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WO2025181030A1 - Thérapie pour maladies pulmonaires inflammatoires - Google Patents

Thérapie pour maladies pulmonaires inflammatoires

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Publication number
WO2025181030A1
WO2025181030A1 PCT/EP2025/054944 EP2025054944W WO2025181030A1 WO 2025181030 A1 WO2025181030 A1 WO 2025181030A1 EP 2025054944 W EP2025054944 W EP 2025054944W WO 2025181030 A1 WO2025181030 A1 WO 2025181030A1
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WO
WIPO (PCT)
Prior art keywords
mir
composition
mirnas
combination
mirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/054944
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English (en)
Inventor
Aina ARENY BALAGUERÓ
Marta CAMPRUBÍ RIMBLAS
Antonio ARTIGAS RAVENTÓS
Daniel Closa Autet
Anna Roig Serra
Anna SOLÉ PORTA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fundacio Institut D'investigacio I Innovacio Parc Tauli
Centro de Investigacion Biomedica en Red CIBER
Original Assignee
Fundacio Institut D'investigacio I Innovacio Parc Tauli
Centro de Investigacion Biomedica en Red CIBER
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Filing date
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Application filed by Fundacio Institut D'investigacio I Innovacio Parc Tauli, Centro de Investigacion Biomedica en Red CIBER filed Critical Fundacio Institut D'investigacio I Innovacio Parc Tauli
Publication of WO2025181030A1 publication Critical patent/WO2025181030A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention is related to the field of inflammatory diseases, in particular inflammatory lung diseases such as Acute Respiratory Distress Syndrome (ARDS).
  • ARDS Acute Respiratory Distress Syndrome
  • Inflammation is an essential component of various common respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis, and acute respiratory distress syndrome (ARDS).
  • COPD chronic obstructive pulmonary disease
  • ARDS acute respiratory distress syndrome
  • Acute Respiratory Distress Syndrome is a common clinical syndrome of diffuse lung inflammation and oedema that leads to acute respiratory failure. ARDS accounts for 10% admissions to intensive care unit and 45% mortality in the severe category. Survivors present a reduced quality of life, turning ARDS in a public health problem.
  • ARDS presents a high patient-to-patient heterogeneity, with pneumonia and sepsis being the most common causes of this syndrome.
  • Pneumonia is the leading cause of infectious disease mortality worldwide, The World Health Organization (WHO) has urged the United Nations member states to develop national strategies to improve early recognition and treatment of sepsis.
  • WHO World Health Organization
  • the inventors have found that the microRNAs miR-297, miR-93-5p, and miRlet-7b have a surprising immunomodulatory effect.
  • the miRNAs of the invention may avoid and excessive inflammatory response while enhancing host immune function and regeneration of the injured tissue.
  • a first aspect of the invention thus refers to a composition comprising a miRNA selected from the group consisting of miR-297, miR-93, miRlet-7b, and combinations thereof.
  • the immunomodulatory effect of the above miRNAs is evidenced in the examples below.
  • the examples show that miR-297, miR-93, and miRlet-7b provide an immunomodulatory effect on THP-1 cells when they are submitted to a LPS treatment and a P. aeruginosa infection by reducing the activation of macrophages.
  • the composition of the first aspect is therefore an immunomodulatory and/or anti-inflammatory composition.
  • composition of the invention can be easily produced by conventional methods, purified and up-scaled for industrial production, without posing relevant regulatory problems (in contrast, for instance, to cell-based or exosomes-based therapies). Further advantages relate to homogenization and standardization of the medicinal product and lack of undesired side effects. Moreover, the composition of the invention also has a regenerative effect. The inventors have thus developed a new therapeutic strategy to restore immune homeostasis and regeneration of a tissue in need thereof which is a ready-to-be-used medicinal product available at any time at bedside.
  • miRNAs disclosed herein have been found to have immunomodulatory and/or anti-inflammatory activity by themselves. Additionally, it has been found that their combination is synergistic, such that the immunomodulatory effect of the combination is higher than what would have been expected from their respective activities taken individually.
  • a second aspect refers to a combination of at least two miRNAs selected from the group consisting of miR- 297, miR-93, and miRlet-7b.
  • a third aspect refers to a composition as defined in the first aspect, or a combination as defined in the second aspect, for use as a medicament. This can be rephrased as the use of a composition or combination as defined in the first or second aspects, for the preparation of a medicament. Also disclosed is a method for treating a subject in need thereof, the method comprising administering to the subject a composition or combination as defined in the first or second aspects.
  • a fourth aspect refers to a composition as defined in the first aspect, or a combination as defined in the second aspect, for use as an immunomodulatory and/or anti-inflammatory agent. This can be rephrased as the use of a composition or combination as defined in the first or second aspects, for the preparation of an immunomodulatory and/or anti-inflammatory agent.
  • a fifth aspect refers to a composition as defined in the first aspect, or a combination as defined in the second aspect, for use in the prevention and/or treatment of inflammation. This can be rephrased as the use of a composition or combination as defined in the first or second aspects, for the preparation of medicament for preventing and/or treating inflammation. Also disclosed is a method for preventing and/or treating inflammation in a subject in need thereof, the method comprising administering to the subject a composition or combination as defined in the first or second aspects.
  • a sixth aspect refers to a composition as defined in the first aspect, or a combination as defined in the second aspect, for use in the prevention and/or treatment of sepsis.
  • composition or combination as defined in the first or second aspects, for the preparation of medicament for preventing and/or treating sepsis.
  • a method for preventing and/or treating sepsis in a subject in need thereof comprising administering to the subject a composition or combination as defined in the first or second aspects.
  • Figure 1 Characterization of MSCs.
  • C) Concentration of particles and protein in Control and LPS-MSCs media, n 3. *p ⁇ 0.05.
  • Figure 2 Characterization of MSCs-derived exosomes.
  • Figure 3 Effect of MSCs-derived exosomes on wound healing and cell proliferation in vitro.
  • n 5. *p ⁇ 0.05; **p ⁇ 0.001 ; ***p ⁇ 0.001 ; ****p ⁇ 0.0001.
  • Figure 5 Effect of MSCs-derived exosomes on animals' physiologic parameters.
  • B) Ratio of lung weight/body weight measured at the end of the experiment (grams/grams; n 6-12). *p ⁇ 0.05; ***p ⁇ 0.001 ; ****p ⁇ 0.0001.
  • Figure 6 Effect of MSCs-derived exosomes on lung permeability in vivo.
  • A) Total protein concentration in pig/ml in the BAL fluid at the end point (n 6-10)
  • B) Total cell count in BAL fluid (n 6-10)
  • C) Percentage of neutrophils in BAL fluid of unilobular lung at the end point (n 6-10). *p ⁇ 0.05; ****p ⁇ 0.0001.
  • Figure 8 Effect of MSCs-derived exosomes on alveolar macrophages inflammation.
  • IL-1 p pro-inflammatory cytokines
  • CXCL-1 chemoattractant mediators
  • Arg-1 and MR M2-phenotype markers
  • Figure 9 Effect of MSCs-derived exosomes on the lung injury score evaluated a 72h. Lung injury score, evaluating haemorrhage, peribronchial infiltration, interstitial edema, pneumocyte hyperplasia, and intraalveolar infiltration, as described in Table 2.
  • B) Expression of target miRNAs in C- and LPS-Exos by qPCR (miRNA expression correlated vs. miR-16-5p expression) (n 3). **p ⁇ 0.01.
  • Figure 12 Comparation of the effect of MSCs-derived exosomes and the one of MIMIX on inflammation in vitro.
  • FIG. 13 Immunomodulatory effect of miR-297, miR-93-5p, let-7b-5p, and the 3 miRNAs simultaneously (MIMIX) on infected THP-1 cells in vitro.
  • Data are presented by the adjusted group means of the expression values and their corresponding 95% confidence intervals: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001.
  • Abbrebiations Extracellular vesicles, EVs; Lipopolysaccharide, LPS; miRNA, miR; PA: Pseudomonas aeruginosa.
  • Y axis represents -Delta Ct.
  • FIG. 14 Immunomodulatory effect of miR-297, miR-93-5p, let-7b-5p, individually, in pairs and the 3 miRNAs simultaneously (MIMIX) on infected THP-1 cells in vitro.
  • Data are presented by the adjusted group means of the expression values and their corresponding 95% confidence intervals: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • any ranges given include both the lower and the upper end-points of the range. Ranges given, such as concentrations and the like, should be considered approximate, unless specifically stated. The term “about” refers to a deviation of plus/minus 10 %, preferably plus/minus 5 %.
  • miRNA or “microRNA”, are used interchangeably in the present disclosure. miRNAs are known as non-coding small RNAs, some of which are known to regulate the expression of protein-coding genes at the post-transcriptional level. As used herein, the term “miRNA” refers to any type of micro -interfering RNA, including but not limited to, endogenous microRNA and artificial microRNA. Typically, endogenous miRNAs are small RNAs that are encoded in the genome which are capable of modulating the productive utilization of mRNA.
  • miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (noncoding RNA); instead, they are processed from primary transcripts known as pri-miRNA to short stem- loop structures called pre-miRNA and finally to functional mature miRNA.
  • a mature miRNA is a single-stranded RNA molecule of about 21-23 nucleotides in length which is complementary to a target sequence and hybridizes to the target RNA sequence to inhibit its translation.
  • miRNA may be provided in the form of a miRNA mimic, which generates a biologically equivalent effect.
  • miRNA mimics are usually modified miRNAs which contain a sequence comprising the same seed region as the original miRNA.
  • the seed sequence or seed region is a conserved heptametrical sequence which is mostly situated at positions 2-7 from the miRNA 5'-end. Even though the base pairing of miRNA and its target mRNA is not usually a perfect match, the "seed sequence” is perfectly complementary.
  • the miRNA mimic will usually have improved resistance and longer half-life while maintaining the biological effect.
  • miRNA is not limited to the mature miRNA and the miRNA mimic derived therefrom but can be used in the form of an miRNA precursor.
  • the herein disclosed miRNAs may encompass the corresponding pri-miRNAs, pre-miRNAs, as well as the mature miRNAs. This is because pri-miRNAs and pre-miRNAs may be processed to render the mature functional miRNAs.
  • Sequence identity including determination of sequence complementarity for nucleic acid sequences and sequence similarity, may be determined by sequence comparison and alignment algorithms known in the field. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and determination of percent identity or similarity between two sequences can be accomplished using a mathematical algorithm.
  • the alignment is a local alignment.
  • a preferred, non- limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • the alignment is optimized by introducing appropriate gaps and percent identity or similarity is determined over the length of the aligned sequences (i.e., a gapped alignment).
  • a gapped alignment i.e., Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res.
  • the alignment is optimized by introducing appropriate gaps and percent identity or similarity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • Inflammatory diseases as used herein are disorders and conditions that are characterized by inflammation.
  • Inflammatory lung diseases are disorders and conditions of the lung that are characterized by inflammation.
  • Non limiting Inflammatory lung diseases are chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis, and acute respiratory distress syndrome (ARDS).
  • COPD chronic obstructive pulmonary disease
  • ARDS acute respiratory distress syndrome
  • Sepsis is a condition where the body's immune system has an extreme response to an infection, causing damage to its own tissues and organs.
  • treatment refers to any type of therapy, which is aimed at terminating, preventing, ameliorating, or reducing the susceptibility to a clinical condition as described herein.
  • treatment refers to obtaining a desired pharmacologic or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effects attributable to the disorder.
  • terapéuticaally effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
  • pharmaceutically acceptable excipients or carriers refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • nanoparticle refers to a particle with at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale (1-1000 nm).
  • shape of the nanoparticles described herein it includes spherical, pseudospherical, spheroid, rod-shaped, polyhedral, etc.
  • the nanoparticle is spherical, spheroid or pseudospherical.
  • the term "size" refers to a characteristic physical dimension.
  • the size of the nanoparticle corresponds to the diameter of the nanoparticle.
  • the set of nanoparticles can have a distribution of sizes around the specified size.
  • a size of a set of nanoparticles can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
  • the diameter is the equivalent diameter of the spherical body including the object.
  • the size refers to the hydrodynamic size.
  • the hydrodynamic size can be determined by methods well known to the skilled person, including Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), field scanning electron microscopy (FESEM), or nanoparticle tracking analysis (NTA).
  • DLS Dynamic Light Scattering
  • TEM Transmission Electron Microscopy
  • FESEM field scanning electron microscopy
  • NTA nanoparticle tracking analysis
  • the hydrodynamic size is determined by NTA, for example, performed with a Nanosight NS300 (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser.
  • nanocapsule refers to a nanoparticle as defined above which are hollow, i.e, have an internal cavity in which the desired substances may be placed.
  • the disclosure provides for miRNAs and compositions thereof with immunoregulatory, antiinflammatory and regenerative effects which are particularly useful for preventing or treating inflammatory diseases, such as ARDS, and sepsis.
  • miRNAs and compositions thereof with immunoregulatory, antiinflammatory and regenerative effects which are particularly useful for preventing or treating inflammatory diseases, such as ARDS, and sepsis.
  • the present disclosure refers to miR-297, miR-93, and miRlet-7b.
  • the miRNAs are human miRNAs, i.e. hsa-miR-297, hsa-miR-93 and hsa-let-7b.
  • miR- 297, miR-93, and miRlet-7b are understood as the mature miRNAs.
  • the mature miRNA is the 5p arm, that is, miR-297-5p, miR-93-5p, and miRlet-7b-5p, or, more in particular, hsa-miR-297- 5p, hsa-miR-93-5p and hsa-let-7b-5p.
  • hsa-miR-297 has accession reference MIMAT0004450.
  • the corresponding hsa-mir-297 precursor has accession reference MI0005775.
  • hsa-miR-93-5p has accession reference: MIMAT0000093.
  • the corresponding hsa-mir-93 precursor has accession reference MI0000095.
  • the mature 3p arm (hsa-miR-93-3p) has accession reference MIMAT0004509.
  • hsa-let-7b-5p has accession reference: MIMAT0000063.
  • the corresponding hsa-let-7b precursor has accession reference MI0000063.
  • the mature 3p arm has accession reference MIMAT0004482.
  • the miRNAs are miRNA mimics. These mimics maintain the original miRNAs' biological effect and usually maintain the original seed sequence but enhanced properties, particularly better binding capacity, stability and/or specificity to the target.
  • the miRNA mimic has at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity or similarity with the original miRNA.
  • the miRNA mimic has 99-100% sequence identity with the seed region of the original miRNA.
  • Non-limiting modifications that may be contained in the miRNA mimics are selected from the group consisting of LNA (locked nucleic acids), phosphorothioate structure (in which the RNA phosphate backbone structure is partially or totally substituted with another element such as sulfur), RNA wholly or partially substituted with DNA, PNAs (peptide nucleic acids), methylation, methoxylation or fluorination (for example in the 2' hydroxyl group of RNA sugar), and combinations thereof.
  • the miRNA mimic is a LNA- enhanced, double or triple strand RNA mimic.
  • the skilled person is able to prepare miRNA mimics, including those containing any of the above modifications, by methods that are well known in the art. Additionally, miRNA mimics are commercially available from various sources.
  • Non-limiting examples of said other modifications are: deoxyadenosine 3'-monophosphate, deoxyguanosine 3'-monophosphate, deoxycytidine 3'-monophosphate, deoxythymidine 3'-monophosphate, adenosine 3'-monophosphate, guanosine 3'-monophosphate, cytidine 3'-monophosphate, uridine 3'-monophosphate, 2'-deoxy guanosine, 2'- deoxy adenosine, 2'-0-methylguanosine, 2'-0-methyl (e.g., 2'-0-methylcytidine, 2'-0-methylpseudouridine, 2'- O-methyluridine, 2'-0-methyladenosine (2prime-O-methyladenosine as
  • the oligonucleotide contains a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2-propenyl, and isopropyl).
  • a 2' lower alkyl moiety e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2-propenyl, and isopropyl.
  • the miRNA mimic may be a linear polynucleotide.
  • linear polynucleotide refers to an oligonucleotide having a 5'-end and a 3'-end. Polynucleotides forming secondary structures are not excluded from the definition of linear polynucleotide, so long as they comprise a 5' and a 3' termini.
  • the miRNA mimic may also be a circular polynucleotide.
  • the term "circular polynucleotide” as used herein refers to closed singular DNA, RNA or DNA/RNA strands, with covalently linked ends.
  • the miRNA mimic can be single- stranded, double- stranded, or triple-stranded.
  • the miRNAs are provided as miRNA precursors, for example as pre-mir (hsa-mir-297, hsa-mir-93, hsa-let-7b precursors) or as pri-miRNAs.
  • the miRNA precursor may also contain modifications selected from those defined above.
  • the disclosure also contemplates providing the miRNAs or their mimics in the form of a polynucleotide encoding said the miRNAs or miRNA mimic. Said polynucleotide is then preferrable functionally linked to a promoter that allows for transcription of the miRNA or mimic thereof.
  • the polynucleotide and the promoter may be contained in a plasmid.
  • the disclosure contemplates host cells that comprise a miRNA, a miRNA mimic, a polynucleotide, or a plasmid as described above. All of the above miRNAs, miRNA mimics or polynucleotides encoding any of them may be prepared by methods well known to the skilled person, for example by recombinant technology, or obtained from commercial sources.
  • the first aspect refers to a composition comprising a miRNA selected from the group consisting of miR-297, miR-93, miRlet-7b, and combinations thereof.
  • the composition comprises miR-297.
  • the composition comprises miR-93.
  • the composition comprises miRlet-7b.
  • the composition comprises miR-297 and miR-93.
  • the composition comprises miR-297 and miRlet-7b.
  • the composition comprises miR-93 and miRlet-7b.
  • the composition comprises miR- 297, miR-93, and miRlet-7b.
  • the composition consists essentially of miR- 297, miR-93, and miRlet-7b.
  • a second aspect refers to a combination of at least two miRNAs selected from the group consisting of miR- 297, miR-93, and miRlet-7b.
  • the combination comprises miR-297.
  • the combination comprises miR-93.
  • the combination comprises miRlet-7b.
  • the combination comprises miR-297 and miR-93.
  • the combination comprises miR-297 and miRlet-7b.
  • the combination comprises miR-93 and miRlet-7b.
  • the combination comprises miR-297, miR-93, and miRlet-7b.
  • the miRNAs contained in the composition of the first aspect or the combination of the second aspect are human, more in particular, they are mature human miRNAs, even more in particular, the 5p arm of said mature human miRNAs.
  • the miRNAs contained in the composition of the first aspect or the combination of the second aspect are miRNA mimics.
  • all embodiments described in the section above for the miRNAs, including those described for miRNA mimics and modifications contained therein, are applicable to the compositions of the first aspect and the combinations of the second aspect.
  • the proportion (ratio) of miR-297, miR-93, and miRlet-7b is 1-10:1-10:1-10, in particular, the ratio is 1-4:1-4:14, more particular the ratio is 1-2:1-2:1-2, for example, the ratio is 1 :1 :1 .
  • the ratio can refer to the weight % ratio or the molar ratio.
  • the amount of miRNAs in the composition of the first aspect or combination of the second aspect is from 50% to100% of the active agents in the composition or combination, or from 60% to 100% of the active agents in the composition or combination, or from 70% to 100% of the active agents in the composition or combination, or from 80% to 100% of the active agents in the composition or combination, or from 90% to 100% of the active agents in the composition or combination, or from 95% to 100% of the active agents in the composition or combination.
  • the amount of miRNAs in the composition of the first aspect or combination of the second aspect is at least 10%, or at least 20%, or at least 30% or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the total composition or combination. In one embodiment, the amount of miRNAs in the composition of the first aspect or combination of the second aspect is 100% of the total composition or combination. In particular embodiments the amount of miRNAs in the composition of the first aspect or combination of the second aspect is from 30 to 80 % of the total composition or combination, for example from 40 to 70% of the total composition or combination.
  • the composition of the first aspect is a pharmaceutical composition.
  • the pharmaceutical composition usually further comprises pharmaceutically acceptable excipients and/or carriers.
  • the pharmaceutical composition comprises miR-297, miR-93, and miRlet-7b.
  • the pharmaceutical composition consists essentially of miR-297, miR-93, and miRlet-7b, together with pharmaceutically acceptable excipients and/or carriers.
  • the pharmaceutical composition consists of miR-297, miR-93, and miRlet-7b, together with pharmaceutically acceptable excipients and/or carriers.
  • the election of the pharmaceutical formulation may well depend upon the route of administration. Any route of administration may be used, such as parenteral, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, transdermal, topical, oral, anal, vaginal, etc.
  • preferred routes of administration of the compositions of the present invention are selected from the group consisting of pulmonary, intratracheal, nasal, parenteral, and intraperitoneal route, preferably, by pulmonary, intratracheal, or nasal route, for which reason the pharmaceutical composition of the invention shall incorporate the suitable pharmaceutically acceptable excipients and/ or carriers.
  • Said pharmaceutical forms of administration can be prepared by conventional methods.
  • the pharmaceutical composition of the invention can be administered using suitable equipment, apparatus or device, which are known by persons skilled in the art, for example, nebulizers, pressurized metered dose inhalers, nasal sprays, dry powder inhalers catheters, cannulas, etc.
  • the pharmaceutical compositions may be in any form, including, among others, tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, aerosols, or dry powdered forms suitable for reconstitution with water or other suitable liquid medium before use, for immediate or retarded release.
  • the pharmaceutical composition of the invention is prepared in the form of an aqueous suspension or solution, in a pharmaceutically acceptable vehicle, such as a saline solution, a phosphate buffered saline solution (PBS), or any other pharmaceutically acceptable vehicle, and administered as an aerosol by means of a nebulizer to the lungs.
  • a pharmaceutically acceptable vehicle such as a saline solution, a phosphate buffered saline solution (PBS), or any other pharmaceutically acceptable vehicle
  • Nebulizers convert drug solutions or suspensions into small droplets that can be deposited in the lungs. They offer the possibility to continuously deliver relatively high doses over an extended period of time.
  • excipients and/or carriers can readily be determined by those skilled in the art according to the type of formulation being prepared.
  • suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid
  • the carrier can be organic, inorganic, or both. Suitable carriers are well known to those of skill in the art and include, without limitation, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles. Carriers may also include, saline, buffer, dextrose, water, glycerol, ethanol, and the combinations thereof. In particular embodiments, the carries may be a polycationic polymer, a vesicle, a liposome, or a nanoparticle.
  • the composition of the present disclosure comprises a carrier capable to optimize the pharmacokinetic properties of the miRNAs, including optimizing delivery to the desired tissue and reducing degradation of the miRNAs.
  • the carrier comprises or consists of a polymer, in particular, poly (lactic-co-glycolic acid) (PLGA).
  • the carrier can be modified with polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PGLA-PEG carrier may be convenient to increase circulating time in blood and also for biodistribution of the nanocapsules. Thus, PGLA pegylation may be desirable for some routes of administration.
  • the carrier additionally extends the shell-life of the product, also in the absence of cold.
  • the carrier is a nanoparticle.
  • the nanocarrier is a nanocapsule.
  • the carrier is a nanocapsule comprising or consisting of PLGA.
  • the PGLA nanocapsules, or a portion thereof can be modified with polyethylene glycol (PEG). .
  • the nanocapsules have a size from 10 to 500 nm. In another embodiment, the nanocapsules have a size from 50 to 450 nm. In a particular embodiment, the nanocapsules have a size from 50 to 350 nm, more in particular from 150 to 350 nm, for example about 250 nm. In particular embodiments, the nanocapsules have a thin PLGA shell of about 40 nm and an empty core suitable to accommodate multimodal drugs. In preferred embodiments these nanocapsules comprise or consist of PLGA and/or PGLA- PEG.
  • PLGA nanocapsules are surprisingly well suited to deliver active agents to the lungs by nebulization. It was found that nanocapsules produced aerosols of appropriate features and were resistant to nebulization. Moreover, the PLGA nanocapsules' biodistribution and accumulation in all the lung lobes make these PLGA nanocapsules excellent candidates for non-invasive pulmonary-targeted therapies. Therefore, these nanocapsules are particularly advantageous to deliver the miRNAs of the present disclosure (or any other medicament) for prevention and/or treatment of lung diseases, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis, and acute respiratory distress syndrome (ARDS).
  • COPD chronic obstructive pulmonary disease
  • ARDS acute respiratory distress syndrome
  • the pharmaceutical composition comprises a therapeutically effective amount of the miRNAs.
  • the pharmaceutical composition comprises a therapeutically effective amount of PGLA nanocapsules comprising the miRNAs of the invention.
  • the therapeutically effective amount for a human subject may be from 0.1 to 1,5 g of nanocapsules comprising the miRNAs of the invention, in particular from 0.5 to 1 g of nanocapsules comprising the miRNAs of the invention.
  • composition of the first aspect or the combination of the second aspect may comprise at least one further active ingredient.
  • the further active ingredient is selected from the group consisting of AnnexinA2, growth factors (KGF, HGF, VEGF), surfactant, antibiotics, corticosteroids, antioxidants, and combinations thereof.
  • the composition or combination is not an extracellular vesicle, in particular, it is not an extracellular vesicle derived from mesenchymal stem cells. Medical uses
  • the third to fifth aspects refer, respectively, to a composition as defined in the first aspect, or a combination as defined in the second aspect, for use as a medicament, for use as immunomodulatory and/or antiinflammatory agent, and for use in the prevention and/or treatment of inflammation.
  • the composition as defined in the first aspect, or the combination as defined in the second aspect is for use in the prevention and/or treatment of a lung disease.
  • a lung disease is for use in the prevention and/or treatment of an inflammatory lung disease, including, for example, lung infections, such as pneumonia, and lung fibrosis.
  • the inflammatory lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, , and acute respiratory distress syndrome (ARDS).
  • COPD chronic obstructive pulmonary disease
  • ARDS acute respiratory distress syndrome
  • the wherein the inflammatory lung disease is ARDS.
  • composition as defined in the first aspect, or the combination as defined in the second aspect is for use in the prevention and/or treatment of acute inflammation, in particular, in the lungs.
  • the disclosure also contemplates in the sixth aspect use in the prevention and/or treatment of sepsis.
  • the composition or combination for use according to any one of the third to sixth aspects is administered locally, more in particular, to the respiratory tract.
  • the composition or combination is administered by pulmonary route.
  • Pulmonary delivery is a worthy strategy for lung disease treatment because of the large absorption surface area of the alveolar region (up to 180 m 2 ) and the natural evasion of first-pass hepatic metabolism.
  • the deposition of therapeutic formulations directly into the lungs allows for improved targeting efficiency, higher bioavailability, and increased accumulation in the target tissue compared to the intravenous route.
  • the composition or combination is administered by pulmonary route by means of a nebulization or any other appropriate system to generate aerosols.
  • compositions or combinations of the invention can be used together with other additional drugs for any of the medical uses mentioned above.
  • Said additional drugs can form part of the same pharmaceutical composition provided by this invention or, alternatively, they can be provided in the form of a separate composition and administered concurrently, sequentially, or separately, in any order within a therapeutically effective interval, with the composition or combination of the invention.
  • the present disclosure also contemplates a composition as defined in the first aspect, or a combination as defined in the second aspect for any of the medical uses defined above, when used in combined therapy with a further active ingredient selected from AnnexinA2, growth factors (KGF, HGF, VEGF), surfactant, antibiotics, corticosteroids, antioxidants, and combinations thereof.
  • MSCs Mesenchymal Stem/Stromal Cells
  • MSCs were isolated from male Sprague-Dawley rats' femora and tibiae under sterile conditions. The marrow was flushed into a dish containing Dulbecco's Modified Eagle Medium (DMEM) (Biowest) culture medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1 % antibiotic/antimycotic and dispersed into a cell suspension. After centrifugation and filtration through 100-pm nylon mesh, cells were resuspended with DMEM supplemented medium and transferred to 75 cm 2 flasks at a density of 1 ⁇ 10 5 cells/cm 2 .
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • MSCs were precultured for 72h at 37°C and 5% CO2 and then, non-adherent cells were removed. MSCs were then washed with PBS 1X and after removal, exosome-free FBS-supplemented DMEM was added to all flasks with or without lipopolysaccharide (LPS) from Escherichia coli 055:B5 (100 ng/ml, Sigma-Aldrich, Spain), to mimic a septic environment, obtaining control MSCs (C-MSCs) and primed MSCs with LPS (LPS-MSCs). The supernatant was collected, and fresh medium was added every 2 days, maintaining the same conditions for 6 days.
  • LPS lipopolysaccharide
  • the minimal characteristics of MSCs include being plastic adherent in standard culture conditions, expressing stromal surface markers (such as CD44, CD90 and CD105) but lacking hematopoietic cell markers, and having the differentiation potential towards adipogenic, osteogenic and chondrogenic lineages in vitro.
  • stromal surface markers such as CD44, CD90 and CD105
  • the immunophenotype of the C- and LPS-MSCs was determined by immunofluorescence evaluating the expression of CD44 (Abeam, Cambridge, UK, ref. ab24504, rabbit, 1 :10), CD90 (Abeam, Cambridge, UK, ref. ab225, mouse, 1 :1000), and CD105 (Abeam, Cambridge, UK, ref. ab156756, mouse, 1 :100).
  • the following secondary antibodies were used to reveal the presence of the primary indicated antibodies: anti-rabbit antibody (Santa Cruz, USA, ref. Sc3917 - rTR, 1 :200) and anti-mouse antibody (Santa Cruz, USA), ref.
  • Ics516140 - mFITC 1 :100.
  • Cell nuclei were stained with Hoechst (Thermo Fisher, Germany) (1 :1000) and the samples were mounted with FluoromountTM Aqueous Mounting Medium (Sigma-Aldrich; Spain).
  • MSCs were counted to assess the percentage of purity using a fluorescence microscope (Eclipse E1000, Nikon) and Imaged software (Imaged 1.40 g; USA). The MSCs' capacity to differentiate into osteogenic, chondrogenic, and adipogenic lineages was also determined.
  • Confluent MSCs were cultured at 37 °C and 5% CO2 with the respective differentiation media, StemProTM Osteogenesis, Chondrogenesis and Adipogenesis Differentiation Kit (Pierce; Thermo Scientific; Spain). The media was changed every 48 h. After 7 days, cells were incubated in Oil Red O solution to stain the intracellular lipid-rich vacuoles and then, the cells were stained with Mayer's hematoxylin and rinsed with current water. After 14 days, the chondrocytes were incubated in 1% Alcian Blue solution and then removed with HOI. After 21 days, osteocytes were stained with 2% Alizarin Red S solution, and washed with distilled water. Undifferentiated MSCs were stained using the same protocols described below. The samples were mounted with DPX Mounting Media (Thermo Scientific, Spain) and imaged using Nikon Eclipse Ti microscope.
  • Exosomes were isolated by differential centrifugations. Previously collected MSCs' culture media (from C- and LPS-MSCs) was centrifuged at 2,000 x g and 10,000 x g for 10 and 30 min, respectively, at 4°C. The 10,000 x g supernatant was filtered through a 0.22 pm filter and ultracentrifuged at 110,000 x g for 2 h and 20 min. The exosome pellets were resuspended in sterile PBS 1 X, obtaining two different pools of exosomes: C-Exos and LPS-Exos.
  • the concentration and the mean size of isolated exosomes were analyzed by NanoSight LM10 machine (NanoSight) by the ICTS "NANBIOSIS” at the ICMAB-CSIC. All the parameters of the analysis were set at the same values for all samples and three 1 min-long videos were recorded in all cases. The background was measured by testing filtered PBS 1X, which revealed no signal.
  • exosomes samples were also verified by cryo-transmission electron microscopy (JEM-2011 operating at 200 kV, UAB service) and the presence of specific surface proteins (CD81, TSG101 and Alix) was detected by Western Blot.
  • Scratch wound assay 1.5 10 5 cells/well of Human Pulmonary alveolar epithelial cells (HPAEpiC) (Sciencell, Innoprot, Spain) were grown until confluence in a 12-well plate with Alveolar Epithelial Cell Medium (AEpiCM) supplemented media (Biowest) (1% penicilin/streptomicin, 1% EpiGS and 2% FBS) and a single scratch wound was made in each well with a 1000 pl pipette tip. Wells were aspirated, rinsed with PBS 1X and re-fed with fresh media followed by 1 ⁇ 10 4 of C- or LPS-Exos/cell. Scratch wounds were imaged at 0 and 24 h after the treatment by phase contrast microscope (Leica Microsystems, Germany) and wound width was assessed through measurement of pixel distance across the wound by Imaged software (Imaged 1.40 g; USA).
  • HPAEpiC Human Pulmonary alveolar epithelial cells
  • AEpiCM Al
  • Cell proliferation assay The CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Madison, Wl, USA) was used. HPAEpiC were seeded at a concentration of 1 ⁇ 10 4 cells/well in a 96-well plate and they received 1 ⁇ 10 4 of C- or LPS-Exos/cell.
  • Immunomodulatory assay The Human monocytic cell line, THP-1 (ATCC, Manassas, VA, USA) was seeded at 1 .5' 10 5 /well in a 24-well plate and exposed to phorbol myristate acetate (PMA) (100 ng/ml) diluted in RPMI 1640 medium (Biowest) supplemented with 10% inactivated FBS, 1% penicillin-streptomycin (p/s), 1% amphotericin and 0.5% L-glutamine for 48 h at 37°C. 5 h before the infection, differentiated cells were primed with fresh RPMI media containing LPS (2 pg/ml) to polarize them into a proinflammatory M1 phenotype. Negative controls received the same media without LPS.
  • PMA phorbol myristate acetate
  • P. aeruginosa (strain PAO1) was seeded on blood agar plates 3 days before the infection of the cells. 24 h later, seeding through the streak plate method was performed. Pre-inoculum was prepared through pick 6 CFU of P. aeruginosa and inoculating them in 10 ml Luria Broth (LB) medium (Thermo Fisher, Spain) in a 50 ml falcon for 16 hours at 37°C and 200 rpm. After this time, 2 ml of pre-inoculum were added to 100 ml of LB medium in a 250 ml bottle.
  • LB Luria Broth
  • the inoculum was incubated at 37°C and 200 rpm until an absorbance of >0.4 was achieved (measured with an OD of 600 nm), which corresponds to the log phase ( «109 CFU P. aeruginosa/ml). The inoculum was then centrifuged for 15 minutes at 1500 x g at RT.
  • THP-1 cells were washed twice with PBS 1X.
  • P. aeruginosa was added with fresh RPMI media with a multiplicity of infection (MOI) of 1 :50.
  • MOI multiplicity of infection
  • the cells were incubated with the bacteria for 1 h at 37°C and then, the cultures were washed 3 times with PBS 1X containing p/s 5X and were re-fed with fresh media followed by 1 HO 4 of C- or LPS-Exos/cell. 24 h after the treatment the cells were recollected with TRizol® reagent (Invitrogen, Spain).
  • TRizol® reagent Invitrogen, Spain.
  • LPS endotoxin lipopolysaccharide
  • recipient animals were administered with C- or LPS-Exos intratracheally by the trans-oral route under sevoflurane anesthesia.
  • Each animal received a single bolus of 1 ⁇ 10 8 particles (determined by Nanosight analysis) suspended in 300 pL of sterile saline.
  • the control groups received the same volume of saline.
  • the animals were continuously supervised, and body weights were recorded every 24 h.
  • Animals were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/kg) and were exsanguinated from the abdominal aorta at 72 h after the induction of ALI. The lungs were removed and weightened.
  • Bronchoalveolar lavage was either performed in unilobular and multilobular lung, while histology was done in unilobular lung and the multilobular lung was frozen for lung tissue analysis. The exact number of animals used for each analysis is indicated in the figure legends.
  • Bronchoalveolar lavage obtention and analysis The BAL was performed by washing the unilobular lung with 5 ml of saline (0.9% NaCI) (5 times).
  • BCA bicinchoninic acid
  • the unilobular lungs were embedded in paraffin and 4 pm-thick histological sections were obtained. They were stained with hematoxylin-eosin (H&E) and evaluated under bright field microscopy using a Nikon Eclipse Ti microscope. The images were evaluated using the Imaged software (Imaged 1.40 g; W. Rasband, NIH, USA).
  • the lung injury score (LIS) was quantified by three blinded investigators using Table 2. The LIS was obtained by the sum of each of the five independent variables (hemorrhage, peribronchial infiltration, interstitial edema, pneumocyte hyperplasia, and intra-alveolar infiltration) and was normalized to the number of fields evaluated. The resulting injury score was a value between zero and 10 (both inclusive).
  • RNA from lung tissue and BAL macrophages was extracted using TRizol® reagent (Thermo Fisher Scientific, Spain) and chloroformisopropanol isolation.
  • the mRNA of pro-inflammatory cytokines (IL-113 and IL- 6) chemoattractant mediators (CCL2 and CXCL-1) and M2-phenotype markers (Arginase-1 and Mannose receptor) were quantified by RT- qPCR using KAPA SYBR® FAST One-Step Kit (Sigma-Aldrich, Merck) and the corresponding primers.
  • the relative expression of target genes was normalized to the housekeeping GAPDH expression by the AC(t) formula.
  • RNA including miRNA was isolated from the MSC-exosomes using the miRNeasy Mini Kit (Invitrogen, Spain), according to the manufacturer protocol. The RNA concentrations and purity was evaluated by NanoDr op ND-1000 spectrophotometer. The miRNA profile was analyzed by miRNA 4.0 Array (IDIBAPS Genomics Platform). The RNA samples extracted from exosomes (amount not specified, the same volume of each sample) were labeled using FlashTag Biotin HSR RNA Labeling Kit (Affymetrix). Afterwards, the biotin-labeled RNA was hybridized onto GeneChip miRNA 4.0 Array for 42 h at 49°C using Affymetrix Hybridization oven.
  • Affymetrix GeneChip Using the Affymetrix GeneChip system, arrays were washed and stained in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneCHip Scanner 3000 System. The data was analyzed with Transcriptome Analysis Console. Differentially expressed miRNAs were then identified through fold change as well as p value calculated by t- test, including correction for multiple testing using the False Discovery Rate (FDR) method (FDR ⁇ 10%). The threshold set for up- and down-regulated genes was a fold change >1 .25 and a p value ⁇ 0.01 .
  • FDR False Discovery Rate
  • candidate miRNAs were quantified by RT-PCR using miRCURY LNA RNA Spike-in, RT and SYBER Green PCR Kit (BioNova cientifica S.L.). MiRNA relative expression was normalized against miR-16-5p as an exosomal endogenous control.
  • THP-1 cells were transfected, 24 h before the LPS activation and P. aeruginosa infection, with selected miRNA mimics (sequence of human origin, Table 3) using TranslT-X2® Dynamic Delivery System (BioNova cientifica S.L.) according to the manufacturer's instructions.
  • a pre-labeled 5' FAM mimic was used as a positive control for the transfection method.
  • the cells were harvested with TRizol® reagent 48 h after the transfection for subsequent RNA isolation and RT-qPCR analysis.
  • MSCs C- and LPS-MSCs were characterized. Both types of MSCs were positive for specific mesenchymal cell linages such as CD105, CD90 and CD44, showing a purity of the 92 ⁇ 5% after being cultured for 6 days ( Figure 1A). Also, they presented plastic adherence, exhibited a spindle-shaped morphology and were capable of differentiating into adipocytes, chondrocytes and osteocytes (Fig. 1 B).
  • LPS-MSCs presented a higher number of exosomes and protein in their culture media, demonstrating an enhanced paracrine activity in comparison with C-MSCs (Fig. 1C).
  • Lung permeability is one of the hallmarks of ALI, which is determined by the protein permeability and cells infiltration in the alveolar compartment, reflected in the BAL.
  • animals showed a significant increase of total proteins concentration in BAL, which was reduced in those animals that were treated with MSCs-derived exosomes (Fig. 6A).
  • the HCI + LPS group also exhibited a noteworthy increase in cell infiltration and neutrophil cell counts, which was diminished in treated animals (Fig. 6B-C).
  • differential cell count revealed a significant reduction of neutrophils proportion in animals that received LPS-Exos in comparison with the ones receiving C-Exos (Fig. 6C). No changes were observed in the total percentage of lymphocytes or macrophages in any group (data not shown).
  • LPS-Exos seem to play a pivotal role.
  • the expression of IL-1 p and CXCL-1 were augmented in HCI + LPS group and its expression was only significantly diminished in the animals that were treated with LPS-Exos (Fig. 8).
  • M2 phenotype markers in the alveolar macrophages, such as Mannose Receptor (MR) and Arginase-1 (Arg-1).
  • MSCs-derived exosomes induce lung tissue regeneration and modulate the inflammatory response in vitro and in vivo.
  • pre-activating MSCs with LPS enhances their paracrine activity and potentiates the therapeutic effect of the exosomes they secrete.
  • LPS treatment environmental stress
  • LPS treatment environmental stress
  • we analyzed the miRNA profile of C- and LPS-Exos to identify some unique miRNAs in LPS- Exos that could explain its superior immunomodulatory and regenerative activity.
  • pre-stimulated THP-1 cells were transfected with the mimics of the miRNAs (miR-297, miR-93-5p and let-7b-5p), independently, combined in pairs or all 3 in combination (MIMIX).
  • Selected miRNAs from LPS-EVs exhibit immunomodulatory effect in P. aeruginosa-infected macrophage-like cells
  • IL-6 only diminished significantly when the injured cells were transfected with miR-93-5p and let-7b-5p (p ⁇ 0.0001 in both cases) (Fig. 13C).
  • MIMIX miRNAs
  • This significant induction of CD206 expression which was absent with individual miRNA transfections, highlights a synergistic effect of the combined miRNAs in promoting macrophage polarization toward an M2 phenotype.

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Abstract

L'invention concerne des combinaisons thérapeutiques ou des compositions comprenant au moins deux miARN choisis dans le groupe constitué par miR-297, miR-93 et miRlet-7b, et leur utilisation dans la prévention et/ou le traitement de maladies pulmonaires inflammatoires telles que le syndrome de détresse respiratoire aiguë.
PCT/EP2025/054944 2024-02-26 2025-02-25 Thérapie pour maladies pulmonaires inflammatoires Pending WO2025181030A1 (fr)

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