WO2015007871A2

WO2015007871A2 - Micrornas and autoimmune-immune mediated inflammatory disease

Info

Publication number: WO2015007871A2
Application number: PCT/EP2014/065450
Authority: WO
Inventors: Alessandra FERRARO; Francesco DOTTA; Manuela Battaglia; Guido SEBASTIANI
Original assignee: FONDAZIONE UMBERTO DI MARIO ONLUS; Ospedale San Raffaele SRL
Current assignee: FONDAZIONE UMBERTO DI MARIO ONLUS; Ospedale San Raffaele SRL
Priority date: 2013-07-17
Filing date: 2014-07-17
Publication date: 2015-01-22
Anticipated expiration: 2016-01-17
Also published as: WO2015007871A3

Abstract

The present invention relates to inhibitor of miR125a-5p and/or miR 642a-5p and/or miR 155-p and/or miR 24-3p and to miRNA 26a-5p and/or a miRNA 30b-5p molecule or equivalent thereof for medical use, in particular for use in the prevention and/or treatment ofautoimmune-immune mediated inflammatory diseases. The invention also relates to a method for the identification of the above miRNAs antagonists or equivalents, a pharmaceutical composition comprising said miRNA, antagonists, miRNAs or equivalents thereof and to a diagnostic method of the above pathology and relative kit.

Description

MicroRNAs and autoimmune-immune mediated inflammatory disease

TECHNICAL FIELD

The present invention relates to inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155- 5p and/or miR 24-3p and to miR A 26a-5p and/or a miRNA 30b-5p molecule or equivalent thereof for medical use, in particular for use in the prevention and/or treatment of autoimmune- immune mediated inflammatory diseases. Further disclosed is a method for the identification of the above miRNAs antagonists or equivalents, a pharmaceutical composition comprising said miRNA inhibitors, antagonists, miRNAs or equivalents thereof. The present invention relates also to a diagnostic method of the above pathology and relative kit.

BACKGROUND ART

MicroRNAs are a class of small endogenous RNAs (19-22nucleotides long) that regulate gene expression post-transcriptionally by mRNA translational repression or decay. They specifically bind the 3 'UTR of mRNA target in a sequence specific fashion, in respect of mRNA secondary structure itself (Bartel D.P. 2009; Djuranovic S. et al 2012). They are transcribed by RNA Polymerase II and subsequently processed in their mature form by two endonucleases: Dgcr8- Drosha in the nucleus and Dicer in the cytoplasm (Kim V.N. et al 2009).

In the light of their function as gene expression regulators, microRNAs have been widely linked to several biological processes (e.g. cell cycle, apoptosis, differentiation and development) and consequently reported to drive or to be associated to alterations in several diseases. MicroRNAs have been also reported to be regulators of immune homeostasis showing specific expression fingerprint(s) also among different cell types of the immune system, including T-regulatory (Treg) cells (Allantaz F. et al 2012; Rouas R. et al 2009). Treg cells mediate immune tolerance by controlling inflammation and self-reactive Tcells by specifically suppressing Tcell responsiveness through anergy induction (Wing K. et al 2010). Recently, it has been demonstrated that Treg cell function also largely depends on microRNAs. Indeed, Dicer- or Dgcr8-deficient Treg cells show decreased levels of Foxp3 together with altered differentiation and function, thus reporting a high degree of dependence on microRNAs mediated regulation (Zhou X. et al 2008; Jeker L. T. et al 2013). More specifically, several microRNAs have been clearly linked to Treg differentiation and suppressive function. For example, miR- 155 has been demonstrated to positively regulate Treg differentiation by targeting SOCS1; in addition, miR- 155 depletion leads to impaired immune system development with a reduced number of Treg cells (Lu F. et al 2009; Kohlhaas S. et al 2009). Moreover, miR-146a inhibition has been reported to decrease the Treg suppression of Thl mediated responses by increased Statl expression, thus revealing a prominent role for this microRNA in controlling the immune response. Type 1 diabetes (TID) is an autoimmune disease mainly characterized by T-cell mediated immune recognition and destruction of beta-cells leading to altered glucose homeostasis (Van Belle TL et al 2011). As it has been previously demonstrated, the deregulation of physiological Treg cells suppressive function more than their peripheral blood frequency, may be a factor influencing the pathogenesis of human TID (Brusko T et al 2007).

The authors previously demonstrated that pancreatic draining lymph nodes (PLN) from patients with TID have Treg cells (CD4⁺CD25^bnght) epigenetically imprinted to have a Treg phenotype but that, for still unknown reasons, are functionally defective in vitro (Ferraro A. et al 2011). The authors hypothesize that in subjects with TID or with islet autoimmunity, there might be post-transcriptional regulations mediated by microRNAs in Treg cells and these regulations may affect Treg cells when residing in the PLN but not when circulating in the periphery.

SUMMARY OF THE INVENTION

Using a customized protocol for microRNA expression profiling by Real Time PCR quantification, which allows to measure up to 384 microRNAs contemporarily in as low as 200 cells, the authors analyzed microRNAs expression in sorted Treg and Tconv (T conventional) cells from PLN and peripheral blood (PB) of non-diabetic (ND) and TID patients.

In the present invention it was found that miR-125a-5p, miR-155-5p and miR-642a-5p are up- regulated in PLN of TID patients. This up regulation is specifically restricted to PLN-residing Treg of TID patients and not to PLN-residing Treg cells of non-diabetic subjects, nor to circulating-Treg cells. It was also found that miR-26a-5p and miR-30b-5p are exclusively down-regulated in the PLN-residing Treg cells of patients with TID.

Moreover it is shown that miR-24-3p is a peripheral Treg specific microRNA of patients with TID. Type 1 diabetes is an autoimmune disease in which inflammation in the target organ plays an undoubted role. Thus, Treg cells isolated from patients with other autoimmune- immune mediated inflammatory diseases may present the same miRNA profile as that described in the present invention. Such diseases include: rheumatoid arthritis, psoriasis, SLE or MS (Multiple Sclerosis), autoimmune thyroiditis.

The above described microRNA alterations may play a critical role in the contribution of TID PLN Treg cells dysfunction (miR-125a-5p, miR-642a-5p, miR-155-5p). According to the microRNA predicted target genes search, the main pathways possibly deregulated by those microRNAs include: impaired Treg migration from PLN to inflammatory sites due to disturbed chemokine receptor signaling (e.g. CCR2, TNFR), increased apoptosis rate (e.g. down- regulation of anti-apoptotic genes), disturbed Treg cell activation and differentiation (e.g. decreased expression of KLF gene family, NFAT5 or TCF7). The decreased expression of miR- 30b-5p and miR-26a-5p in Treg cells isolated from PLN of TID patients may affect cytokine signaling by specifically targeting SOCS6 and SMAD1.

Finally, the altered miRNA expression profile found in Treg cells from TID PLN was not found in Treg cells circulating in the PB of the same TID patients. In the circulating Treg cells hsa-miR-24-3p up-regulation was detected. According to predicted and already validated target, this differential expression may lead to an impaired Treg cell activation (IL2R, IL1R and TNFR) or to impaired apoptosis (NAIF1, BCL2L11). Moreover, miR-24-3p increased expression may lead to an unbalance of FOXP3 expression, since it has been previously demonstrated the specific targeting of this microRNA to FOXP3 3'UTR. The differential expression of mir-24 in circulating Treg cells from TID patients represents a novel bio marker for diagnosing, staging and following-up of patients affected by TID.

It is now widely accepted that a defect in immune regulation, at a certain stage, occurs in patients with TID and other autoimmune diseases. What is still controversial is at which level immune regulation is impaired. The authors have clearly shown that Treg cells isolated from the target organ of patients with long lasting TID (e.g. the PLN) are dysfunctional at least in vitro as compared to those isolated from the periphery (Ferraro et al Diabetes 2011). Thus, the identification of miRNA differentially expressed only in the Treg cells isolated from the PLN of TID patients open new possibilities in the development of innovative therapeutic agents, which may restore Treg cell function once are attracted in the PLN. Given the up-regulation of miR- 125a-5p, miR-642a-5p, miR-155-5p in TID PLN Treg cells, their inhibition, individual or in combination, may restore miRNAs homeostatic levels. This step may be accomplished using modified oligos or by specific exosomes delivery. Inhibitors of miR-125a-5p, miR-642a-5p, miR-155-5p may be antagonists of the expression and/or the function of said miRs.

The same approach may be applied for the other two microRNAs found to be down-regulated (miR-26a-5p and miR-30b-5p). In this case, a specific miRNA replacement or mimic using oligos mimicking microRNA sequences may be applied to restore miRNA basal levels.

Finally, the function of Treg cells isolated from the periphery of patients with TID has been reported with conflicting results. Some investigators have found defective in vitro Treg cell function. Others claim that the defect is in the responder cells, which are insensitive to Treg- mediated regulation. The authors, over the last 10 years of research, have observed a slight reduced in vitro Treg cell function and this can be ascribed to the over-expression of hsa-miR- 24-3p. Thus, mir-24-3p inhibition, by modified oligos or by specific exosomes delivery, may restore the function of circulating Treg cells leading to a better disease control. It is therefore an object of the present invention at least one compound selected from the group consisting of:

a) an inhibitor of miR 125a-5p and/or of miR 642a-5p and/or of miR 155-5p and/or of miR 24- 3p;

b) a miR A 26a-5p and/or a miR A 30b-5p molecule and/or an equivalent thereof and/or a source thereof;

c) a polynucleotide coding for the compound a) and/or b);

d) a recombinant expression vector comprising said polynucleotide;

e) a host cell genetically engineered expressing said polynucleotide,

for use in the treatment and/or prevention of an autoimmune-immune mediated inflammatory disease.

Said equivalent is preferably a mimic or an isomiR.

The autoimmune-immune mediated inflammatory disease is preferably selected from the group consisting of: type 1 diabetes, islet autoimmunity, rheumatoid arthritis, psoriasis, SLE, multiple sclerosis or autoimmune thyroiditis.

The inhibitor, the equivalent or the source as above defined is preferably a nucleic acid molecule.

Said nucleic acid molecule is preferably an R A molecule, optionally comprising at least one modified building block, preferably the modified building block is selected from nucleobase- modified building blocks, sugar-modified building blocks, backbone-modified building blocks and combinations thereof.

The inhibitor as above defined is preferably selected from:

a single-stranded or double-stranded nucleic acid molecule, an siRNA molecule, an antisense oligonucleotide, derivatives and mixtures thereof.

In the inhibitor as above defined the nucleic acid molecule has preferably sufficient complementarity to miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p to form a hybrid under physiological conditions.

Preferably, miR 125a-5p has essentially the sequence of SEQ ID NO: 1.

Preferably, miR 642a-5p has essentially the sequence of SEQ ID NO: 2.

Preferably, miR 155-5p has essentially the sequence of SEQ ID NO: 3.

Preferably, miR 24-3p has essentially the sequence of SEQ ID NO: 5.

In a preferred aspect, the inhibitor as above defined is a nucleic acid molecule comprising at least 20 nucleotides complementary to at least one sequence selected from SEQ ID NO: 1 , 2, 3 or 5, or a variant thereof. In another preferred aspect, the inhibitor as above defined is a nucleic acid molecule having at least 85 % complementarity to at least one sequence selected from SEQ ID NO: 1 , 2, 3 or 5, or a variant thereof.

In another preferred embodiment, the inhibitor as above defined comprises the sequence SEQ ID NO: 91 and/or SEQ ID NO: 92 and/or SEQ ID NO: 93 and/or SEQ ID NO: 94, more preferably said inhibitor has essentially the sequence of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 or SEQ ID NO: 94.

The inhibitor of the invention also comprises the corresponding RNA sequences of the above defined inhibitors.

In a yet preferred embodiment, the inhibitor as above defined is a nucleic acid molecule having

100% complementarity to at least one sequence selected from SEQ ID NO: 1, 2, 3 or 5.

Preferably, the inhibitor as above defined has essentially the following sequences:

5'-tcacaggttaaagggtctcaggga-3' (SEQ ID NO: 91)

5'-caagacacatttggagagggac-3'(SEQ ID NO: 92)

5 '-acccctatcacgattagcattaa-3 '(SEQ ID NO: 93)

5'-ctgttcctgctgaactgagcca-3'(SEQ ID NO: 94)

The miRNA 26a-5p and/or a miRNA 30b-5p molecule, an equivalent or the source thereof as above defined is preferably characterized by being an oligonucleotide comprising at least 20 nucleotides of any of the sequences SEQ ID NO: 6 or SEQ ID NO: 7 or a variant thereof.

Said miRNA 26a-5p and/or miRNA 30b-5p molecule, equivalent or source thereof preferably comprises the sequence of SEQ ID NO: 6 and/or SEQ ID NO: 7, more preferably has essentially the sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

Another object of the invention is a method for identifying an inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p comprising:

a) contacting a cell with a candidate molecule;

b) assessing at least one of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p activity or expression;

c) assessing at least one of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p activity or expression in the absence of the candidate molecule and d) comparing the activity or expression in step (b) with the activity or expression in step

(c), wherein a decrease between the measured activities or expression of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p in step (b) compared to step (c) indicates that the candidate molecule is an inhibitor of miR125a-5p and/or miR- 642a-5p and/or miR 155-5p and/or miR 24-3p. A further object of the invention is a method for identifying a miRNA 26a-5p and/or a miRNA 30b-5p equivalent or source thereof comprising:

a) contacting a cell with a candidate molecule;

b) assessing at least one of miRNA 26a-5p and/or a miRNA 30b-5p activity or expression; c) assessing at least one of miRNA 26a-5p and/or a miRNA 30b-5p activity or expression in the absence of the candidate molecule and

d) comparing the activity or expression in step (b) with the activity or expression in step (c), wherein an increase between the measured activities or expression of miRNA 26a- 5p and/or a miRNA 30b-5p in step (b) compared to step (c) indicates that the candidate molecule is an equivalent or source of miRNA 26a-5p and/or a miRNA 30b-5p.

In a preferred embodiment of the above methods, the cell is contacted with the candidate molecule in vitro or in vivo.

In another preferred embodiment, the candidate molecule is a protein, a peptide, a polypeptide, a polynucleotide, an oligonucleotide or a small molecule.

In the methods of the invention, assessing the expression of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or of miRNA 26a-5p and/or a miRNA 30b-5p preferably comprises Northern blotting or RT-PCR.

In the methods according to the present invention, assessing the activity of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or of miRNA 26a-5p and/or a miRNA 30b-5p comprises assessing expression or activity of a gene regulated by miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or miRNA 26a-5p and/or a miRNA 30b-5p as indicated in Table 2.

The genes indicated in table 2 have essentially one of the sequences of SEQ ID NO: 8 to SEQ ID NO: 90.

Another object of the invention is a pharmaceutical composition comprising at least one compound as above defined and excipients and/or adjuvants for use in the treatment and/or prevention of an autoimmune-immune mediated inflammatory disease.

In a preferred embodiment of the above pharmaceutical composition, the inhibitor as defined above and/or the miRNA 26a-5p and/or a miRNA 30b-5p molecule and/or an equivalent and/or a source thereof as above defined is/are comprised in a vector.

A further object of the invention is a method for the diagnosis and/or prognosis of an autoimmune-immune mediated inflammatory disease in a subject or to identify a subject at risk to develop an autoimmune-immune mediated inflammatory disease comprising the following steps: a) measuring the amount of miR 24-3p in a biological sample isolated from the subject, and b) comparing the measured amount of step a) with an appropriate control amount of miR 24-3p, wherein if the amount of miR 24-3p in the biological sample is higher than the control amount, this indicates that the subject is affected by an autoimmune-immune mediated inflammatory disease or is at risk of developing an autoimmune-immune mediated inflammatory disease. Said autoimmune-immune mediated inflammatory disease is preferably type 1 diabetes.

The biological sample is preferably a blood sample.

Another object of the invention is s kit for the diagnosis and/or prognosis of an autoimmune- immune mediated inflammatory disease comprising:

- means to detect and/or measure the amount of miR 24-3p; and optionally

- control means.

Control means can be used to compare the amount or the increase of the miR 24-3p to a value from a control sample. The value may be obtained for example, with reference to known standard, either from a normal subject or from normal population.

The means to measure the amount miR24-3p are preferably at least one probe or and/or primer. The kits according to the invention can further comprise customary auxiliaries, such as buffers, carriers, markers, etc. and/or instructions for use.

A further object of the invention is miR 24-3p for use in a method for the diagnosis and/or prognosis of an autoimmune-immune mediated inflammatory disease, wherein said autoimmune-immune mediated inflammatory disease is preferably type 1 diabetes.

Another object of the invention is a method of treatment of and/or prevention of an autoimmune-immune mediated inflammatory disease, comprising administering in a subject in need thereof an effective amount of at least one compound selected in the group consisting of: a) an inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p as above defined;

b) a miRNA 26a-5p and/or a miRNA 30b-5p molecule and/or an equivalent thereof and/or a source thereof as above defined;

c) a polynucleotide coding for the compound a) and/or b);

d) a recombinant expression vector comprising said polynucleotide;

e) a host cell genetically engineered expressing said polynucleotide,

wherein the autoimmune-immune mediated inflammatory disease is preferably selected from the group consisting of: type 1 diabetes, islet autoimmunity, rheumatoid arthritis, psoriasis, SLE, multiple sclerosis or autoimmune thyroiditis. In the present method for the diagnosis and/or prognosis of an autoimmune- immune mediated inflammatory disease in a subject or to identify a subject at risk to develop of an autoimmune- immune mediated inflammatory disease the appropriate control amount is the amount of the same miR in an age/sex matched healthy individual or from a patient affected by another disorder or pathology.

In the present invention, the subject diagnosed with an autoimmune-immune mediated inflammatory disease by the above method, may be treated with an appropriate therapy, e.g. with immune-suppressive molecules, immune tolerance inductive molecules, anti-cytokines, B- cell modulators.

In the present invention, the subject diagnosed with type 1 diabetes by the above method, may be treated with an appropriate therapy, e.g. with ciclosporin, prednisone, azathioprine, antithymocyte globulin, Tacrolimus, mycophenolate mofetil, Teplizumab, Otelixizumab, anti- CD20, anti-ILl or anti-ILIRA, low dose IL-2 and sirolimus, CTLA4Ig, DiaPep277, Proinsulin therapeutic tolerance induction mechanisms, thymic Treg cell based therapy, GLP1 analogs. In the present invention, the expression "measuring the amount" can be intended as measuring the amount or concentration or level of the respective miRNA and/or DNA thereof, preferably semi-quantitative or quantitative. The term "amount", as used in the description refers but is not limited to the absolute or relative amount of miRNA and/or DNA thereof, and any other value or parameter associated with the same or which may result from these. Methods of measuring miRNA and DNA in samples are known in the art. To measure nucleic acid levels, the cells in a test sample can be lysed, and the levels of miRNA in the lysates or in RNA purified or semi-purified from lysates can be measured by any variety of methods familiar to those in the art. Such methods include hybridization assays using detectably labeled DNA or RNA probes (i.e., Northern blotting) or quantitative or semi-quantitative RT-PCR methodologies using appropriate oligonucleotide primers. The expert in the art knows how to design appropriate primers. Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out using, for example, tissue sections, or unlysed cell suspensions, and detectably labeled ( e.g., fluorescent, or enzyme-labeled) DNA or RNA probes. Additional methods for quantifying miRNA include RNA protection assay (RPA), cDNA and oligonucleotide microarrays, representation difference analysis (RDA), differential display, EST sequence analysis, and serial analysis of gene expression (SAGE).

In the present invention, miR inhibitors are e.g. antagonists of the expression of the miR or molecules able to functionally inactivate the action of the miR on the target gene as e.g. molecules able to bind to the miR by sequence complementarity or antagomir. MiR inhibitors are preferably single-stranded, chemically modified nucleic acid molecules that regulate gene expression by binding to and inhibiting a specific mature miRNA. Preferably they are small nucleic acid molecules, e.g. 19-22 nucleotides long.

In the present invention a nucleic acid molecule that has sufficient complementarity to a miR to form a hybrid under physiological conditions is e.g. a nucleic acid molecule which is at least 85% complementary to the sequence of the above miR.

In the context of the present invention, physiological conditions are preferably a temperature range of 20-40 °C and/or atmospheric pressure of 1 and/or pH of 6-8 and/or glucose concentration of 1-20 mM and/or atmospheric oxygen concentration.

In the present invention miR inhibitors and/or antagomirs are chemically engineered oligonucleotides which are used to silence endogenous microRNAs. An antagomir is a small synthetic RNA that is perfectly complementary to the specific miRNA target with either mispairing at the cleavage site of Ago2 or some sort of base modification to inhibit Ago2 cleavage. Usually, antagomirs have some sort of modification to make it more resistant to degradation. It is unclear how antagomirization (the process by which an antagomir inhibits miRNA activity) operates, but it is believed to inhibit by irreversibly binding the miRNA. Antagomirs are used to constitutively inhibit the activity of specific miRNAs.

In the context of the present invention, the variants of the sequence of SEQ ID NO:l, 2, 3, 5 6 or 7 are preferably at least 85%, preferred 90% and more preferred 95% homologue to the SEQ ID NO: 1, 2, 3 or 5. This means that in the variants e.g. up to three nucleotides can be replaced by other nucleotides, preferably only two and more preferred only one nucleotide is replaced. The term homology is understood as identity. This means that e.g. at least 85% of the nucleotides are identical whereas the remainder of the nucleotides may be changed.

Variants may also comprise the above mentioned sequences and a modified oligonucleotide conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. A preferred moiety is a cholesterol moiety or a lipid moiety. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain variants, a conjugate group is attached directly to a modified oligonucleotide. In certain variants, a conjugate group is attached to a modified oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (SMCC), 6-aminohexanoic acid (ALEX or AHA), substituted CI -CIO alkyl, substituted or un- substituted C2-C10 alkenyl, and substituted or un- substituted C2-C10 alkynyl. In certain variants, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. In certain variants, the compound comprises a modified oligonucleotide having one or more stabilizing groups that are attached to one or both termini of a modified oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect a modified oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps. Suitable cap structures include a 4',5'-methylene nucleotide, a l-(beta-D-erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, an L- nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 3',4'-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'- 3'-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, and a non-bridging methylphosphonate moiety 5'-amino-alkyl phosphate, a 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5 '-inverted nucleotide moiety, a 5'- 5'-inverted abasic moiety, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and/or non-bridging 5'-phosphoramidate, a phosphorothioate, and a 5'-mercapto moiety.

The compounds as above defined can be provided within a delivery vehicle, optionally wherein the delivery vehicle is selected from a viral vector, microspheres, liposomes, colloidal gold particles, lipopolysaccharides, polypeptides, polysaccharides, or pegylation of viral vehicles. Preferably they are introduced into the body of the person to be treated as a nucleic acid within a vector which replicates into the host cells and produces the oligonucleotides.

As used herein, the term "host cell genetically engineered" relates to host cells which have been transduced, transformed or transfected with the polynucleotide or with the vector described previously. As representative examples of appropriate host cells, one can cite bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells such as Sf , animal cells such as CHO or COS, plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. Preferably, said host cell is an animal cell, and most preferably a human cell. The introduction of the polynucleotide or of the vector described previously into the host cell can be effected by method well known from one of skill in the art such as calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. The polynucleotide may be a vector such as for example a viral vector. Another object of the invention is a composition comprising a transformed host cell expressing the compound as above defined. The man skilled in the art is well aware of the standard methods for incorporation of a polynucleotide into a host cell, for example transfection, lipofection, electroporation, microinjection, viral infection, thermal shock, transformation after chemical permeabilisation of the membrane or cell fusion.

In the present invention, the antisense oligonucleotides are preferably antisense DNA- and/or RNA-oligonucleotides, and the derivatives of the antisense oligonucleotide are e.g. modified antisense oligonucleotide as e.g. antisense 2'-0-methyl oligo-ribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid LNA(R) bases, morpholino antisense oligonucleotides, PPAR-gamma agonists, antagomirs.

Since miRNAs target their mRNA by Watson-Crick base-pairing it is preferred that the inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p is an antisense oligonucleotide, which is complementary to the miRNA and base pairs with the miRNA in competition with the endogenous mRNA target. For the purpose of the invention, the sequence of the antisense oligonucleotide is 50% identical to the complement of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or its seed sequence, preferably 60%, 70%, 80%, 90%, or 95% and most preferably 100% identical to the complement of the miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or its seed sequence. Moreover, particularly preferred are antisense oligonucleotides which are chemically modified to improve the thermal stability of the duplex between the antisense oligonucleotide and the miRNA. Preferred chemical modifications comprise, for example, bicyclic high-affinity RNA analogues in which the furanose ring in the sugar-phosphate backbone is chemically locked in an RNA mimicking N-type conformation by the introduction of 2'-0,4'-C-methylene bridge (LNA(R)-antimiRs). Other preferred chemical modified oligonucleotides include morpholinos, 2'-0-mettiyl, 2'-0-methoxyethyl oligonucleotides and cholesterol-conjugated 2'- O-methyl modified oligonucleotides (antagomirs).

Inhibitors in context of the invention also comprise any substance that is able to inhibit miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p either by inhibiting the expression or by inhibiting the silencing function of the microRNA. Thus, any compound interfering with the microRNA pathway, for example by inhibiting the function of the proteins Pasha, Drosha, Dicer or Argonaut family proteins can be an inhibitor according to the invention. Furthermore any compound inhibiting the expression of the precursor microRNA of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p, such as, for example inhibitors of polymerase II or III are candidate inhibitor of miRNA expression. The mature miRNA also serves as a target for the design of inhibitors of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p function. Nucleic acids having perfect or mismatched complementarity to the microRNA may be used to inhibit, or to compete with the binding of the endogenous miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p with its target mRNA. How to design such miRNA inhibitors is well known in the art.

Inhibitors of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p, modified oligonucleotide complementary to a miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p, or precursor thereof, described herein as well as miRNA 26a-5p and/or a miRNA 30b-5p molecule, an equivalent or a source thereof may be prepared as a pharmaceutical composition, in particular for the treatment of autoimmune-immune mediated inflammatory diseases. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). An additional suitable administration route includes chemoembolization. In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect.

In certain embodiments, a pharmaceutical composition of the present invention is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.).

The compound as above defined may be administered as a pharmaceutical composition comprising a pharmacologically acceptable carrier and diluent. Administration may be carried out by known methods, wherein the inhibitor is introduced into the desired target cell in vitro or in vivo. Suitable administration methods include injection, viral transfer, use of liposomes, e.g. cationic liposomes, oral intake and/or dermal application.

For pharmaceutical applications, the composition may be in the form of a solution, e.g. an injectable solution, emulsion, suspension or the like. The composition may be administered in any suitable way, e.g. by injection, infusion, oral intake and/or by dermal application. The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used which is capable of increasing the efficacy of the RNA molecules to enter the target cells. Suitable examples of such carriers are liposomes. The compound as above defined is administered in a pharmaceutically effective dosage, which may be in the range of 0.001 pg/kg body weight to 1 mg/kg body weight depending on the route of administration and the type or severity of the disease. The inhibitor of the present invention may comprise a single type of inhibitor molecule or a plurality of different inhibitor molecules, e.g. a plurality of different siRNA molecules and/or antagomirs. For example, an inhibitor of miR-125a-5p, e.g. an antagomir, may be combined with an inhibitor of miR-642a-5p, e.g. an antagomir.

The miRNA 26a-5p and/or a miRNA 30b-5p molecule, an equivalent or a source thereof may be administered as a monotherapy or in combination with a further different medicament, particularly a medicament suitable for the prevention or treatment of autoimmune-immune mediated inflammatory disease as described above.

In the context of the present invention, miRNA 125a-5p, miRNA 642a-5p, miRNA 155-5p, miRNA 24-3p, miRNA 26a-5p and miRNA 30b-5p molecules are intended as e.g. miRNA precursors or a mature miRNAs.

In the context of the present invention, for a source it is intended e.g. a RNA or DNA molecule encoding for said miRNA, for said miRNA precursor, for said mature miRNA, for said miRNA mimic or equivalent.

In the context of the invention, a miRNA molecule or an equivalent or a mimic or an isomiR thereof may be a synthetic or natural or recombinant or mature or part of a mature miRNA or a human miRNA or derived from a human miRNA as further defined in the part dedicated to the general definitions. A human miRNA molecule is a miRNA molecule which is found in a human cell, tissue, organ or body fluids (i.e. endogenous human miRNA molecule). A human miRNA molecule may also be a human miRNA molecule derived from an endogenous human miRNA molecule by substitution, deletion and/or addition of a nucleotide. A miRNA molecule or an equivalent or a mimic thereof may be a single stranded or double stranded RNA molecule. Preferably a miRNA molecule or an equivalent, or a mimic thereof is from 6 to 30 nucleotides in length, preferably 12 to 30 nucleotides in length, preferably 15 to 28 nucleotides in length, more preferably said molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

A miRNA molecule or an equivalent or a mimic or an isomiR thereof may have 70 % identity over the whole mature sequence of the miR (SEQ ID NO: 6 or 7 or orthologous or orthologs thereof), preferably identity is at least 75 %, 80 %, 85 %, 90 %, 95 %, 97%, 98 %, 99% or 100 %.

An equivalent of a miRNA molecule may be an isomiR or a mimic. A precursor sequence may result in more than one isomiR sequences depending on the maturation process. A mimic is a molecule which has a similar or identical activity with a miRNA molecule. In this context a similar activity is given the same meaning as an acceptable level of an activity.

Each of the miRNA molecules or equivalents or mimics or isomiRs thereof as identified herein has an acceptable level of an activity of a given miRNA they derive from. An acceptable level of an activity is preferably that said miRNA or equivalent or mimics or isomiRs thereof is still able to exhibit an acceptable level of said activity of said miRNA. An acceptable level of an activity is preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, or more than 100%), such as 200% or 300% or more of the activity of the miRNA they derive from. A source of a miRNA molecule or a source of an equivalent of a miRNA molecule, mimic, isomiR may be any molecule which is able to induce the production of a miRNA molecule or of an equivalent thereof such as a mimic or isomiR as identified herein and which comprises a hairpin-like structure and/or a double stranded nucleic acid molecule. The presence of a hairpin- like structure, may be assessed using the RNA shapes program (Steffen P. et al 2006) using sliding windows of 80, 100 and 120 nt or more. The hairpin- like structure is usually present in a natural or endogenous source of a miRNA molecule whereas a double-stranded nucleic acid molecule is usually present in a recombinant or synthetic source of a miRNA molecule or of an equivalent thereof.

A source of a miRNA molecule or of an equivalent or a mimic or an isomiR thereof may be a single stranded optionally within a hairpin like structure, a double stranded RNA or a partially double stranded RNA or may comprise three strands, an example of which is described in WO2008/10558. As used herein partially double stranded refers to double stranded structures that also comprise single stranded structures at the 5' and/or at the 3' end. It may occur when each strand of a miRNA molecule does not have the same length. In general, such partial double stranded miRNA molecule may have less than 75% double stranded structure and more than 25% single stranded structure, or less than 50% double stranded structure and more than 50% single stranded structure, or more preferably less than 25%, 20 % or 15% double stranded structure and more than 75%, 80%, 85% single stranded structure. Alternatively, a source of a miRNA molecule or of an equivalent or a mimic or an isomiR thereof is a DNA molecule encoding a precursor of a miRNA molecule or of an equivalent or a mimic or an isomiR thereof. The invention encompasses the use of a DNA molecule encoding a precursor of a miRNA molecule that has at least 70% identity with said sequence SEQ ID NO: 6 or 7. Preferably, the identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. Preferably in this embodiment, a DNA molecule has a length of at least 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 130, 150, 200, 250, 300, 350, 400 nucleotides or more and has at least 70% identity with a DNA sequence as as SEQ ID NO: 6 or 7, variants or orthologs thereof.

The induction of the production of a given miRNA molecule or of an equivalent thereof or of a mimic or an isomiR thereof is preferably obtained when said source is introduced into a cell using one assay as defined below. Cells encompassed by the present invention are later on defined. A preferred source of a miRNA molecule or of an equivalent thereof or of a mimic or an isomiR thereof is a precursor thereof, more preferably a nucleic acid encoding said miRNA molecule or an equivalent thereof or of a mimic or an isomiR thereof. A preferred precursor is a naturally-occurring precursor. A precursor may be a synthetic or recombinant precursor.

A preferred source includes or comprises an expression construct comprising a nucleic acid, i.e. DNA encoding said precursor of said miRNA, more preferably said expression construct is a viral gene therapy vector selected from gene therapy vectors based on an adenovirus, an adeno- associated virus (AAV), a herpes virus, a pox virus and a retrovirus. A preferred viral gene therapy vector is an AAV or lentiviral vector. Other preferred vectors are oncolytic viral vectors. Such vectors are further described herein below. Alternatively, a source may be a synthetic miRNA molecule or a chemical mimic as further defined in the part dedicated to general definitions.

The detection of the presence of a miRNA molecule or of an equivalent thereof such as a mimic or an isomiR of a miRNA molecule or equivalent thereof may be carried out using any technique known to the skilled person. The assessment of the expression level or of the presence of such molecule is preferably performed using classical molecular biology techniques such as (real time Polymerase Chain Reaction) qPCR, microarrays, bead arrays, RNAse protection analysis or Northern blot analysis or cloning and sequencing. The skilled person will understand that alternatively or in combination with the quantification of a miRNA molecule or of an equivalent thereof, the quantification of a substrate of a corresponding miRNA molecule or of an equivalent thereof of any compound known to be associated with a function of said miRNA molecule or of said equivalent thereof or the quantification of a function or activity of said miRNA molecule or of said equivalent thereof using a specific assay is encompassed within the scope of the invention.

A miRNA molecule or an equivalent thereof or a mimic or an isomiR thereof may be used as such as a naked molecule, with or without chemical modifications, or encapsulated into a particle or conjugated to a moiety. A preferred composition comprises a miRNA molecule or an equivalent thereof or a mimic or an isomiR thereof encapsulated into a nanoparticle or a liposomal structure. A miRNA molecule or equivalent thereof or a mimic or an isomiR thereof may be an aptamer-miRNA hybrid. An aptamer-miRNA is defined as a miRNA linked to an RNA (or DNA) oligonucleotide, the latter adopting a conformation that targets the aptamer- miRNA hybrid molecule to a cell-surface protein (e.g. cyclic RGD peptide (cyclic arginine(R)- glycine(G)-aspartic acid(D) peptide). The aptamer-tagged miRNA can be linked to e.g. polyethylene glycol, which increases the chimera's circulating half-life (Dassie, J.P., et al. 2009).

The present invention will now be illustrated by means of non-limiting examples in reference to the following figures.

Fig.l- Expression of miR-125a-5p, miR-155-5p and miR-642a-5p in sorted Treg cells.

Taqman array quantitative RT-PCR analysis on 200 sorted Treg cells from ND PB (n=8), ND PLN (n=3), TID PB (n=4) and TID PLN (n=4).

The graphs show the up-regulation of miR-125a-5p (a), miR-155-5p (b) and miR-642a-5p (c) in PLN Treg cells of TID patients compared to PB. Results from PB and PLN of non-diabetic (ND) subjects are also reported. Expression values for each sample are reported as 2^A-dCT calculated using the mean of three different housekeeping small RNAs (RNU6, RNU44, RNU48).

2-tailed paired t-test was applied on dCt values of TID PB and TID PLN Treg (p<0,05). The different colors in TID PB and TID PLN Treg cells refer to the same donor whose samples belong to.

Fig.2- Expression of miR-125a-5p, miR-155-5p and miR-642a-5p in sorted Tconv cells.

Taqman microRNA single assay quantitative RT-PCR analysis on 200 sorted Tconv cells from ND PB (n=8), ND PLN (n=3), TID PB (n=4), TID PLN (n=4).

The graphs show the expression of miR-125a-5p (a), miR-155-5p (b) and miR-642a-5p (c) among PB and PLN Tconv cells of TID patients. Results from the same samples of non- diabetic (ND) subjects are also reported. Expression values for each sample are reported as 2^Λ- dCT calculated using the mean of three different housekeeping small RNAs (RNU6, RNU44, RNU48).

2-tailed paired t-test was applied on dCt values of TID PB and TID PLN Tconv (p<0,05). The different colors in TID PB and TID PLN Tconv cells refer to the same donor whose samples belong to.

Fig.3- Hsa-miR-146a-5p quantitative RT-PCR analysis on 200 sorted Treg (a) and Tconv cells (b) from D PB (n=8), ND PLN (n=3), TID PB (n=4), TID PLN (n=4). The graph shows statistically differential expression of miR-146a between PB and PLN Treg cells of non-diabetic subjects and between PB and PLN Treg cells of TID patients (a) but not in Tconv cells (b).

Expression values for each sample are reported as 2^A-dCT calculated using the mean of three different housekeeping small RNAs (RNU6, RNU44, RNU48).

2-tailed unpaired t-test was applied on normalized dct values of NM PB and NM PLN Treg while 2-tailed paired t-test was applied on dCt values of ND-PB vs ND-PLN and TID PB vs TID PLN Treg. (p< 0,05). Triangle's colors in TID PB and TID PLN Treg cells refer to the same donor.

Fig.4- Hsa-miR-26a-5p quantitative RT-PCR analysis on 200 sorted Treg (a) and Tconv cells (b) from ND PB (n=8), ND PLN (n=3), TID PB (n=4) and TID PLN (n=4).

The graphs show the down-regulation of miR-26a-5p in PLN Treg cells of TID patients compared to PLN Tregs from non-diabetic subjects (a), not observed in Tconv cells (b). Expression values for each sample are reported as 2^A-dCT calculated using the mean of three different housekeeping smallRNAs (RNU6, RNU44, RNU48).

2-tailed unpaired t-test was applied on dCt values of ND PLN vs TID PLN Treg and Tconv (p<0,05). The different colors in TID PB and TID PLN Treg/Tconv cells refer to the same donor whose samples belong to.

Fig.5- Hsa-miR-30b-5p quantitative RT-PCR analysis on 200 sorted Treg (a) and Tconv cells (b) from ND PB (n=8), ND PLN (n=3), T 1 D PB (n=4) and T 1 D PLN (n=4).

The graphs show the down-regulation of miR-26a-5p in PLN Treg cells of TID patients compared to PLN Tregs from non-diabetic subjects (a), not observed in Tconv cells (b).

2-tailed unpaired t-test was applied on dCt values of ND PLN vs TID PLN Treg and Tconv

(p<0,05). The different colors in TID PB and TID PLN Treg/Tconv cells refer to the same donor whose samples belong to.

Fig.6- Expression of miR-24-3p in sorted Treg and Tconv cells. Taqman array quantitative RT- PCR analysis on 200 sorted Treg (a) and Tconv cells (b) from ND PB (n=8), ND PLN (n=3), T 1 D PB (n=4) and TID PLN (n=4).

The graphs show the up-regulation of miR-24-3p in Treg cells from PB of TID patients compared to PB from non-diabetic subjects (a) but not in Tconv cells (b). 2-tailed unpaired t-test was applied on dCt values of ND PB vs T1D PB Treg and Tconv (p<0,05). The different colors in T1D PB and T1D PLN Treg/Tconv cells refer to the same donor whose samples belong to.

Fig. 7- mirl25a-5p is up-regulated in Treg cells isolated from pancreatic lymph-nodes (PLN) of diabetic NOD mice as compared to those isolated from peripheral blood (PB) or spleen (SPL), as in humans.

Fig. 8- miPvl46a is a Treg-PLN specific miRNA irrespective of disease stage also in NOD mice.

DETAILED DESCRIPTION OF THE INVENTION

Materials and Methods

Donor and sample collection

Peripheral blood (PB) was collected prior to transplantation from 4 patients with Typel diabetes (T1D) undergoing pancreas or pancreas & kidney transplant at the San Raffaele Hospital. The same patients donated pancreatic lymph node (PLN) and non-pancreatic lymph node that were collected during the surgical procedure using scissors. In addition 3 PB and 3 PLN samples were collected from non-diabetic (ND) donors who underwent pancreas surgery for non-malignant conditions. PB was also collected from 5 healthy controls.

Cell isolation and sorting

Lymphocytes were extracted from PLNs by mechanical dissociation. PB mononuclear cells were isolated by density-gradient centrifugation on Lympho prep.

200 Tconv (CD4+CD25-CD127+) and 200 Treg (CD4+CD25++CD1271ow/-) cells were sorted by Flow Cytometry for each sample and re-suspended in 5μ1 of PBS and stored at -80°C until ready to proceed with downstream reactions.

MicroRNA expression profiling

200 sorted cells re-suspended in 5μ1 of PBS were thawed on ice, centrifuged 16000g for lmin at 4°C and then lysed at 95°C for 5min. Cell lysate was momentarily placed on ice prior to Reverse Transcription (RT). Sample lysates were then processed for microRNA reverse transcription using human Megaplex RT-stem-loop microRNA Pool A v2.1 specific reaction which allowed the cDNA production using 384 specific microRNA primers. Each reverse transcription reaction was made of: 1.33μ1 Megaplex human RT-stem loop primers PoolA v2.1 (lOx), 0.33μ1 dNTPs (lOOmM), 2.50μ1Μώΐΐ8ΰΓΛε RT 50U/ul, 1.33μ1 10X RT buffer, 1.50μ1 MgCl₂ (25mM), 0.17μ1 RNAse Inhibitor 20U/ul, 0.33μ1 Nuclease free H₂0 and 5μ1 200 sorted cells lysate. The reaction underwent the following temperatures and cycles: 40 cycles (16°C x2min, 42°C xlmin, 50°C xlsec), 85°C x5min. The resulting cDNA was then subjected to specific pre-amplification reaction in order to enhance expression output using specific primers. Each reaction was composed of: 20^1Taqman preamp Master Mix 2x, 4.16 μΐ Preamp Primers Pool A v2.1, 12.5μ1 Nuclease free Η₂0 and 4.16 μΐ of microRNAs specific cDNA. The reaction underwent the following temperatures and cycles: 95°C xlOmin, 55°C x2min, 72°C x2min, 12 cycles (95°C xl5sec, 60°C x4min), 99,9°C xlOmin. Preamp product was then diluted 1 :4 by adding 0.1X Tris-EDTA pH 8.0. Pre-amplified cDNA was then stored at -20°C before performing quantitative Real- Time reaction (qPCR).

Taqman microRNA array cards (Panel A v2.1) were used in order to evaluate the expression of 384 microRNAs. The microRNA array card is made of 8 ports through which the TaqMan Universal PCR Master Mix containing the sample is loaded. The reaction mix was composed of: 450μ1 Taqman Universal PCR Master Mix II 2X, 350μ1 Nuclease free H₂0 and ΙΟΟμΙ of specific pre-amplified cDNA.

Each port of Taqman array cards was loaded with ΙΟΟμΙ reaction mix. The microRNA array card was then centrifuged twice at 1200rpm for lmin. RT-qPCR was set at the following temperature and cycles: 95°Cxl0min, 40cycles (95°Cxl5sec, 60°Cxlmin). For the following selected microRNAs, single assay evaluation using TaqMan microRNA single assay qPCR on pre-amplified products was performed: hsa-miR-125a-5p (ID002198), hsa-miR-642a-5p (ID001592), hsa-miR-155-5p (ID 002623), hsa-miR-146a-5p (ID 00468), hsa-miR-24-3p (ID 000402), hsa-miR-30b-5p (ID 000602) and hsa-miR-26a-5p (ID 000405). Mature microRNA sequences are reported in Table3.

The reaction for microRNAs single assay qPCR was composed of: ΙΟμΙ Taqman Universal PCR Master Mix II 2X, Ιμΐ MicroRNAs Single Assay Primers 20X, 6^1Nuclease free H₂0 and 2^1preamplified cDNA. Temperatures and cycles were the same applied to perform the microRNAs profiling. All the reagents were from Life Technologies. The RT and pre- amplification reactions were performed on Veriti Thermal cycler and the TaqMan Array cards and qPCR single assay evaluation using ViiA7^® instrument (all from Life Technologies).

Data and statistical analysis

Results were collected and exported using VIIA7 RUO Software v2.1 and analysed by using Expression suite software 2.1, through the 2^"ddct or 2^"dCT technique.

The measurement of the expression level of each microRNA is reported as Cycles to Threshold (Ct) of PCR, a relative value that represents the cycle number at which the amount of amplified DNA reaches the threshold level. Because of the possible technical variability between experiments the Ct were normalized (dCt) using endogenous controls (small RNAs RNU6A, R U48, RNU44). Endogenous controls were chosen based on their stability value (M-value) as the lowest among a wider set of candidates (Vandesompele J. et al 2002).

By comparing the normalized expression of two conditions it is possible to calculate the fold change. For the control sample, ddCt equals zero and 2^{" ddct} equals one (2°=1), so that the fold change in gene expression relative to the control equals one by definition. For the study sample, evaluation of 2^{" ddct} indicates the fold change in microR A expression relative to the control sample

MicroRNAs were considered differentially expressed both the following conditions were met: cutoff fold change <0.5 or >2.0 and cutoff p-value from 2-tailed paired or unpaired "Student's t test" (t-test) on dCt <0.05. Paired t-test was applied when samples were dependent, (e.g. PB and PLN from the same patient).

Undetermined values were set to a maximum Ct of 40. MicroRNAs with undetermined Ct value were excluded from the analysis as well as the microRNAs with a Ct values above 35.

Each amplification plot for every microRNAs was manually checked in order to avoid false positive Ct. Moreover, only microRNAs with a Ct below 35 and with a good efficiency amplification plot in all sample replicates were taken into consideration for fold change calculation and subsequent statistical analysis.

Correlation analysis among samples was performed using GraphPad Prism 5.0 software. Briefly, the microRNAs expression values, reported as 2^"dCT, of two different samples were plotted together. Spearman R Test was performed to evaluate the correlation level between two samples followed by p-value calculation.

Hierarchical Clustering analysis reporting the microRNAs expression values as heat map visualization was performed using Tibco Spotfire 5.0 software. The following conditions were applied for the calculation of clustering and dendogram: Euclidean clustering with complete linkage and average value calculations. Only expressed and valid microRNAs in all samples replicates were included in the hierarchical clustering analysis.

MicroRNAs bioinformatic analysis and target genes prediction

Bioinformatic computational analysis for differentially expressed microRNAs was performed using several online algorithms.

The in-silico analyses performed used the following criteria and workflow: miRBASE vl8.0 (http://www.mirbase.org/) was used to define genomic loci of microRNAs or those clustered together each one or to verify specific microRNAs nomenclature and sequences; Tarbase (http://diana.imis.athenainnovation.gr/DianaTools/index.php ?r=tarbase/index) was firstly interrogated to get the previously already validated microRNAs target genes which was compared to literature available data (Vergoulis T. et al 2012); two online microRNA target gene prediction algorithms were used to discover new potential targets: Targetscan 6.2 (http://www.targetscan.org/) and PICTAR (http://pictar.mdc-berlin.de ). The following functional categories were considered in order to include target genes predicted by the two algorithms: Treg specific function, T-cell function, apoptosis, cytokines and chemokines signaling pathways, adhesion or migration function.

Mice

11-week old normoglycemic Non Obese Diabetic (NOD) and diabetic NOD mice (Makino S. et al 1980) were bred and housed in specific pathogen-free condition. Peripheral blood (PB), spleen (SPL) and pancreatic draining lymph nodes (PLN) were collected to sort T regulatory (Treg).

Cell isolation and sorting

SPL and PLN isolated from the above mentioned mice were mashed to yield single cell suspensions. Peripheral blood was red cell lysed with water. Cells were then stained with anti- CD45 -CD4 -CD25 mAbs (BD, Becton Dickinson, San Jose, CA) [ and FACS sorted with a FACSVantage (BD) as Treg cells (i.e., CD45+CD4+CD25bright). The isolated cells isolated were suspended in 5 μΐ PBS and immediately frozen at -80°C.

Selected microRNA expression

Sorted cells re-suspended in 5μ1 of PBS were thawed on ice, centrifuged 16000g for lmin at 4°C and then lysed at 95°C for 5min. Cell lysate was momentarily placed on ice prior to Reverse Transcription (RT). Sample lysates were then processed for microRNA reverse transcription using human Megaplex RT-stem-loop microRNA Pool A v2.1 specific reaction which allowed the cDNA production using 384 specific microRNA primers. Each reverse transcription reaction was made of: 1.33μ1 Megaplex Rodent RT stem loop primers PoolA v2.1 (lOx), 0.33μ1 dNTPs (lOOmM), 2.50μ1 Multiscribe RT 50U/ul, 1.33μ1 10X RT buffer, 1.50μ1 MgC12 (25mM), 0.17μ1 RNAse Inhibitor 20U/ul, 0.33μ1 Nuclease free H20 and 5μ1 200 sorted cells lysate. The reaction underwent the following temperatures and cycles: 40 cycles (16°C x2min, 42°C xlmin, 50°C xlsec), 85°C x5min. The resulting cDNA was then subjected to specific pre-amplification reaction to enhance expression output using specific primers. Each reaction was composed of: 20.8μ1 Taqman preamp Master Mix 2x, 4.16 μΐ Preamp Primers Pool A v2.1, 12.5μ1 Nuclease free H20 and 4.16 μΐ of microRNAs specific cDNA. The reaction underwent the following temperatures and cycles: 95°C xlOmin, 55°C x2min, 72°C x2min, 12 cycles (95°C xl5sec, 60°C x4min), 99,9°C xlOmin. Preamp product was then diluted 1 :4 by adding 0.1X Tris-EDTA pH 8.0. Pre-amplified cDNA was then stored at -20°C before performing quantitative Real-Time reaction (qPCR).

For the following selected microRNAs, conserved between mouse and human, single assay evaluation using TaqMan microRNA single assay qPCR on pre-amplified products was performed: hsa-miR-125a-5p (ID002198), hsa-miR-146a-5p (ID 00468), hsa-miR-24-3p (ID 000402), hsa-miR-30b-5p (ID 000602) and hsa-miR-26a-5p (ID 000405). The reaction for microRNAs single assay qPCR was composed of: Ι ΟμΙ Taqman Universal PCR Master Mix II 2X, Ι μΐ MicroRNAs Single Assay Primers 20X, 6.5μ1 Nuclease free H20 and 2.5μ1 preamplified cDNA. Temperatures and cycles were the same applied to perform the microRNAs profiling. All the reagents were from Life Technologies. The RT and pre- amplification reactions were performed on Veriti Thermal cycler and the TaqMan Array cards and qPCR single assay evaluation using ViiA7® instrument (all from Life Technologies).

Results

T conventional cells (Tconv) (CD4⁺CD25^"CD 127⁺) and Treg cells (CD4⁺CD25⁺⁺CD127^"/low) were sorted from PB and PLN of 4 T1D patients and from PB of 8 non-diabetic (ND) subjects. Three of these 8 subjects donated also PLNs.

The expression profiling analysis of 384 microRNAs was performed on 200 sorted cells from the samples described above (Tconv or Tregs) using Taqman Array cards technology. Given the small amount of cells, the authors directly loaded the cell lysates on Reverse Transcriptase reaction; this step allowed them to retrieve the maximum efficiency. Although the method showed a high level of sensitivity and specificity, it may lead to some degree of variability on expression output, in terms of number of detected microRNAs. Therefore, to avoid data analysis artefacts the authors have exclusively taken into consideration only those microRNAs which were detected in all the replicates of the same samples.

As the authors previously reported, Treg cells residing within PLN of T1D patients are functionally defective; moreover, although the frequency of Treg cells determined by TSDR analysis (Treg specific de-methylation assay) was similar among PB and PLN of diabetic and non-diabetic subjects, the frequency of FOXP3⁺ Treg cells determined by flow cytometry was reduced only in PLN of T1D (Ferraro A. et al. 2011). Therefore, the authors' first objective was to identify those microRNAs which may mediate post-translational alterations and which may contribute to Treg dysfunction. The authors firstly focused on those microRNAs, which were differentially expressed between PB and PLN of T1D patients but not between the same samples of non-diabetic subjects. To this aim, the authors evaluated the microRNA expression profiling in the sorted cells and selected those that fulfilled the cutoff fold change and the statistical criteria.

According to such selection criteria the authors identified 3 microRNAs, namely: miR-125a-5p, miR-642a-5p and miR-155-5p. These 3 microRNAs were significantly up-regulated in PLN- residing Treg cells as compared to those from PB of TID patients (p<0.05, paired t-test on dcT). The same differential expression was not observed among samples coming from non- diabetic subjects, thus highlighting TID and PLN specific differential expression of these microRNAs (FIG.l).

To verify the possibility that the microRNA differential expression observed was not specifically restricted to Treg cells only, the authors evaluated the expression of these 3 microRNAs in sorted Tconv cells obtained from the same samples used for whole profiling, using single assay Real Time PCR. The authors did not detect any differential expression for the microRNAs of interest (FIG.2).

These data indicate that the up-regulation of miR-125a-5p, miR-155-5p and miR-642a-5p in PLN of TID patients is specifically restricted to PLN-residing Treg of TID patients and not to PLN-residing Treg cells of non-diabetic subjects, nor to circulating-Treg cells.

Among potentially interesting microRNAs with a relevant expression pattern, the authors identified also miR-146a-5p. Although the authors did not detect any difference between non- diabetic and TID samples, the authors observed the up-regulation of miR-146a-5p exclusively in PLN-residing Treg cells as compared to PB-residing for both TID patients and non-diabetic subjects (FIG.3A). Similarly to miR-125a-5p, miR-155-5p and miR-642a-5p, the authors investigated whether miR-146a-5p differential expression was specific for Treg cells. miR- 146a-5p expression was determined in Tconv sorted cells using single assay Real Time PCR and the authors did not detect the same differential expression the authors observed in Treg cells (FIG.3B). These data suggests that miR-146a-5p is a PLN-Treg associated microRNA (irrespective of the disease state). Next, the authors looked for those microRNAs whose expression was significantly different among the same type of samples but which differed by the presence of an inflammatory milieu such as in TID patients (ND-PB vs T1D-PB and ND- PLN vs T1D-PLN). Therefore, the authors firstly compared ND PLN and TID PLN Treg cells and detected the decreased expression of 2 microRNAs in Tregs from TID PLN: miR-26a-5p an miR-30b-5p (Fig.4A and 5A). In contrast no differential expression for these microRNAs was observed in Tconv cells (Fig.4B and 5B). These data demonstrate that miR-26a-5p and miR-30b-5p are down-regulated only in the PLN-residing Treg cells of patients with TID. Finally, by comparing ND-PB and T1D-PB Treg cells the authors detected miR-24-3p differentially expressed between the two set of samples. Specifically, the authors observed an up-regulation of miR-24-3p in PB Treg cells obtained from T1D patients and not from non- diabetic subjects. This differential expression, similarly to what observed for other microRNAs, was specific for Treg cells, since such difference was not detected in sorted Tconv cells (Fig.6). Thus, miR-24-3p is a peripheral Treg specific microRNA of patients with T1D. Table 1 summarizes the authors' key findings.

Table 1: key findings of the present invention

f^~~~n^~^ [77^~T [77^~1 r^~ j-y— — i -j— -j ^~ — - ^. .— ^~-| To gain insight into functional alterations that may be caused by microRNA differential expression observed in Tregs cells, the authors performed a computational prediction analysis of microRNAs target genes. To this end the authors used two different algorithms: Targetscan 6.2 and PICTAR. Both algorithms preferentially predict target genes, firstly focusing on their seed sequences evolutionary conservation among vertebrates and then on the contribution of the flanking sequences near the predicted seed match, finally assigning them a specific prediction score. Based on the results retrieved employing these two algorithms, the authors generated a list of target genes specifically belong to at least one of the following functional categories: Treg specific function, T-cell function, apoptosis, cytokine and chemokine signaling pathways and adhesion or migration functions. The authors listed the target genes selected using the two prediction algorithms reporting also those genes predicted and already experimentally validated (Table 2).

Table 2: Differentially expressed microRNAs target gene predictions

In the table are listed the microRNA target genes predicted using Targetscan6.2 and PICTAR algorithms. Official microRNA name, Chromosomic Loci (chromosome number, nucleotides portion, DNA strand) as well as miRBASE identification number for each microRNA, are reported. Each predicted target gene is identified by sequence accession number (RefSeq on http://www.ncbi.nlm.nih.gov/gene/) and official gene name. Specific reference is reported for those predicted target genes with a confirmed experimental target validation.

Table 3: MicroRNA sequences

MicroRNA Nanie/miRbase ID Mature seauence

hsa-miR-125a-5p/ MI0000469 ucccugagacccuuuaaccuguga SEQ ID NO: 1 hsa-miR-642a-5p/ MI0003657 gucccucuccaaaugugucuug SEQ ID NO: 2 hsa-miR-155-5p/ MI0000681 uuaaugcuaaucgugauaggggu SEQ ID NO: 3 hsa-miR-146a-5p/ MI0000477 ugagaacugaauuccauggguu SEQ ID NO: 4 hsa-miR-24-3p/ MI0000080 uggcucaguucagcaggaacag SEQ ID NO: 5 hsa-miR-26a-5p/ MI0000083 uucaaguaauccaggauaggcu SEQ ID NO: 6 hsa-miR-30b-5p/ MI0000441 uguaaacauccuacacucagcu SEQ ID NO: 7 Table 4: Examples of Inhibitors sequences

To test whether the miRNA- signature identified in Tregs cells sorted from PB and PLN of non- diabetic donors and in patients with T1D patients was also conserved in an autoimmune diabetes animal model (i.e., the NOD mouse), the expression levels of selected miRNAs were analyzed in Tregs cells from peripheral blood, spleen and pancreatic draining lymph nodes of normoglycemic and diabetic NOD mice.

The inventors first tested the miRNA that was up-regulated in Treg cells isolated from PLN of patients with T1D as compared to their circulating counterpart (i.e., mirl25a-5p) (table 1). The expression of miR-125a-5p in murine samples revealed the exact same expression pattern observed in human samples with an up-regulation only in Treg cells isolated from PLN of diabetic NOD mice (Figure 7).

Inventors then tested miR-146a-5p, whose expression in human samples was increased exclusively in PLN-residing Treg cells as compared to PB residing Treg cells irrespective of disease stage, thus being categorized as a PLN-Treg specific miRNA (table 1). The analysis of mouse samples showed the same expression pattern with a specific upregulation of this miRNA in Treg cells derived from PLN as compared to those isolated from PB in both normoglycemic and diabetic NOD mice, as it occurs in humans (Figure 8).

REFERENCES

- Allantaz F, Cheng DT, Bergauer T, Ravindran P, Rossier MF, Ebeling M, Badi L, Reis B, Bitter H, DAsaro M, Chiappe A, Sridhar S, Pacheco GD, Burczynski ME, Hochstrasser D, Vonderscher J, Matthes T. - Expression profiling of human immune cell subsets identifies miRNA-mRNA regulatory relationships correlated with cell type specific expression. PLoS One. 2012;7(l):e29979

- Bartel DP. MicroRNAs: target recognition and regulatory functions Cell. 2009 Jan 23;136(2):215-33.

Boissart C, Nissan X, Giraud-Triboult K, Peschanski M, Benchoua AmiR-125 potentiates early neural specification of human embryonic stem cells. Development. 2012;139(7): 1247-57.

- Brusko T, Atkinson M. Treg in type 1 diabetes. Cell Biochem Biophys. 2007;48(2-3): 165-75.- Ceppi M, Pereira PM, Dunand-Sauthier I, Barras E, Reith W, Santos MA, Pierre P. MicroRNA- 155 modulates the interleukin- 1 signaling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci U S A. 2009;106(8):2735-40

Djuranovic S, Nahvi A, Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science. 2012;336(6078):237-40

- Fayyad-Kazan H, Rouas R, Fayyad-Kazan M, Badran R, El Zein N, Lewalle P, Najar M, Hamade E, Jebbawi F, Merimi M, Romero P, Burny A, Badran B, Martiat P MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and impact of differentially expressed microRNAs on expression of two genes essential to their function.J Biol Chem. 2012. 287(22): 18584.

- Ferraro A, Socci C, Stabilini A, Valle A, Monti P, Piemonti L, Nano R, Olek S, Maffi P, Scavini M, Secchi A, Staudacher C, Bonifacio E, Battaglia M. Expansion of Thl7 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes. Diabetes. 2011;60(11):2903-13.

- Guo S, Lu J, Schlanger R, Zhang H, Wang JY, Fox MC, Purton LE, Fleming HH, Cobb B, Merkenschlager M, Golub TR, Scadden DT. MicroRNA miR-125a controls hematopoietic stem cell number. Proc Natl Acad Sci U S A. 2010;107(32): 14229-34.

Hafher M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, Rothballer A, Ascano M Jr, Jungkamp AC, Munschauer M, Ulrich A, Wardle GS, Dewell S, Zavolan M, Tuschl T. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR- CLIP. Cell. 2010;141(1):129-41.

- Hou J, Wang P, Lin L, Liu X, Ma F, An H, Wang Z, Cao X. MicroRNA- 146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAKI, and IRAK2. J Immunol. 2009;183(3):2150-8.

Jeker LT, Zhou X, Blelloch R, Bluestone JA. DGCR8-Mediated Production of Canonical Micrornas Is Critical for Regulatory T Cell Function and Stability PLoS One. 2013;8(5):e66282.

- Jiang S, Zhang HW, Lu MH, He XH, Li Y, Gu H, Liu MF, Wang ED. MicroRNA-155 functions as an OncomiR in breast cancer by targeting the suppressor of cytokinesignalingl gene. Cancer Res. 2010;70(8):3119-27.

- Jurkin J, Schichl YM, Koeffel R, Bauer T, Richter S, Konradi S, Gesslbauer B, Strobl H. miR- 146a is differentially expressed by myeloid dendritic cell subsets and desensitizes cells to TLR2-dependent activation. J Immunol. 2010;184(9):4955-65

- Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol.

2009;10(2): 126-39. - Kohlhaas S, Garden OA, Scudamore C, Turner M, Okkenhaug K, Vigorito E. Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory T cells. J Immunol. 2009;182(5):2578-82.

- Krek A, Griin D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus C, Stoffel M, Rajewsky N. Combinatorial microRNA target predictions. Nat Genet.

2005;37(5):495-500. (PICTAR)

Lai A, Navarro F, Maher CA, Maliszewski LE, Yan N, O'Day E, Chowdhury D, Dykxhoorn DM, Tsai P, Hofmann O, Becker KG, Gorospe M, Hide W, Lieberman J. miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to "seedless" 3TJTR microRNA recognition elements. Mol Cell. 2009;35(5):610-25.

- Lewis BP, Burge CB, Bartel DP Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets- Cell. 2005;120(l): 15-20. (TARGETSCAN)

- Louafi F, Martinez-Nunez RT, Sanchez-Eisner T. MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor- {beta} . J BiolChem.

2010;285(53):41328-36.

Lu LF, Thai TH, Calado DP, Chaudhry A, Kubo M, Tanaka K, Loeb GB, Lee H, Yoshimura A, Rajewsky K, Rudensky AY. Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity. 2009;30(1):80-91.

- Luzi E, Marini F, Sala SC, Tognarini I, Galli G, Brandi ML. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res. 2008;23(2):287-95.

Makino S, Kunimoto K, Muraoka Y, Mizushima Y, Katagiri K, Tokino Y. Breeding of a non- obese, diabetic strain of mice. Jikken Dobutsu. 1980 29(1):1-13.

- McCallie B, Schoolcraft WB, Katz-Jaffe MG. Aberration of blastocyst microRNA expression is associated with human infertility. Fertil Steril. 2010;93(7):2374-82.

- O'Connell RM, Rao DS, Chaudhuri AA, Boldin MP, Taganov KD, Nicoll J, Paquette RL, Baltimore D. Sustained expression of microRNA- 155 in hematopoietic stem cells causes a myeloproliferative disorder. J Exp Med. 2008;205(3):585-94.

- Rouas R, Fayyad-Kazan H, El Zein N, Lewalle P, Rothe F, Simion A, Akl H, Mourtada M, El Rifai M, Burny A, Romero P, Martiat P, Badran B. Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression. Eur J Immunol. 2009;39(6): 1608-18.

Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008;455(7209):58-63.

Taganov KD, Boldin MP, Chang KJ, Baltimore D NF-kappaB-dependent induction of microRNA miR-146, an inhibit or targeted to signaling proteins of innate immune responses. ProcNatlAcad Sci U S A. 2006;103(33):12481-6.

- Van Belle TL, Coppieters KT, von Herrath MG. Type 1 diabetes: etiology, immunology, and therapeutic strategies. Physiol Rev. 2011;91(1):79-118.

- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F.

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002 18;3(7)

- Vergoulis T, Vlachos IS, Alexiou P, Georgakilas G, Maragkakis M, Reczko M, Gerangelos S, Koziris N, Dalamagas T, Hatzigeorgiou AG. TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support. Nucleic Acids Res. 2012; 40(Database issue):D222- 9.

- Xu G, Fewell C, Taylor C, Deng N, Hedges D, Wang X, Zhang K, Lacey M, Zhang H, Yin Q, Cameron J, Lin Z, Zhu D, Flemington EK. Transcriptome and targetome analysis in MIR155 expressing cells using RNA-seq. RNA. 2010; 16(8): 1610-22.

- Zhao X, Tang Y, Qu B, Cui H, Wang S, Wang L, Luo X, Huang X, Li J, Chen S, Shen N.

MicroRNA- 125 a contributes to elevated inflammatory chemokine RANTES levels via targeting KLF13 in systemic lupus erythematosus. Arthritis Rheum. 2010;62(l l):3425-35.

- Zhou X, Jeker LT, Fife BT, Zhu S, Anderson MS, McManus MT, Bluestone JA. Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity J Exp Med. 2008;205(9):1983-91.

Claims

1- At least one compound selected from the group consisting of:

a) an inhibitor of miR 125a-5p and/or of miR 642a-5p and/or of miR 155-5p and/or of miR 24-3p;

b) a miRNA 26a-5p and/or a miRNA 30b-5p molecule and/or an equivalent thereof and/or a source thereof;

c) a polynucleotide coding for the compound a) and/or b);

d) a recombinant expression vector comprising said polynucleotide;

e) a host cell genetically engineered expressing said polynucleotide,

2- The compound for use according to claim 1, wherein the autoimmune-immune mediated inflammatory disease is selected from the group consisting of: type 1 diabetes, islet autoimmunity, rheumatoid arthritis, psoriasis, SLE, multiple sclerosis or autoimmune thyroiditis.

3- The inhibitor, the equivalent or the source thereof for use according to claim 1 or 2, being a nucleic acid molecule.

4- The inhibitor according to any of claim 1-3, wherein said inhibitor is selected from:

5- The inhibitor according to any one of claims 1-4 wherein the nucleic acid molecule has sufficient complementarity to miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p to form a hybrid under physiological conditions.

6- The inhibitor according to any one of claims 1 to 5 being a nucleic acid molecule comprising at least 20 nucleotides complementary to at least one sequence selected from

SEQ ID NO: 1, 2, 3 or 5, or a variant thereof.

7- The inhibitor according to any one of previous claim being a nucleic acid molecule having at least 85 % complementarity to at least one sequence selected from SEQ ID NO: 1, 2, 3 or 5, or a variant thereof.

8- The inhibitor according to any one of previous claim, comprising the sequence SEQ ID NO:

91 and/or SEQ ID NO: 92 and/or SEQ ID NO: 93 and/or SEQ ID NO: 94, preferably said inhibitor having essentially the sequence of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 or SEQ ID NO: 94. 9- The miRNA 26a-5p molecule, miR A 30b-5p molecule, equivalent or source thereof according to any one of claims 1-3, characterized by being an oligonucleotide comprising at least 20 nucleotides of any of the sequences SEQ ID NO: 6 or SEQ ID NO: 7 or a variant thereof.

10- A method for identifying an inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155- 5p and/or miR 24-3p comprising:

e) contacting a cell with a candidate molecule;

f assessing at least one of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p activity or expression;

g) assessing at least one of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p activity or expression in the absence of the candidate molecule and h) comparing the activity or expression in step (b) with the activity or expression in step (c), wherein a decrease between the measured activities or expression of miR125a-5p and/or miR-642a-5p and/or miR 155-5p and/or miR 24-3p in step (b) compared to step (c) indicates that the candidate molecule is an inhibitor of miR125a-5p and/or miR- 642a-5p and/or miR 1 5-5p and/or miR 24-3p.

11- A method for identifying a miRNA 26a-5p and/or a miRNA 30b-5p equivalent or source thereof comprising:

e) contacting a cell with a candidate molecule;

f) assessing at least one of miRNA 26a-5p and/or a miRNA 30b-5p activity or expression; g) assessing at least one of miRNA 26a-5p and/or a miRNA 30b-5p activity or expression in the absence of the candidate molecule and

h) comparing the activity or expression in step (b) with the activity or expression in step (c), wherein an increase between the measured activities or expression of miRNA 26a- 5p and/or a miRNA 30b-5p in step (b) compared to step (c) indicates that the candidate molecule is an equivalent or source of miRNA 26a-5p and/or a miRNA 30b-5p.

12- The method according to claim 10 or 11, wherein the cell is contacted with the candidate molecule in vitro or in vivo.

13- The method according to any one of claims 10-12, wherein the candidate molecule is a protein, a peptide, a polypeptide, a polynucleotide, an oligonucleotide or a small molecule.

14- The method according to any of claims 10-13, wherein assessing the activity of miR 125a- 5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or of miRNA 26a-5p and/or a miRNA 30b-5p comprises assessing expression or activity of a gene regulated by miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p and/or miR A 26a-5p and/or a miRNA 30b-5p as indicated in Table 2.

15- A pharmaceutical composition comprising at least one compound as defined in any one of claims 1 to 9 and excipients and/or adjuvants for use in the treatment and/or prevention of an autoimmune-immune mediated inflammatory disease.

16- The pharmaceutical composition according to claim 15 wherein the inhibitor as defined in any one of claim 1 to 8 and/or the miRNA 26a-5p and/or miRNA 30b-5p molecule, and/or the equivalent thereof and/or the source thereof as defined in claims 1-3 or 9 is/are comprised in a vector.

17- A method for the diagnosis and/or prognosis of an autoimmune-immune mediated inflammatory disease in a subject or to identify a subject at risk to develop an autoimmune- immune mediated inflammatory disease comprising the following steps:

a) measuring the amount of miR 24-3p in a biological sample isolated from the subject, and b) comparing the measured amount of step a) with an appropriate control amount of miR 24-3p, wherein if the amount of miR 24-3p in the biological sample is higher than the control amount, this indicates that the subject is affected by an autoimmune- immune mediated inflammatory disease or is at risk of developing an autoimmune-immune mediated inflammatory disease.

18- The method according to claim 17 wherein the autoimmune-immune mediated inflammatory disease is type 1 diabetes.

19- The method according to claim 18 or 19 wherein the biological sample is a blood sample.

20- A kit for the diagnosis and/or prognosis of an autoimmune-immune mediated inflammatory disease comprising:

- means to detect and/or measure the amount of miR 24-3p; and optionally

- control means.

21- miR 24-3p for use in a method for the diagnosis and/or prognosis of an autoimmune- immune mediated inflammatory disease, wherein said autoimmune-immune mediated inflammatory disease is preferably type 1 diabetes.

22- A method of treatment of and/or prevention of an autoimmune-immune mediated inflammatory disease, comprising administering in a subject in need thereof an effective amount of at least one compound selected in the group consisting of:

a) an inhibitor of miR 125a-5p and/or miR 642a-5p and/or miR 155-5p and/or miR 24-3p as defined in any one of claims 1-8; b) a miRNA 26a-5p and/or a miRNA 30b-5p molecule and/or an equivalent thereof and/or a source thereof as defined in any one of claims 1-3 or 9;

c) a polynucleotide coding for the compound a) and/or b);

d) a recombinant expression vector comprising said polynucleotide;

e) a host cell genetically engineered expressing said polynucleotide,

wherein the autoimmune-immune mediated inflammatory disease is preferably selected from the group consisting of: type 1 diabetes, islet autoimmunity, rheumatoid arthritis, psoriasis, SLE, multiple sclerosis or autoimmune thyroiditis.