WO2025181030A1 - Therapy for inflammatory lung diseases - Google Patents

Abstract

The invention refers to therapeutical combinations or compositions comprising at least two miRNAs selected from the group consisting of miR-297, miR-93, and miRlet-7b, and their use in preventing and/or treating inflammatory lung diseases such as acute respiratory distress syndrome.

Description

Therapy for inflammatory lung diseases

This application claims the benefit of European Patent Application EP24382207.9 filed on February 26th, 2024.

Technical Field

The present invention is related to the field of inflammatory diseases, in particular inflammatory lung diseases such as Acute Respiratory Distress Syndrome (ARDS).

Background Art

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). In particular, Acute Respiratory Distress Syndrome (ARDS) 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.

Currently, there are no therapies that directly reverse the pathophysiology and injury mechanisms underlying acute inflammatory lung diseases such as ARDS. This imposes the need to envision new therapeutic approaches targeting the excessive inflammatory acute response which can be translated into the clinical practice without undesired side effects and at reasonable cost.

Summary of Invention

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. In particular, 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.

The 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.

Each of the miRNAs disclosed herein (miR-297, miR-93, and miRlet-7b) 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. 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 sepsis. Also disclosed is a method for preventing and/or treating sepsis 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.

Brief description of the figures

Figure 1 : Characterization of MSCs. A) Immunophenotype of Control- and LPS-MSCs, staining of CD105, CD90 and CD44 markers. B) Representative images of MSCs differentiation towards adipogenic, osteogenic and chondrogenic lineages in vitro. C) Concentration of particles and protein in Control and LPS-MSCs media, n=3. *p < 0.05.

Figure 2: Characterization of MSCs-derived exosomes. A) Representative cryo-TEM images of C- and LPS- Exos, 12kX magnification. B) Detection of Alix, TSG101 and CD81 surface markers in C- and LPS-Exos by Western Blot. Cell lysate (C. Lys.) as negative control.

Figure 3: Effect of MSCs-derived exosomes on wound healing and cell proliferation in vitro. A) Percentage of wound closure in HPAEpiC 24 h after being treated with C- and LPS-Exos. B) Percentage of cell viability of HPAEpiC 24 h after being treated with C- and LPS-Exos, considering that non-treated cells (Control) had a 100% of cellular viability. n= 5. *p < 0.05; **p < 0.001 ; ***p < 0.001 ; ****p < 0.0001.

Figure 4: Effect of MSCs-derived exosomes on inflammation in vitro. Representation of mRNA expression of IL-113 (A), IL-6 (B) and IL-8 (C) in THP-1 cells activated with LPS and infected by P. aeruginosa and treated with C- or LPS-Exos. The relative expression of target genes was normalized to RPL37a expression; n= 6. *p < 0.05; **p < 0.001; ***p < 0.001 ; ****p < 0.0001.

Figure 5: Effect of MSCs-derived exosomes on animals' physiologic parameters. A) Body weight every 24 h, considering 100% as the starting body weight for each group. 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 7: Effect of MSCs-derived exosomes on lung tissue inflammation. mRNA expression of pro- inflammatory cytokines (IL-113 and IL-6) and chemoattractant mediators (CCL-2 and CXCL-1) in lung tissue at 72 h (mRNA expression correlated vs. GAPDH); (n= 5-10). *p < 0.05; **p < 0.001 ; ***p < 0.001. Figure 8: Effect of MSCs-derived exosomes on alveolar macrophages inflammation. mRNA expression of pro- inflammatory cytokines (IL-1 p), chemoattractant mediators (CXCL-1) and M2-phenotype markers (Arg-1 and MR) in alveolar macrophages at 72 h (mRNA expression correlated vs. GAPDH); (n= 5-10). *p < 0.05; **p < 0.01.

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.

Figure 10: miRNA expression profiling on total miRNAs isolated from C-Exos (n=2) and LPS-Exos (n=2). A) Volcano plot representing the differential miRNA expression (up- and downregulated miRNAs) between both types of exosomes (FC>1.25, FDR 10%, p < 0.01). 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 11 : Effect of target miRNA's mimics on inflammation in vitro. Representation of mRNA expression of IL-1 p (A), IL-6 (B) and IL-8 (C) in pre-activated and infected THP-1 cells transfected with miRNA's mimics separatedly or in combinantion (MIMIX). The relative expression of target genes was normalized to RPL37a expression; n= 3-5. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 12: Comparation of the effect of MSCs-derived exosomes and the one of MIMIX on inflammation in vitro. Representation of mRNA expression of IL-1 p (A), IL-6 (B) and IL-8 (C) in pre-activated and infected THP-1 cells treated with C- or LPS-Exos or transfected with miRNA's mimics in combination (MIMIX). The relative expression of target genes was normalized to RPL37a expression; n= 3-5. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 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. mRNA expression of pro-inflammatory cytokines, chemoattractant mediators and M1 and M2 phenotype markers: A) IL-1 p, B) IL-8, C) IL-6, D) TNF-o, E) CD86, and F) CD206. 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.

Figure 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. mRNA expression of pro-inflammatory cytokines, chemoattractant mediators and M1 and M2 phenotype markers: A) IL-1 p, B) IL-6, C) IL-8, and D) CD206. 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. Abbrebiations: Extracellular vesicles, EVs; Lipopolysaccharide, LPS; miRNA, miR; PA: Pseudomonas aeruginosa. Detailed description of the invention

Definitions

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more.” Unless indicated otherwise, definite articles used herein, such as "the” also include the plural of the noun.

For purposes of the present invention, 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 %.

The terms "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.

In the present disclosure, 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.

In the present disclosure, 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. As would be apparent to the skilled person, 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions* 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced. Similarity in sequence alignment is the resemblance between two sequences when compared. This fact is dependent on the identity of sequences. Similarity depicts the extent to which the residues are aligned. Hence, similar sequences contain similar properties.

The comparison of sequences and determination of percent identity or similarity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, 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. In another embodiment, 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). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res.

25(17):3389-3402. In another embodiment, 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. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

"Inflammatory diseases” as used herein are disorders and conditions that are characterized by inflammation.

"Inflammatory lung diseases” as used herein 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). "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.

The expression "treatment”, as used herein, refers to any type of therapy, which is aimed at terminating, preventing, ameliorating, or reducing the susceptibility to a clinical condition as described herein. Thus, "treatment," "treating," and their equivalent terms refer 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.

The expression "therapeutically effective amount” as used herein, 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.

The expression "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.

The term "nanoparticle” as used herein refers to a particle with at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale (1-1000 nm). As regards the shape of the nanoparticles described herein, it includes spherical, pseudospherical, spheroid, rod-shaped, polyhedral, etc. In a particular embodiment the nanoparticle is spherical, spheroid or pseudospherical.

As used herein, the term "size" refers to a characteristic physical dimension. For example, in the case of a nanoparticle that is substantially spherical, the size of the nanoparticle corresponds to the diameter of the nanoparticle. When referring to a set of nanoparticles as being of a particular size, it is contemplated that the set of nanoparticles can have a distribution of sizes around the specified size. Thus, as used herein, 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. In addition, when not perfectly spherical, the diameter is the equivalent diameter of the spherical body including the object. In preferred embodiments 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). In a preferred embodiment of the present disclosure the hydrodynamic size is determined by NTA, for example, performed with a Nanosight NS300 (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser.

The term "nanocapsule” as used herein refers to a nanoparticle as defined above which are hollow, i.e, have an internal cavity in which the desired substances may be placed.

As outlined above, 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

The present disclosure refers to miR-297, miR-93, and miRlet-7b. In particular embodiments, the miRNAs are human miRNAs, i.e. hsa-miR-297, hsa-miR-93 and hsa-let-7b. In the sense of the present disclosure, miR- 297, miR-93, and miRlet-7b are understood as the mature miRNAs. In particular embodiments, 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. However, the disclosure does not rule out the 3p arms. 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 (hsa-let-7b-3p) has accession reference MIMAT0004482.

All accession references mentioned above refer to miRbase database release 22.1 (in effect on 19.02.2024).

In preferred embodiments, 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. In particular embodiments, 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. In preferred embodiments 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. In a particular embodiment the miRNA mimic is a LNA- enhanced, double or triple strand RNA mimic. For any given miRNA, 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.

The above modifications are but some usually found in miRNA mimics. However, the state of the art included many other possible modifications that could be considered to for the MiRNA mimics. 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 referred in the sequence listing), 2'-0- methylguanosine) ribonucleotide, 2'- amino, 2'-thio and 2'-fluoro modified ribonucleotide, 2'-fluoro-cytidine, 2'- fluoro-uridine, 2'- fluoro-guanosine, 2'-fluoro-adenosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino- adenosine, 2'-amino-guanosine, 2'-amino-butyryl-pyrene-uridine, 2'-amino-adenosine, 5-iodo-uridine, ribothymidine, 5-bromo-uridine, 2-aminopurine, 5-methyl-cytidine , 5-fluoro-cytidine, and 5- fluoro-uridine, 2,6- diaminopurine, 4-thio-uridine, 5-amino-allyl-uridine, 5-(2-amino) propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine; derivatization of the 6 position, for instance 6-(2-amino)propyl uridine; derivatization of the 8-position for adenosine and/or guanosines, for instance 8- bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, a 2'-methoxy modification (e.g., 2'-0-methylcytidine, 2'-0-methylpseudouridine, 2'-0- methylguanosine, 2'-0-methyluridine, 2'-0-methyladenosine, 2'-0-methyl), PEGylation, GalNAc (N- acetylgalactosamine) modification, peptide nucleic acid (PNA), Morpholino nucleic acid, glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acids (HNA), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'- methoxycarboxymethyluraci 1, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5- oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil and/or 2,6-diaminopurine.

Other modifications may include modifications to the phosphate backbone such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. In some embodiments, 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).

The miRNA mimic may be a linear polynucleotide. The term "linear polynucleotide”, as used herein, 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.

In some embodiments, 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. In such cases, 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. Additionally, 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.

Compositions and combinations

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. In one embodiment of the first aspect, the composition comprises miR-297. In another embodiment, the composition comprises miR-93. In another embodiment, the composition comprises miRlet-7b. In another embodiment, the composition comprises miR-297 and miR-93. In another embodiment, the composition comprises miR-297 and miRlet-7b. In another embodiment, the composition comprises miR-93 and miRlet-7b. In a particular embodiment, the composition comprises miR- 297, miR-93, and miRlet-7b. In another particular embodiment, 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. In one embodiment of the second aspect, the combination comprises miR-297. In another embodiment, the combination comprises miR-93. In another embodiment, the combination comprises miRlet-7b. In another embodiment, the combination comprises miR-297 and miR-93. In another embodiment, the combination comprises miR-297 and miRlet-7b. In another embodiment, the combination comprises miR-93 and miRlet-7b. In a particular embodiment, the combination comprises miR-297, miR-93, and miRlet-7b.

In particular embodiments, 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. Preferably, the miRNAs contained in the composition of the first aspect or the combination of the second aspect are miRNA mimics. In any case, 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.

In one embodiment of the first or second aspects, 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 . In these embodiments the ratio can refer to the weight % ratio or the molar ratio.

In one embodiment, 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.

In one embodiment, 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.

In one particular embodiment, the composition of the first aspect is a pharmaceutical composition. The pharmaceutical composition usually further comprises pharmaceutically acceptable excipients and/or carriers. In a particular embodiment, the pharmaceutical composition comprises miR-297, miR-93, and miRlet-7b. In another particular embodiment, the pharmaceutical composition consists essentially of miR-297, miR-93, and miRlet-7b, together with pharmaceutically acceptable excipients and/or carriers. In another particular embodiment, 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. However, 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. In any case, 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. In a particular embodiment, 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. 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.

The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared. Examples of 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 binders, lubricants, polymers and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

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.

In preferred embodiments, 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. In particular embodiments, the carrier comprises or consists of a polymer, in particular, poly (lactic-co-glycolic acid) (PLGA). In some embodiments, the carrier can be modified with polyethylene glycol (PEG). 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.

In some embodiments, the carrier is a nanoparticle. In particular embodiments, the nanocarrier is a nanocapsule. Preferably the carrier is a nanocapsule comprising or consisting of PLGA. In particular embodiments, the PGLA nanocapsules, or a portion thereof, can be modified with polyethylene glycol (PEG). .

In one embodiment, 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.

Interestingly, the inventors have found that 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).

Usually, the pharmaceutical composition comprises a therapeutically effective amount of the miRNAs. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of PGLA nanocapsules comprising the miRNAs of the invention. In one embodiment, 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.

Although the miRNAs of the invention have shown a significant therapeutic effect when used by themselves (as sole active ingredients), the composition of the first aspect or the combination of the second aspect may comprise at least one further active ingredient. In one embodiment the further active ingredient is selected from the group consisting of AnnexinA2, growth factors (KGF, HGF, VEGF), surfactant, antibiotics, corticosteroids, antioxidants, and combinations thereof.

In one embodiment of the first aspect or the combination of the second aspect, 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.

All embodiments described in the sections above for the miRNAs, including miRNA mimics and modifications contained therein, as well as for the compositions of the first aspect and combinations of the second aspect, are applicable to the medical uses of the third to sixth aspects.

In a particular embodiment of the third to fifth aspects, 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. In particular, it 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. More in particular, the inflammatory lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, , and acute respiratory distress syndrome (ARDS). In a very particular embodiment, the wherein the inflammatory lung disease is ARDS.

In another embodiment of the third to fifth aspects, 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 acute inflammation, in particular, in the lungs. As disclosed above, the disclosure also contemplates in the sixth aspect use in the prevention and/or treatment of sepsis.

In particular embodiments, 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. In a preferred embodiment 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 m²) and the natural evasion of first-pass hepatic metabolism. Furthermore, 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. In one particular embodiment, the composition or combination is administered by pulmonary route by means of a nebulization or any other appropriate system to generate aerosols.

The 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. Thus, 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.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

1. Immunomodulatory and regenerative effect of the miRNAs

Materials and Methods:

Isolation, culture and preconditioning of Mesenchymal Stem/Stromal Cells (MSCs):

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 cm² flasks at a density of 1 ■ 10⁵cells/cm². 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.

Characterization of control and preconditioned MSCs:

Based on the standardization by the International Society for Cellular Therapy [ref], 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.

Isolated MSCs proved their ability to adhere to plastic. 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 Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich; Spain). MSCswere 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, StemPro™ 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 isolation and characterization:

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.

The quality of exosomes samples was 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.

In vitro MSC-derived exosomes therapeutic evaluation:

Scratch wound assay: 1.5 10⁵ 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 ■ 10⁴ 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).

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 ■ 10⁴ cells/well in a 96-well plate and they received 1 ■ 10⁴ of C- or LPS-Exos/cell. 24 h after, cells were washed with PBS 1X and incubated with the CellTiter 96® AQueous One Solution Reagent containing a tetrazolium compound [3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] for 1-4 h at 37°C in a humidified cell culture incubator. The amount of formazan generated was quantified by measuring the absorbance at 490 nm on a microplate reader.

Immunomodulatory assay: The Human monocytic cell line, THP-1 (ATCC, Manassas, VA, USA) was seeded at 1 .5' 10⁵/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.

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. 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.

5 h after the LPS-priming, 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. 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 HO⁴ of C- or LPS-Exos/cell. 24 h after the treatment the cells were recollected with TRizol® reagent (Invitrogen, Spain). The expression of the housekeeping RPL37a and interleukin (IL)- 113, IL-

6 and IL- 8 mRNA was evaluated by qPCR using KAPA SYBR® FAST One-Step Kit (Sigma-Aldrich, Merck) and the corresponding primers. The relative expression of target genes was normalized to RPL37a expression by the AC(t) formula.

In vivo MSC-derived exosomes therapeutic evaluation:

Animals: Male Sprague-Dawley rats (Charles River, France) weighing 200-225 g at the beginning of the experiment were used. They were kept under controlled environmental conditions (temperature, relative humidity), 12:12 light-dark cycle, enrichment material placed inside the cages and free access to food (A04 Scientific Animal Food & Engineering, Panlab) and water. The study was performed in accordance with the European Community Directive 86/609/EEC and Spanish guidelines for experimental animals approved by the Animal Research Ethics Committee of Autonomous University of Barcelona and the Animal Experimentation Committee of Generalitat de Catalunya. Development of acture lung injury (AU) pre-clinical model and exosomes administration: ALI was induced as in our previous study (13). The animals received an intratracheal instillation of 300 pL of HCI (0.1 M at pH = 1.4), and 2 h later, an intratracheal instillation of the endotoxin lipopolysaccharide (LPS; Escherichia coll 055: B5, Sigma, St. Louis, MO, USA, 30 pg/g of body weight) dissolved in 500 pL of saline under sevoflurane anesthesia. Nine hours after the endotracheal LPS administration (or saline, in the case of control animals), 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 ■ 10⁸ particles (determined by Nanosight analysis) suspended in 300 pL of sterile saline. The control groups received the same volume of saline. During the experiment, 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 (BAL) 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).

Differential and total cell count analysis: After treating the total BAL cells with ammonium chloride potassium (ACK) they were Fc blocked with CD16/CD32 antibody and then stained with the antibody mix (Table 1) at 4 °C in the dark for 30 min. The cell pool was analysed by a FACSCantoll cytometer for different cell leukocyte subsets counts measurement and classification. CountBright™ Absolute Counting Beads (Invitrogen) were added to each sample with for absolute cells count. Data was analyzed by using FlowJo. Cell subset populations were gated as followed after selecting singlets (Table 1); total myeloid cells: CD45+ and CD11b+; Classical monocytes: CD45+ CD11b+ His48+; Non-classical monocytes: CD45+ CD11b+ His48- Neutrophils: CD45+ CD11b+ His48+ (distinguished by higher side scatter, SSC- A), Natural killers (NK): CD45+ CD11b+ CD161+.

Table 1. List of conjugated antibodies used in the flow cytometry analysis.

Antibody Fluororchrome Source Reference Dilution

CD45 BioLegend 202214

CD11b Per-CP-Cy5.5 BioLegend 201820 1 :200

His48 eBioscience 15268119

CD161 APC Biolegend 205606 1 :100

Total protein collection and quantification and alveolar macrophages obtaining: The BAL was centrifuged for 5 min at 500 x g and the supernatant of the BAL was recollected for total protein measurement using the bicinchoninic acid (BCA) protein assay (Pierce™ BCA Protein Assay Kit, ThermoFisher Scientific), according to the manufacturer's protocol. The pellet was resuspended in RPM1 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 pig/ml streptomycin and was incubated at 37°C. Adhered cells correspond to alveolar macrophages, that were collected.

Histological studies: 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).

Table 2. Lung injury score system

Parameter Score per field

Haemorrhage :

Peribronchial infiltration i 0-1

Interstitial edema j

Pneumocyte hyperplasia 0-3

Intraalveolar infiltration 0-3

Inflammation in lung homogenates and alveolar macrophages: Total 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.

Exosomal miRNA isolation, microarray and quantification:

Total 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. 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 . To confirm the results from microRNA profiling, 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.

Transfection of miR mimics:

The same in vitro model of infection that has been explained above was used. The 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.

Table 3. miRNAs sequences

Exosomal miRNA Accession number mi Rbase (release 22.1)

Statistics for in vitro and in vivo studies:

All the data were analysed using GraphPad Prism 7 software and expressed as the mean ±standard error of the mean (SEM). One-way ANOVA with Newman-Keuls multiple-comparison test was applied to compare more than two groups and two-way ANOVA followed by Bonferroni's multiple comparison test was used to analyse data with more than one variable. All statistical tests conducted are two-sided and p < 0.05 is considered significant.

Results:

Characterization of isolated 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).

Regarding the activity of the MSCs, 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).

Characterization of MSCs-derived exosomes: Size distribution evaluated by Nanosight indicates that the average size of C-Exos is 169,8 nm (average of mode size 138 nm) and 181.4 nm (average of mode size 146,2 nm) for LPS-Exos. The EVs released by C- and LPS-MSC were analysed by cryo-TEM and the obtained images showed vesicles with a size and morphology compatible with exosomes (Fig. 2A). Finally, Western Blot analysis confirmed the presence of exosome-specific surface markers such as Alix, TSG101 and CD83 in both pools, sustaining that these vesicles were exosomes. In addition, the absence of calnexin, an endoplasmic reticulum marker, confirmed that the potential contamination of exosomes populations with vesicles from other cellular compartments was minimal (Fig. 2B).

Effect of MSCs-derived exosomes on wound healing and cell proliferation in vitro:

Scratch wound assays were performed to determine the effect of MSC-derived exosomes on regeneration. The LPS-Exos treatment showed a significantly accelerated wound healing process compared to the C-Exos treatment in HPAEpiC monolayer. Cells that were treated with LPS-Exos were able to regenerate the wound a 17,1 % more in comparison with non-treted cells, while the cells that received C-Exos only healed the wound a 5,2% more (Fig. 3A).

In addition, a cell viability assay was performed. In this case, both C-Exos and LPS-Exos significantly increased cell proliferation (by 5.5% and 9.4%, respectively) with respect to untreated cells (normal growth was set as 100%) (Fig. 3B). However, LPS-Exos still produced a major effect.

Effect of MSCs-derived exosomes on inflammation in P. aeruginosa-infected THP-1 in vitro:

The effect of MSCs-derived exosomes was determined in activated macrophages both types of exosomes were able to significantly decrease the mRNA expression of IL-113 (Fig. 4A), which was induced by LPS and P. aeruginosa infected macrophages at 24 h after treatment. However, in the case of IL-6 and IL-8 only LPS- Exos significantly reduced their expression in infected macrophages after 24 h of treatment (Fig. 4B and 4C).

Effect of MSCs-derived exosomes in body and lung weight in an ALI pre-clinical model:

The variation in body weight over the 72 h is similar among all groups of animals, even so, we observed that the injured animals that did not receive treatment showed a greater loss and a lower recovery of body weight. Animals that received both types of exosomes presented attenuated weight loss and a better recovery at 72 h in comparison with injured and non-treated animals (Fig. 5A). Regarding the lung weight, animals that received both C- or LPS-Exos administration significantly had reduced lung weight/body weight ratio (signal of lung damage), which was significantly increased in the injured and non-treated animals (Fig. 5B).

Effect of MSCs-derived exosomes on BAL in an ALI pre-clinical model:

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. In our pre-clinical model of ALI, 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). In line with these results, 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). Interestingly, 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).

Effect of MSCs-derived exosomes on inflammation in an ALI pre-clinical model:

We measured the expression of several pro-inflammatory chemokines and chemoattractant mediators in lung tissue homogenates. Compared to the HCI + LPS group at 72 h, animals treated with both types of exosomes had a significant decrease in the expression of IL-1 p and IL-6, as well as, in CCL2 and CXCL1 levels, whose are the mediators of recruitment of monocytes and neutrophils, respectively (Fig. 7). No changes were observed between both types of exosomes.

In relation to the attenuation of the alveolar macrophage-mediated inflammatory response, 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).

However, both types of exosomes enhanced the expression of M2 phenotype markers in the alveolar macrophages, such as Mannose Receptor (MR) and Arginase-1 (Arg-1).

Effect of MSC-derived exosomes on lung damage restoration:

To further assess the effect of MSCs-derived exosomes on the improvement of ALI, we evaluated several histological lung sections and quantified the representative hallmarks for ALI using lung injury score (LIS). Animals that received C- or LPS-Exos recovered the lung structure compared to the injured non-treated animals, but not significantly. Even though multifocal lesions were still present in the lungs of animals administered with MSC-exosomes, they showed a reduced edema, hemorrhage, intraalveolar cell infiltration and alveolar epithelial type II cells (ATI I) hyperplasia compared to HCI + LPS group, which was evidenced with a lower LIS (Fig.9). The reduction of lung lesions in treated animals was evidenced by large areas of undamaged tissue with normal architecture. miRNA expression profile of MSC-derived exosomes:

In the results explained above, we have proved that MSCs-derived exosomes induce lung tissue regeneration and modulate the inflammatory response in vitro and in vivo. Yet, we have determined that pre-activating MSCs with LPS enhances their paracrine activity and potentiates the therapeutic effect of the exosomes they secrete. Accordingly, we hypothesize that exposing MSCs to environmental stress (LPS treatment) may induce a change in MSCs, including their secretome and thus, the composition of MSC-exosomes cargo. In this direction, 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. Following probe screening and data normalization, we found 15 significantly up-regulated miRNAs in LPS-Exos compared to C-Exos (FC >1.25, FDR < 10%), represented in the volcano plot (Fig.10A). Among them, seven miRNAs were particularly relevant (miR-297, miR-466-c, miR672, miR-326, miR-93, let-7b and miR-702). Finally, we further validated the presence of only three of all the miRNAs by qPCR (miR-297, miR-93-5p and let-7b-5p) (Fig. 10B). Effect of the exosomal miRNAs on inflammation in P. aeruginosa-infected THP-1 in vitro:

To study the role of the selected exosomal miRNAs, 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).

The analysis of the expression of the mRNA of pro-inflammatory markers revealed that the combination of the 3 miRNAs together (MIMIX) has a higher effect on diminishing the expression of IL-1 p, IL-6 and IL-8 than each miRNA separately in infected macrophages (Fig. 11), suggesting that a synergic activity of administered miRNAs.

Furthermore, if we compare the effect of the 3 miRNAs together with the effect that the LPS-Exos showed in vitro, we observe that the overexpression of the MIMIX in THP-1 cells has a similar or higher effect on immunomodulating the activation of the macrophages when they are submitted to a LPS treatment and a P. aeruginosa infection (Fig. 12).

Selected miRNAs from LPS-EVs exhibit immunomodulatory effect in P. aeruginosa-infected macrophage-like cells

To evaluate the therapeutic effect of the 3 selected miRNAs, we transfected them into macrophage-like cells infected with P. aeruginosa and assessed the expression of various pro-inflammatory mediators and specific macrophage phenotype markers. The overexpression of miR-297, miR-93-5p, and let-7b-5p reduced significantly the expression of IL-1 p (p < 0.0001) (Fig. 13A) and the expression of IL-8 (p = 0.016, p = 0.0001, and p = 0.009, respectively) (Fig. 13B) induced by P. aeruginosa infection. However, the expression of 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). Regarding TNF-a expression, it was significantly decreased when the cells were transfected with let-7b-5p (p = 0.004), although transfection with miR-93-5p also reduced this cytokine's expression, but not significantly (p = 0.068) (Fig. 13D).

Furthermore, the overexpression of miR-93-5p, and let-7b-5p reduced the expression of CD86 compared to infected and non-transfected cells, but these differences were not statistically significant (p = 0.1603 and p = 0.0674, respectively) (Fig. 13E). Concerning M2 macrophages phenotype, injured cells significantly presented increased CD206 levels compared to the non-infected control (p < 0.0001). No differences were found in the expression of CD206 after transfection with each of the miRNA individually (Fig. 13F).

The combination of selected miRNAs (MIMIX) exerts a synergic immunomodulatory effect on macrophage-like cells

To further study the synergistic effect of miR-297, miR-93-5p, and let-7b-5p, THP-1 cells were also transfected with the 3 miRNAs simultaneously (MIMIX). The transfection of MIMIX significantly enhanced the therapeutic effect compared to individual miRNA transfections. Specifically, MIMIX significantly reduced IL-113 (p = 0.009, p = 0.0001, and p = 0.007 vs. miR-297, miR-93-5p, and let-7b-5p, respectively) (Fig. 13A), IL-8 (p = 0.028 and p = 0.043 vs. miR-297 and let-7b, respectively) (Fig. 13B), and IL-6 (p < 0.0001 vs. miR-297) (Fig. 13C). TNF-a expression was also significantly reduced with MIMIX compared to miR-297 alone (p = 0.017) (Fig. 13D). Notably, MIMIX transfection resulted in a significant increase in CD206 expression compared to both the infected control (p < 0.0001) and cells transfected with individual miRNAs (p = 0.0003, p < 0.0001, and p = 0.0017 vs. miR-297, miR-93-5p, and let-7b-5p, respectively) (Fig. 13F). 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.

These findings support that the synergistic action of the 3 miRNAs in the MIMIX combination is crucial to mitigating the M1 macrophage phenotype induced by P. aeruginosa and enhancing the M2 phenotype, demonstrating their therapeutic potential.

There is no synergistic effect when miR-297, miR-93-5p, and let-7b-5p are transfected in pairs: To ensure that the MIMIX is the option with better therapeutic effect, THP-1 cells were also transfected with these miRNAs in pairs.

All miRNA pair combinations (miR-297 + miR-93-5p; miR-93-5p + let-7b-5p; LPS + PA + miR-297 + let-7b-5p) significantly decreased IL-1 p (p < 0.0001 in all cases) and IL-6 expression (p = 0.0213, p = 0.0033, and p = 0.0004, respectively) compared to the infected control cells (Fig. 14A and 14B). However, the observed reduction was not greater than the individual effects of miRNAs when transfected separately. The transfection of miR-297 and let-7b together significantly reduced the expression of IL-1 p and IL-6 compared to cells transfected with miR-297 alone. Yet, this reduction did not indicate a true synergistic effect, as the combination did not outperform the individual effects of miR-93-5p or let-7b when transfected independently. In the case of IL-8, a significant reduction in expression was only observed when cells were transfected with the combination of miR-297 and miR-93-5p, compared to the infected control cells (p = 0.0113) (Fig. 14C). However, once again, this effect was not superior to the individual miRNAs or to the MIMIX condition. Pairwise transfections of the selected miRNAs did not significantly increase CD206 expression (Fig. 14D), reinforcing the fact that the three miRNAs need to be administered simultaneously to achieve a higher expression of CD206 and, consequently, a better polarization of macrophages toward an M2 phenotype.

Conclusions

Overall, these results show that the overexpression of miR-297, miR-93-5p and let-7b-5p can mimic the immunomodulatory effect of the MSC-derived exosomes. Also, they could exert regenerative effects as specified in the bibliography, as, by the moment, we have only proved regenerative effects of the MSCs- derived exosomes. These mimics could be encapsulated inside the already characterized PLGA NCs (suitable for pulmonary drug delivery), obtaining a cost-effective, widely accessible, ready-to-be-used medical product, that in the future could be easily administered (but not restricted to) to patients with ARDS.

Citation List

Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68

Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77 Altschul, et al. (1990) J. Mol. Biol. 215:403-10

Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. of Myers and Miller, CABIOS (1989).

Claims

Claims

1. A combination comprising at least two miRNAs selected from the group consisting of miR-297, miR-93, and miRlet-7b.

2. The combination according to claim 1, comprising miR-297, miR-93, and miRlet-7b.

3. The combination according to any one of claims 1-2, wherein miRlet-7b is miRlet-7b-5p, and/or miR-93 is miR-93-5p.

4. The combination according to any one of claims 1-3, wherein the miRNAs are miRNA mimics.

5. A composition comprising a combination as defined in any one of claims 1-4.

6. The composition according to the preceding claim, which is a pharmaceutical composition and further comprises pharmaceutically acceptable excipients and/or carriers.

7. The composition according to the preceding claim, wherein the carrier comprises poly (lactic-co-glycolic acid) (PLGA).

8. The composition according to the preceding claim, wherein the carrier comprises PLGA-PEG.

9. The composition according to any of the claims 6-8, wherein the carrier is a nanocapsule, in particular one having hydrodynamic size from 50 to 450 nm, more in particular about 250 nm.

10. A combination as defined in any one of claims 1-4, or a composition as defined in any one of claims 5-9, for use as a medicament.

11 . A combination as defined in any one of claims 1-4, or a composition as defined in any one of claims 5-9, for use in the prevention and/or treatment of inflammation.

12. The composition for use according to claim 11, which is for the prevention and/or treatment of an inflammatory lung disease, in particular, an inflammatory lung disease selected from the group consisting of chronic obstructive pulmonary disease (COPD), asthma, pneumonia, lung fibrosis, and acute respiratory distress syndrome (ARDS).

13. A combination as defined in any one of claims 1-4, or a composition as defined in any one of claims 5-9, for use in the prevention and/or treatment of sepsis.

14. The composition for use according to any one of claims 10-13, that is administered to the respiratory tract.

15. The composition for use according to the preceding claim, that is administered by pulmonary route, in particular by nebulization.