Novel mirnas for treating osteoarthritis, combinations thereof, compositions comprising said mirnas and related methods

Abstract

The present disclosure refers to novel RNA molecules based on miRNAs which are useful for the treatment of osteoarthritis (OA), by modulating the expression of OA-related genes, as well as combinations and compositions comprising them, and methods related thereto.

Description

[0001] NOVEL MIRNAS FOR TREATING OSTEOARTHRITIS, COMBINATIONS THEREOF, COMPOSITIONS COMPRISING SAID MIRNAS AND RELATED METHODS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of biotechnology. In particular, the invention refers to modified micro-RNAs with therapeutic activity. More particularly, the invention refers to synthetic micro-RNAs designed for treating osteoarthritis and combinations thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Osteoarthritis (OA) is a chronic degenerative joint disease, most frequently affecting the knee, which leads to a marked loss of quality of life for sufferers due to loss of mobility and pain. It is a multifactorial disease caused by a complex interaction between mechanical, biological, and genetic factors that lead to the progressive loss of articular cartilage. Although it has been considered a disease associated with aging, its incidence in young people is constantly increasing due to environmental factors associated with modern life (malnutrition, over-exercise injuries, aging).

[0006] Today, OA is the most common musculoskeletal medical condition worldwide. About 500 million people suffer from OA, and 20% of the cases correspond to knee osteoarthritis. This causes major social, economic, and public health problems. Therefore, it is urgent to generate new treatments that revolutionize the field, considering the severe economic damage caused by this pathology as it affects increasingly younger people and because of the increase of the adult population in the world.

[0007] The main clinical indicators of the development and progression of OA are synovitis, cartilage destruction, subchondral bone sclerosis and fistula formation, all of which cause the clinical symptoms of the disease that begin with inflammation, progress to disabling pain and eventually loss of joint function. However, the changes that occur at the cellular level are not fully understood; this and its complexity in terms of multifactorial causes mean that there are still no effective treatments to reverse the progression of the disease.

[0008] All current therapeutic strategies are focused on mitigating symptoms, mainly pain, up to the extreme of knee replacement surgery with a prosthesis.

[0009] The key structural event in the development of OA is the loss of the hyaline cartilage that separates the synovial space from the bone tissue. The cells responsible for the formation of this cartilage are chondrocytes, cells derived from mesenchymal progenitors. Cartilage loss is associated with a catabolic microenvironment in the knee where osteocyte activity predominates, and chondrocytes acquire a hypertrophic state, leading to ossification of the basal cartilage layers and progressive cartilage loss.

[0010] The present invention is aimed at restoring homeostasis in the joint. To this end, the invention seeks to promote: i) the differentiation of chondrocytes that generate hyaline cartilage; ii) recovery of dysfunctional chondrocytes; and iii) reduction of inflammation.

[0011] The development consists of defined micro-RNA (miRNA) sequences, customized and designed by the inventors from clinical data and bioinformatics data of the changes of different miRNAs during the development of the pathology and its treatment with advanced therapies (stem cells, Platelet Rich Plasma and proIotherapy). miRNAs are a group of small, non-coding RNAs, which, through an evolutionarily conserved mechanism, can inhibit protein expression by degrading messenger RNAs with sequences partially complementary to them. miRNAs thus modify gene expression without altering the gene sequence. Hundreds of miRNAs have been studied in OA and shown to have differential expression in pathology, yet they are often used for diagnostic purposes.

[0012] The use of miRNAs for therapeutic purposes has been proposed for several pathologies, although to this date no miRNA-based therapy has been approved. Their permissive repression nature gives them simultaneously a great advantage and a high risk to be considered as therapeutic strategies. The main adverse effect is based on the fact that a miRNA can have dozens of targets because of its low restriction to inhibit complementary messenger RNA sequences. This makes it too risky for systemic delivery strategies since it is not known which cells an administered miRNA will encounter on the way to its target and what the effects on them will be. Some examples of this are: the clinical trial with miR- 34, which was discontinued due to immune cytotoxic effects (Peltier et al., 2016; Hong et al., 2020), as well as miR 122 which was discontinued due to adverse effects during phase 2 (Zhang et al., 2021).

[0013] To overcome this issue, several more specific and controlled delivery strategies have been developed based on nanomaterials, such as dendrimers and hydrogels, that minimize the release of active molecules far from their target. Other functions intended for these systems are to help protect the active molecules from degradation, to facilitate the entry of the compounds they contain into the cell or to achieve a slow release of the dose. While this approach has solved some problems related to the lack of specificity inherent to miRNA-based therapy, there is another problem associated with toxicity. If the dose of pre-miRs (i.e., the precursor molecule to a mature miRNA) that a cell receives is too high, its internal processing machinery will stop processing endogenous sequences that are indispensable for cell maintenance, resulting in a failure of regulation that has associated cytotoxic effects. To address this problem, it was proposed to deliver mature miRNA molecules, but their biological activity is very low since, during the processing phase, the proteins that exert the active cleavage effect of the target mRNA are recruited. By introducing processed miRNA molecules, the mRNA cleavage machinery is less effective.

[0014] Correspondingly, there is an unmet need for additional strategies for developing miRNAs that allow for a more selective interfering activity while exhibiting low toxicity and maintaining high activity.

[0015] SUMMARY OF THE INVENTION

[0016] Correspondingly, it is a first aspect of the invention to provide a RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR- 3p pairing targets osteoarthritis-related genes and it is selected from:

[0017] • a miR-5p with a nucleotide sequence as set forth in any one of SEQ ID NO: 1-16; and

[0018] • a miR-3p with a nucleotide sequence as set forth in any one of SEQ ID NO: 17-32.

[0019] More specifically, the RNA molecule of the first aspect invention comprises a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:

[0020] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 17;

[0021] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 18;

[0022] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 19; and

[0023] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 20.

[0024] In an embodiment of the invention, the nucleotide loop consists of one of the nucleotide sequences of SEQ ID NO: 33-34. In an embodiment of the invention, the RNA molecule further comprises a two-nucleotide overhang 3’ terminus with the sequence TH, wherein H is selected from A, T and C.

[0025] It is a second aspect of the invention to provide a combination of RNA molecules comprising at least one RNA molecule of the first aspect of the invention.

[0026] In an embodiment, the combination of the second aspect comprises a first RNA molecule of the first aspect of the invention, and at least one other RNA molecule selected from a second RNA molecule of the first aspect of the invention and another miR different from the RNA molecule of the first aspect.

[0027] In an embodiment, the combination of the second aspect comprises a first RNA molecule of the first aspect of the invention and at 1 , 2, 3, 4, or 5 further RNA molecules each selected from an RNA molecule of the first aspect of the invention and a miR miR different from the RNA molecule of the first aspect.

[0028] It is yet another aspect of the invention to provide a pharmaceutical composition comprising either at least one RNA molecule according to the first aspect of the invention or a combination of the second aspect, and at least one pharmaceutically acceptable excipient.

[0029] It is yet another aspect of the present invention to provide a method for treating OA in a subject in need thereof, comprising administering a RNA molecule according to the first aspect of the invention, a combination according to the second aspect of the invention, or a composition according to the third aspect of the invention to said subject.

[0030] It is yet another aspect of the present invention to provide a use of an RNA molecule of the first aspect, a combination of the second aspect, or a composition of the third aspect, for the manufacture of a medicament for the treatment of OA.

[0031] It is yet another aspect of the present invention to provide an RNA molecule of the first aspect, a combination of the second aspect, or a composition of the third aspect, for use in treating OA.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1.

[0034] A Examples of Pre-miR, mature sequences, and their putative top targets are shown.

[0035] Those natural sequences of miR were modified to create miR-Ds. B. Schematic representation of the rationale of formation of the pre-miR-Ds. Loop secondary structure of pre-miR-30 are shown as example. Modified mature miRNA sequences were in the pre-miR structure of miR-30 skeleton creating a hybrid sequence. C Final sequence (left), secondary structure (middle) and top 5 targets of modified pre- miR are shown. The sequence represents the mature miR sequence with sequences of the loop in the pre-miR. All principal sequences in the miRs are in 5'p chain because our modified pre-miR expresses more efficiently those chains, 3'p chain was prepared to express siRNA (100% homology sequence) against relevant targets for OA thar are absent in natural miRs sequences.

[0036] Figure 2. Resume of bioinformatic analysis and cytokine determination in synovial fluid. A) Bioinformatic cluster analysis of miR expression and functions involved in critical parameters associated with OA: Each spot represents a miR and the colors inside represent the biological functions associated with each miR. Hexagonal clusters represent a biological process associated whit development of OA. miRs linked to three or more parameters and with at least two biological functions associated with the development of OA were selected for further analysis. B) Representative Cytokine array analysis in synovial Fluid from patients from clinical study are shown. The presence of relevant cytokines was grouped using Gene Ontology algorithms regarding molecular functions in OA. Knowing what cytokines explain cellular processes associated with OA and how those cytokines are modulated during OA and in OA-treated patients, miR associated with the modulation of those cytokines were searched to include an additional layer or analyses to bioinformatics.

[0037] Figure 3. miR levels associated with clinical improvements. After bioinformatic analysis, the inventors continued working with ten miR candidates for OA treatments. Each of those miR were measured in synovial fluids of patients. Quantitative miR presence was measured using TSDR-mediated flow cytometry approaches. Data obtained were analyzed and compared with clinical parameters of patients in different treatments groups of clinical study. Panel A show an example of 4 miRs analysis regarding WOMAC clinical data in group “Stem cell”. Data show that miR22, miR214 and miR204 increasing currency after treatment but not miR 21 1 , was associated with improved patient pain and mobility (low WOMAC). B) matrix chart summarizing information of miR involvements in different aspect of OA development. Data were collected mainly by three principal sources. Our bioinformatic analysis from public data sets; a systematic bibliographic search of function validations of the miR in OA, and our in-house validations of miRs occurrence and function using our clinical study data.

[0038] Figure 4. Schematic representation of the isolation process of human hypertrophic chondrocytes (HAC) from knees of patients undergoing knee replacement surgery for advanced OA. Right panel: representative microphotograph of HAC cell culture. In the lower panel, flow cytometry analysis shows phenotypic markers of mature chondrocytes.

[0039] Figure 5. A. Schematic representation of Proof of concept of the use of natural selected miRs in chondrogenic differentiation. B. Gene expression analysis by qPCR of genes relevant to the restoration of hyaline cartilage or C. master transcription factors of the chondrogenic differentiation process on HAC cells in the presence of different natural miRNAs miRs. The miRs were transfected using liposome-based technologies. Statistical analysis was performed using ANOVA with post-hoc analysis of Dunnett’s method. Each bar represents media + SE. each dot represents an individual patient with qPCR measures performed in triplicate. *p<0.05; **p<0.01 ; ***p<0.001

[0040] Figure 6. Proof of concept and comparative improvements of modified miR-D vs natural miRNAs in A. Expression of relevant transcription factors for the chondrogenic differentiation on HAC and B. Gene expression ratio analysis of genes relevant to the restoration of hyaline cartilage. Ratios represents comparations between biological relevant and opposite genes during progression of OA. Statistical analysis was performed using paried T-Test, each dot represents an individual patient with qPCR measures performed in triplicate. *p<0.05; **p<0.01 ; ***p<0.001

[0041] Figure 7 Proof of concept and comparative improvements of modified miR-D vs natural miRNAs in chondrogenic extracellular matrix production by Confocal microscopy analysis on HAC cells transfected with miRNA-92a or its modified analogue miR-D1 on the expression of essential proteins of the extracellular matrix of chondrocytes.

[0042] Figure 8. Proof of concept of qPCR validation of siRNA effects on modified miRDs. Statistical analysis was performed using paried T-Test, each dot represents an individual patient with qPCR measures performed in triplicate. All analysis show a statistical significance p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention is based on a rational design for a series of microRNAs (miRNAs or miRs) useful for the treatment of osteoarthritis (OA). The present inventors have relied on information obtained from clinical assays of their own, as well as on computational analysis, to prepare synthetic RNA sequences optimized for the treatment of OA. The inventors have selected sequences able to modulate key processes of the differentiation, homeostasis and survival of chondrocytes to increase in situ and sustainably the production of collagen-ll, thus promoting articular regeneration, and prepared new synthetic RNA molecules based thereon.

[0044] The problem with using miRNAs is the great number of potential targets thereof, since miRNA molecules have a seeding sequence of 6-8 base pairs followed by a loose sequence. This latter sequence allows them to exhibit a loose specificity, which evolutionarily was selected as an advantage by acting not only on one messenger RNA (mRNA) but on a family of them, functionally affecting several cellular processes. The problem with this is the number of targets (>100) that each molecule possesses, which makes unwanted effects uncontrollable when thinking of them as therapy. On the other hand, small-interfering RNAs (siRNAs) use the same cellular machinery as miRNAs, but the siRNA sequence is 100% complementary to the mRNA target, which makes them very specific but also ineffective in regulating complex processes.

[0045] A pre-miR, as a person of skill in the art will readily appreciate, is an RNA molecule which serves as a precursor to a miRNA. Pre-miRs are single stranded RNA molecules with a hairpin structure, with a mature miRNA comprised within each arm of the hairpin, named 3p and 5p (or miR-3p and miR-5p, respectively) according to their orientation, and which are, at least partially, complementary to each other. Typically, only one of these mature miRNAs is used as the main molecule for the RNA interference (RNAi) activity of the whole construct, while the other (termed “passenger chain”) is usually eliminated by the cellular system. The term “miR-5p / miR-3p pairing” will be used throughout this description to refer to the miR-3p and miR-5p sequences comprised within a particular pre-miR, taken together.

[0046] The present inventors have developed several miR-5p / miR-3p pairings, designed from miRNAs involved in key OA processes which were modified to increase the number of complementary bases (8-12) by reducing the number of targets in the passenger chain, which are all validated as important in processes related to OA. Thus, these pairings maintain the advantages of being specific and, at the same time, modifying different biological processes. The rationale behind the design of these pairings is as follows:

[0047] Table 1 . Description of the 5p and 3p used in the development of the different embodiments of the synthetic RNA molecules created by the inventors.

[0048] Correspondingly, it is a first aspect of the invention to provide an RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR- 3p pairing targets osteoarthritis-related genes and it is selected from: • a miR-5p with a nucleotide sequence as set forth in any one of SEQ ID NO: 1-16, and

[0049] • a miR-3p with a nucleotide sequence as set forth in any one SEQ ID NO: 17-32.

[0050] In an embodiment, the RNA molecule comprises a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:

[0051] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 17;

[0052] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 18;

[0053] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 19; and

[0054] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 20.

[0055] The RNA molecules of the invention are essentially pre-miRs which, once processed by the cellular system, release the corresponding mature miRNAs to exert the intended therapeutic effect.

[0056] The term “synthetic miR-5p / miR-3p pairing” is to be understood as referring to a miR- 5p / miR-3p pairing which has been modified with respect to the miR-5p / miR-3p pairing present in the naturally occurring pre-miR on which it is based.

[0057] The synthetic miR-5p / miR-3p pairing comprised in the RNA molecule of the invention targets osteoarthritis-related genes. Throughout this description, this is intended to mean that either the miR-5p or the miR-3p branches of the miR-5p / miR-3p pairing, or both, are able to interfere with the expression of genes which modulate processes related to osteoarthritis, such as inflammation, cartilage regeneration, etc. Non-limiting examples of such genes include IL1-R, HDAC4, Runx2, MEF2C, among others.

[0058] The inventors have also optimized the structures of the aforementioned RNA molecules or pre-miRs to increase the processing efficiency thereof by the cellular system. To this end, the pre-miRs were obtained by introducing the novel synthetic miR-5p / miR-3p pairings referred to above in the backbone of hsa-pre-miR-30, as shown in Fig. 1 .

[0059] Correspondingly, in a preferred embodiment, the RNA molecule of the invention comprises a nucleotide loop flanked by the miR-5p / miR-3p pairing, wherein the nucleotide loop consists of the nucleotide sequence of SEQ ID NO: 33, which corresponds to the nucleotide loop of hsa-pre-miR-30, or SEQ ID NO: 34 which corresponds to the nucleotide loop of hsa-pre-miR-24.

[0060] These optimized pre-miRs comprising the novel miR-5p / miR-3p pairings of the invention and the nucleotide loop from hsa-pre-miR-30 will be referred to as miR-D1 , miR-D2, miR- D3 and miR-D4 (comprising pairings D1 to D4 described above, respectively)

[0061] In a particularly preferred embodiment, the RNA molecule comprises a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:

[0062] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 17;

[0063] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 18;

[0064] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 19; and

[0065] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 20; wherein the nucleotide loop consists of the nucleotide sequence of any one of SEQ ID NO: 33-34 and the RNA molecule further comprises a two-nucleotide overhang 3’ terminus with the sequence TH, wherein H is selected from A, T and C.

[0066] The RNA molecules of the invention may be combined with each otherto provide a higher activity for treating OA. The RNA molecules may also be combined with other pre-miRs to achieve the intended therapeutic effects.

[0067] Thus, it is a second aspect of the invention to provide a combination of RNA molecules, comprising at least one RNA molecule of the first aspect of the invention.

[0068] In an embodiment, the combination of the second aspect comprises a first RNA molecule of the first aspect of the invention, and at least one other RNA molecule selected from a second RNA molecule of the first aspect of the invention and another miR known in the art.

[0069] In an embodiment, the combination of the second aspect comprises a first RNA molecule of the first aspect of the invention and 1 , 2, 3, 4, or 5 further RNA molecules each selected from an RNA molecule of the first aspect of the invention and a miR different from the RNA molecule of the first aspect. Preferably, the combination of the second aspect comprises a 1 or 2 further RNA molecules selected from an RNA molecule of the first aspect of the invention and a miR different from the RNA molecule of the first aspect.

[0070] With the teaching provided herein, the person skilled in the art would understand that the different embodiments as described above of the RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing can be combined with each other or with other miR different from the RNA molecule of the first aspect, depending on their necessary needs.

[0071] One of the main advantages provided by the present invention with respect specifically to OA is the "off the shelf concept, which, in advanced regenerative therapies, is a major problem that needs to be solved. Most therapies for regenerative medicine in general and for OA in particular use autologous stem cells or platelet-rich plasma that are injected into the joint and are expected to differentiate into chondrocytes. These therapies have the problem of the personalization of the medicine, they are self-transplantation and therefore are far from being able to be thought of on a large scale. An alternative to this strategy is to use extracellular vesicles of stem cells that contain bioactive molecules with functions similarto those of the cells that give rise to them. Although this method solves the problem of injecting live cells, it has other limitations since scaling up production is very complex, batch-to-batch heterogeneity is a problem not yet solved, and defining the content or formulation is impossible.

[0072] The present invention solves these problems and improves the effects by simplifying the process to chemical mediators essential to produce in situ differentiation of stem cells already residing in the joint. Much of the biological effects of the vesicles, PRP and stem cells are due to the miRNAs that are loaded or released by them. The pre-miRs of the present invention allow for the injection of only a small amount of chemically defined, stabilized sequences specifically designed to modulate signaling pathways and processes that complement each other to achieve cartilage regeneration.

[0073] In order to be administered to a subject in need of a therapy involving RNAi, the RNA molecules or the combination of the invention must be formulated appropriately for their proper delivery to the target tissue or cells.

[0074] Correspondingly, it is a third aspect of the invention to provide a pharmaceutical composition comprising at least one RNA molecule of the first aspect or a combination of the second aspect, and at least a pharmaceutically acceptable excipient. The pharmaceutical composition of the invention may comprise a pharmaceutically acceptable carrier for delivering RNA to cells. Such carriers are known in the art, and include, without limitation, dendrimers, liposomes, cationic polymers, rigid nanoparticles, viral vectors, hydrogels, fusion peptides, extracellular vesicles, among others.

[0075] Considering the ability of RNA molecules of the invention modulate signaling pathways and processes related to OA, it is yet another aspect of the invention to provide a method for treating OA in a subject in need thereof, comprising administering an RNA molecule according to the first aspect of the invention, a combination according to the second aspect of the invention, or a composition according to the third aspect of the invention to said subject.

[0076] A person of skill in the art will be able to establish the therapeutically effective amount to administer to the subject in view of the severity of OA the subject to be treated is experiencing, the RNA molecules to be administered and the dosage form selected, among other factors.

[0077] In a preferred embodiment of this aspect of the invention, the administered RNA molecule, combination or composition comprise an RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:

[0078] • a miR-5p with a nucleotide sequence as set forth in any one of SEQ ID NO: 1-16; and

[0079] • a miR-3p with a nucleotide sequence as set forth in any one of SEQ ID NO: 17-32.

[0080] More preferably, the administered RNA molecule, combination or composition comprise an RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:

[0081] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 17;

[0082] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 18;

[0083] • a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 19; and a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 20.

[0084] In a particularly preferred embodiment of this aspect of the invention, the method comprises administering a combination or a pharmaceutical composition comprising three RNA molecules, wherein:

[0085] • a first RNA molecule comprises a miR-5p / miR-3p pairing selected from a miR- 5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 17; and a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 20;

[0086] • a second RNA molecule comprises a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 18; and

[0087] • a third RNA molecule comprises a miR-5p / miR-3p pairing selected from a miR- 5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 19, a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 21 . with the proviso that when the first RNA molecule comprises a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR-3p with a nucleotide sequence as set forth in SEQ ID NO: 20.

[0088] The RNA molecules of the invention may be prepared by known techniques in the art. For instance, the RNA molecules may be prepared by chemical synthesis as typically carried out with a DNA synthesizer.

[0089] EXAMPLES

[0090] The invention will now be further described based on the following examples. It is to be understood that these examples are intended for illustrative purposes only, and by no means should be construed to be limiting the scope of the invention, which is only defined by the appended claims.

[0091] Example 1 - Selection of miRN As for OA From an extensive bioinformatics search 17 miRNA candidates were selected, that are simultaneously associated with:

[0092] 1 . Signaling pathways involved in the differentiation of col-2-producing chondrocytes.

[0093] 2. Production of factors that promote cartilage regeneration.

[0094] 3. Inhibition of factors that impede cartilage growth.

[0095] 4. Inflammation mediating factors.

[0096] 5. Factors associated with pain.

[0097] Briefly, the search was carried out by looking for coincidences of the miRNAs in different databases. One of them is miRNet ca) , which allowed to obtain a map based on published miRNA reported to be deregulated in knee arthrosis, knee inflammation, general inflammation and trauma, but not in arthritis. From this information, a list of miRNAs based on function profile was prepared (KEGG analysis). Then, this list was compared to another list generated from public access bioinformatic resources (NCBI GEO Datasets) of assays reporting deregulated expression of miRNAs in knee OA from various sample types, from synovial fluid to cartilage or peripheral blood. Once coinciding miRNAs from both lists were identified, their targets were analyzed by referring to mirdb.org and targetscan.org databases. Those miRNAs with the most relevant targets were selected for a search in scientific papers for their relevance in independent assays, so as to create a signaling and expression map for the OA pathway. Finally, based on their relevance and number of targets, they were assigned a value in a matrix. Those with a higher score were selected for in vitro testing.

[0098] The inventors carried out a miRNA expression cluster analysis where the expression of miRNAs reported in previous studies was analyzed in an unsupervised manner. miRNAs simultaneously present in more than one cluster, or miRNAs reported in previous literature and / or whose functional relevance was relevant to the model, were selected to be determined in synovial fluid samples from an in-house clinical study (NTC05416255). Using an adapted TSDR technique, the presence of miRNAs 211 , 214, 204, 222, could be determined. The inventors then associated the quantitative presence of these miRNA with clinical improvement variables such as WOMAC index and joint functionality in patients under different treatments (proIotherapy, platelet-rich plasma, and stem cells) (Fig. 3A).

[0099] Additionally, the presence of relevant cytokines and synovial fluids in patients with advanced OA was determined by ELISA, using a proteome Profiler Human Cytokine Array kit (R&D system, ARY005B), and they were organized according to their biological functions to understand which processes are most affected by overexpressed cytokines in patients (Fig. 2). With these data the inventors constructed a matrix containing the biological functions related to the progression and regression of pathology, the signaling mechanisms and the miRNAs that best explain the modulation of these pathways and processes. In turn, a level of complexity was added by determining whether the participation of miRNAs in these processes was identified only by bioinformatics (putative targets or appearance in arrays), whether they were also validated in the literature, and finally whether this validation was corroborated by the inventors’ own data as described above (Fig. 3B).

[0100] Example 2 - Design and preparation of modified miRNAs to obtain customized cocktails

[0101] From the results of the bioinformatics analysis performed in Example 1 , the inventors then proceeded to optimize the sequences of hsa-miR-31 , hsa-miR-204, hsa-miR-92a and hsa- miR-140 to reduce the off-targets of the passenger sequence while preserving the desired targets for the main sequence.

[0102] Additionally, the miRNAs were incorporated into the backbone of hsa-pre-miR-30 (SEQ ID NO: 35), so as to ensure a more efficient processing and a greater effectiveness of the corresponding miRNAs (FIG 1).

[0103] Correspondingly, pre-miRs miR-D1 (SEQ ID NO: 36), miR-D2 (SEQ ID NO: 37), miR-D3 (SEQ ID NO: 38), and miR-D4 (SEQ ID NO: 39) were synthesized using an DNA synthesizer. Briefly, the first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing RNA chain in the reaction column by traditional chemical synthesis reactions.

[0104] Example 3 - Proof of concept

[0105] In order to validate the use of the selected miRNAs in the chondrogenic differentiation required for hyaline cartilage recovery, and to compare their effects with respect to synthetic miRNAs, the inventors developed a physiologically relevant method in humans (Fig. 4A). Most current strategies to validate the use of molecules in joint regeneration are based on the use of chondrocytes differentiated from stem cells, or from joint tissue explants. The present inventors were able to isolate hypertrophic chondrocytes from knees with severe OA and maintain them in culture to test the therapeutic effect of miRNAs on them. The advantage of this strategy is that it acts directly on the cells that are the target of the ex vivo therapy, and it evaluates their reprogramming from a real pathological state. Each isolate corresponds to a patient who underwent knee replacement surgery for severe OA. Each human hypertrophic articular chondrocyte (HAC) isolate is a unique case and represents the actual variability in patients.

[0106] In this regard, HAC cells were used as a study model for proof of concept that: 1) miRNAs selected in this development enhance and reprogram damaged joint cells by increasing the expression of proteins and transcription factors critical for cartilage production; 2) that the effect of the cocktail of the natural selected miRNAs is superior to that of the individual miRNAs; and 3) that the modified miRNAs have potentiating and unexpected effects with respect to the cocktail of natural miRNAs.

[0107] The miRNAs were incorporated into the HACs by transfection with commercially available lipidic nanoparticles (Lipofectamine 3000 (L3000015) Thermo Fisher). 100 nM of total miRNA + 2 pl of Lipofectamine 3000 were mixed in an antibiotic-free medium (200 pl final volume), and at 2-4 h, fresh HAC complete medium was added to each plate, and the cells were incubated for another 48-96 h until functional validations were performed (FIG 5A).

[0108] Cells transfected with empty liposomes or with scramble sequences of miRNAs were used as controls. Post-transfection the cells were separated into three groups. One of them was lysed for Western blot analysis to determine the presence of proteins associated with the targeted effects for the miRNAs. Another group was subjected to a protocol for purification of RNAs which were then back-transcribed into cDNA and analyzed for gene expression by real-time PCR. Finally, a group of cells were fixed for immunostaining and confocal microscopy analysis to evaluate the production and localization of key proteins in extracellular matrix generation.

[0109] As a representation of the proof of concept, the results obtained with the natural miR-140, miR-204, miR-222, miR-31 , and miR-92a are shown in figure 5 (FIG 5B, C). The individual miRNAs were always transfected at a concentration of 100 nM. First, the inventors analyzed by qPCR the expression of genes relevant to the formation of the extracellular matrix that gives rise to hyaline cartilage by HACs where the distinct responses of genes associated with the formation (COL2, Aggrecan) and degradation (MMP-13) and COL10 as a non-hyaline cartilage related collagen are observed (Fig. 5B). The expression of paradigmatic transcription factors for anabolic (SOX9, WNT5) and catabolic (RUNX2) processes was also studied (Fig. 5C). The data show a trend in the reprogramming of chondrocyte gene expression towards anabolic profiles of hyaline cartilage formation after transfection with the miRNAs specialty with miRNAs 140 and 92a. In order to demonstrate the increased activity of modified miRNAs compared to naturally occurring ones, the inventors conducted a comparison of their chondrogenic effects. For transcription factor expression, miR-D2 and miR-D3 did not show a significant improvement over natural occurring miRNAs. However, miR-D1 and miR-D4 exhibited enhanced chondrogenic effects compared to natural miRNA-31 and miRNA-92a. It is important to note that miRNA-31 had no predicted effects in this context, highlighting the novel effects acquired by the modified miRNAs (FIG 6A).

[0110] Regarding the modulation of extracellular matrix-related genes, miR-D1 demonstrated an unexpectedly increase in the expression of hyaline cartilage production genes compared to miRNA-92a, which was already higher than that of other natural miRNAs. In this scenario, miR-D2 and miR-D3 did not enhance the effects of the natural miRNAs, while miR-D2 significantly improved the expression of COL2 (FIG 6B).

[0111] One of the most important proteins during joint degradation is the aggrecanase ADAMTS4, which degrades aggrecan by destabilizing the extracellular matrix. By confocal microscopy study, it was observed that the addition of the miRNA-D1 almost eliminates the expression of this protein in hypertrophic chondrocytes from OA patients who express it constitutively because of the pathology (Fig. 7B). Finally, to validate the qPCR data the expression of aggrecan was determined by confocal microscopy in HACs exposed to the miRNA-92a and the modified miR-D1 , observing a remarkable increase in the extracellular expression of aggrecan. In summary, the results show the effectiveness of the cocktail using the modified miRNAs and validate the effectiveness of the analysis to find the best candidates.

[0112] Finally, to demonstrate that the modifications made to the miRNAs are functional and relevant for treatment, the expression of genes that are targets of the siRNAs were anazyed from the passenger strands in the miRDs (see FIG1). This allows the evaluation of the effects of the modified miRNAs, their correct processing, and their functionality. It was confirmed that all four miRDs exhibit siRNA effects as stipulated in their modifications. An example of this is the reduction in expression of the inflammatory receptor IL1 R in HACs following treatment with miD1 , orthe decrease in RUNX2 expression after treatment with miRD4, which was initially selected to mitigate pain but also demonstrates a significant effect on chondrogenic differentiation after modification.

[0113] REFERENCES 1- Hong, D. S. et al. Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours. Br. J. Cancer 122, 1630-1637 (2020).

[0114] 2- Peltier, H. J., Kelnar, K. & Bader, A. G. Effects of MRX34, a liposomal miR-34 mimic, on target gene expression in human white blood cells (hWBCs): qRT-PCR results from a first-in-human trial of microRNA cancer therapy. Ann. Oncol. 27, vi531 (2016).

[0115] 3- Zhang S, Cheng Z, Wang Y, Han T. The Risks of miRNA Therapeutics: In a Drug Target Perspective. Drug Des Devel Ther. 2021 Feb 22;15:721-733. doi: 10.2147 / DDDT.S288859. Erratum in: Drug Des Devel Ther. 2021 Mar 30;15:1423. PMID: 33654378; PMCID: PMC7910153

[0116] SEQUENCES LISTING

Claims

CLAIMS1 . An RNA molecule comprising a nucleotide loop flanked by a synthetic miR-5p / miR-3p pairing, wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:• a miR-5p with a nucleotide sequence as set forth in any one of SEQ ID NO: 1-16; and• a miR-3p with a nucleotide sequence as set forth in any one of SEQ ID NO: 17-32.

2. The RNA molecule of claim 1 , , wherein the miR-5p / miR-3p pairing targets osteoarthritis-related genes and it is selected from:• a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 1 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 17;• a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 2 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 18;• a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 3 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 19; and• a miR-5p with a nucleotide sequence as set forth in SEQ ID NO: 4 and a miR- 3p with a nucleotide sequence as set forth in SEQ ID NO: 20.

3. The RNA molecule of claim 1 , wherein the nucleotide loop consists of the nucleotide sequence selected from any one of SEQ ID NO: 33-34.

4. The RNA molecule of claim 1 or 2, further comprising a two-nucleotide overhang 3’ terminus with the sequence TH, wherein H is selected from A, T and C.

5. A combination comprising a first RNA molecule of any one of claims 1-4, and at least one selected from:• a second RNA molecule of any one of claims 1-4; and• a miRNA molecule that is different from the RNA molecule of any one of claims 1-4.

6. A pharmaceutical composition comprising either at least one RNA molecule according to any one of claims 1-4, or a combination according to any one of claims 5, and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the at least one pharmaceutically acceptable excipient comprises at least one selected from dendrimers, liposomes, cationic polymers, rigid nanoparticles, viral vectors, hydrogels, fusion peptides, and extracellular vesicles.

8. A method for treating osteoarthritis (OA) in a subject in need thereof, comprising administering an RNA molecule according to any one of claims 1-4, a combination according to claim 5, or a composition according to any one of claims 6-7 to said subject.

9. Use of an RNA molecule according to any one of claims 1 -4, a combination according to claim 5, or a composition according to any one of claims 6-7, for the manufacture of a medicament for the treatment of OA.

10. An RNA molecule according to any one of claims 1 -4, a combination according to claim 5, or a composition according to any one of claims 6-7 , for use in treating OA.

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