microbubble.

48. 48. The composition of any one of claims 46-47, further comprising one or more prodrugs.

49. A medicament for the treatment of dysregulated immune responses comprising a miRNA-based particle according to any one of claims 1 to 45 or a composition according to any one of claims 46 to 48.

50. The pharmaceutical agent of claim 49, wherein the dysregulated immune response is acute respiratory distress syndrome.

51. The pharmaceutical agent of claim 49, wherein the dysregulation of the immune response is myocardial dysfunction in sepsis.

52. 50. The pharmaceutical composition of claim 49, wherein the dysregulation of the immune response is any one or a combination of acute kidney injury, acute encephalopathy, hemodynamic instability, liver dysfunction, intestinal dysfunction, and muscle dysfunction.

53. An RNA collector consisting of a synthetic or naturally occurring RNA molecule, which has multiple binding sites for target miRNAs and captures the target miRNAs.

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