Extracellular nanovesicles from mesenchymal stem cells induced to glycolytic metabolism for the treatment of osteoarthritis

Abstract

The invention relates to a composition enriched in extracellular nanovesicles (sEVs) from umbilical mesenchymal cells, wherein the composition consists of a group of sEVs with increased expression of the AGRN and HSPG2 proteins, and a medium or vehicle. The invention also relates to a method for obtaining the composition, which comprises culturing umbilical mesenchymal cells (UC-MSCs) in an induction medium supplemented with 0.01-100 µg / ml oligomycin, for 2-48 hours, and subsequently isolating the nanovesicles secreted by the MSCs into the medium; testing the increased expression of the AGRN and HSPG2 proteins; and resuspending the nanovesicles in a medium or vehicle to obtain the composition of the invention. The invention further relates to the use of the composition to treat osteoarticular diseases or injuries.

Description

[0001] Extracellular nanovesicles from mesenchymal stem cells induced to glycolytic metabolism for the treatment of osteoarthritis

[0002] Descriptive report

[0003] Previous background

[0004] The WHO considers osteoarthritis (OA) a disease that affects the population's quality of life, and that by 2050, more than 579 million people worldwide will suffer from it. It is the disease that most reduces the quality of life of patients per year lived.

[0005] According to global statistics, more than 50% of adults over the age of 60 suffer from OA. Currently, there are no effective FDA / EMA-approved treatments available for OA patients that can halt or reverse disease progression, and only provide pain relief.

[0006] From a therapeutic perspective, there are no treatments that modify, delay, or restore articular cartilage function. Therefore, the current strategy is based on palliative medications with low effectiveness that seek to control pain, inflammation, and stiffness. According to the guidelines provided by the Ministry of Health (MINSAL) through its "2009 Clinical Guideline for Medical Treatment in People Aged 55 and Older with Mild or Moderate Hip and / or Knee Osteoarthritis," OA management focuses on a multimodal approach that includes patient education and self-management, non-pharmacological management, and pharmacological treatments consisting of simple analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, analgesic opioids, and intra-articular (IA) drugs. The last-line therapeutic option remains prosthetic surgery to replace the native joint with an artificial prosthesis.

[0007] This therapeutic problem represents an opportunity for the introduction of new therapies that pursue a structural purpose with an emphasis on regenerative or chondroprotective rather than merely symptomatic action. In this regard, gene therapy has been proposed as an alternative solution; however, viral strategies entail high immunogenicity, high production costs, and problems associated with genetic alterations in other cellular targets, while non-viral strategies have low transfection efficiencies and poor cell specificity. In this regard, ex vivo gene delivery offers further advantages, such as TissueGene-C (TG-C), which involves the use of allogeneic human chondrocytes and irradiated GP2-293 cells (derived from HEK-293) that overexpress TGF-β1.This procedure has proven effective and safe in phase 1 and 2 clinical studies, has been approved for the treatment of patients with moderate knee osteoarthritis in South Korea, and is recently in phase 3 in the US. However, despite the remarkable results of these clinical trials, the molecular mechanisms underlying the effect of TG-C, as well as the potential side effects associated with long-term cell line injection, remain unknown.

[0008] On the other hand, mesenchymal stem cells (MSCs) have been the focus of attention of physicians and scientists for over a decade as a potential biological therapy for the treatment of OA due to their chondrogenic potential, immunosuppressive and anti-inflammatory effects, and safety in autologous and allogeneic administration. However, although preclinical data have demonstrated that intra-articular (IA) treatment with MSCs can prevent or even reverse cartilage degradation in animal models of OA, clinical results in patients have been inconclusive, and market approvals have been limited.Recently, our group has successfully conducted a Phase I / II Randomized Controlled Clinical Trial (NCT02580695) of IA administration of allogeneic umbilical cord-derived MSCs (UC-MSCs) in patients with OA, reporting a long-standing symptomatic benefit at 12 months follow-up, a finding also replicated in recent studies.

[0009] Currently, OA treatment is being addressed through research that develops and studies potential compounds capable of acting as DMOADs (disease-modifying osteoarthritis drugs). Generally speaking, these experimental compounds act on one of the three axes of disease pathogenesis: articular cartilage regeneration, subchondral bone remodeling, or synovial inflammation management, thus limiting their therapeutic potential. In contrast, MSC therapy has been shown to act on at least two axes—cartilage regeneration and inflammation—which provides an advantage over other therapies. Despite this, the most recent systematic reviews of clinical trials with MSCs conclude that, although the therapy is beneficial in some patients with OA, the regenerative effectiveness of MSCs as a treatment modality remains uncertain, and market authorizations have been limited.From the above, the researchers deduce the idea of ​​​​boosting MSCs in their regenerative properties to provide an effective treatment for OA. They have worked on this "cellular potentiation" hypothesis in recent years in their laboratory, using oligomycin as an inhibitor of oxidative phosphorylation to increase glycolysis and thus enhance its regenerative and anti-inflammatory effects. The findings that the stimulation of glycolytic metabolism in UC-MSCs - called GlycoStem by the inventors - increases their anti-inflammatory potential in vitro and regenerative potential in an animal model of OA have been robustly demonstrated, evidence that has generated both intellectual property (PCT / IB2020 / 061190) and scientific articles that are already under review in various international journals.Since it is impossible to consider the development of a new medicinal product without a precise understanding of its mechanism of action (MoA), we have established that the invention exerts at least part of its biological effect through its small extracellular vesicles (sEV), mainly exosomes, which constitute this invention.

[0010] As previously described by the inventors and other research groups, sEVs are nanovesicles secreted by almost all cells that act as important mediators of intercellular communication capable of generating phenotypic changes in the acceptor cell, thus influencing different physiological and pathological conditions. Today, it is known that sEVs can enhance tissue regeneration, participate in immune modulation, and function as potential alternatives to stem cell therapy. The inventors have demonstrated that the induction of glycolytic metabolism in UC-MSCs using oligomycin and its analogues promotes their regenerative (PCT / IB2020 / 061190), immunosuppressive, and anti-inflammatory potential53 in vitro and in vivo. Recently, the inventors have established that the mechanism of action by which the thus treated cell is exerting its biological effects in vivo, would be mediated by its secreted sEVs.This is not at all obvious or expected, since sEVs constitute less than 10% of the exchange products with the environment generated by cells, so if a cell has an effect, it cannot necessarily be established that the same effect is obtained by using its exosomes (sEVs).

[0011] The inventors' results demonstrated the potential for the development of an sEV-based product for a new class of biological therapy, acellular biopharmaceuticals, capable of generating a transformative impact on the treatment of OA. The inventors' previous results show that pretreatment with oligomycin in MSCs, for short or long periods, has no toxic effects on the cells, does not alter the MSC immunophenotype, and also maintains glycolytic activity for at least three days. This last finding is critically important from the perspective of manufacturing the sEV-based product, since vesicles are isolated from the cell supernatant after 48 hours. Furthermore, pre-incubation of MSCs with oligomycin does not alter the rate of direct secretion of sEVs into the cell culture medium, while also preserving their size distribution.Furthermore, after isolating -sEVs from MSCs treated with oligomycin, it was confirmed that, in terms of concentration and identity, there are no major variations with respect to sEVs derived from native MSCs, and importantly, in terms of size according to that described by the ISEV (International Society of Extracellular Vesicles), it was observed that there is an enrichment of the exosomal fraction (> 200nm) contained in the total sEV fraction. Likewise, we have observed that soluble factors from MSCs treated with oligomycin as in the present invention, promote the expression of hyaline cartilage markers and the survival of chondrocytes from patients with OA.These results allowed us to propose -sEV of the invention as a bioactive mechanism, which shows a very similar profile to UC-MSC-sEV in terms of secretion rate, size distribution, as well as size and post-isolation characterization, using a standard protocol validated by our group. Furthermore, our data show that UC-MSC-sEVs present a standard morphology by electron microscopy and are capable of internalizing into target cells such as chondrocytes from OA patients. An interesting fact that impacts the production of -sEVs according to the invention is that no significant differences in yield, size, and characterization are observed between fresh and frozen cells, which are directly thawed for the purposes of obtaining sEVs.

[0012] On the other hand, at the functional level, in order to compare the anti-inflammatory effect of UC-MSC and their sEV derivatives, we used as an experimental approach an in vitro assay on murine macrophages activated with lipopolysaccharide (LPS), to obtain pro-inflammatory MI macrophages. Our results show that both Glycostem and GlycoStem-sEVs possess a greater anti-inflammatory capacity than UC-MSCs or their sEV derivative, in terms of promoting the generation of anti-inflammatory M2 macrophages and inhibiting pro-inflammatory MI phenotypes. Interestingly, the effects exerted by -sEVs according to the invention were very similar to their cellular counterpart, a result that allows the inventors to propose that a large part of the effect obtained by said cells is based on their sEVs.

[0013] No document has been found in the prior art that associates the use of oligomycin as a booster of MSCs or their extracellular derivatives for the treatment of OA. Likewise, as explained above, to date there are no documents that disclose the use of oligomycin as an MSC booster for the production of sEVs or exosomes for the treatment of OA. These new results complement the previous developments of the same inventors, such as those protected in the PCT application (PCT / IB2020 / 061190), referring to the use of MSCs pretreated with oligomycin for the treatment of OA. For the person skilled in the art, the advantages of an acellular therapy, which is a new technology protected in this invention, will be evident, based on the determination that the OA treatment effect can also be obtained with only sEVs from MSCs treated with oligomycin, which was not anticipated in the prior art.

[0014] sEVs, including exosomes, are secreted by almost all cells as part of their normal physiology or during the acquisition of abnormalities. Depending on their cell of origin, sEVs can contain many of the cell's constituents, such as nucleic acids, lipids, metabolites, and cytosolic and cell surface proteins. Due to their efficient ability to transfer the encoded cargo within them and trigger phenotypic changes in recipient cells, they have generated substantial interest from the scientific and medical community as a novel therapeutic strategy. Undoubtedly, one of the main clinical advantages of sEVs is that, since they are "naturally" secreted by cells, the lipid and protein composition of their membrane is very similar to that of their parent cell, making them inherently non-immunogenic.Other advantages that sEVs have over their cells of origin are that they are less likely to become trapped in the microvasculature due to their nanometric size, their practically zero risk of malignancy per se, since being inert compounds they do not have the capacity to divide, and their greater efficacy in vivo due to their greater stability. Finally, from a marketing point of view, they can be stored at -80°C, or if they are lyophilized, they can be kept at room temperature for several months, without loss of their therapeutic efficacy, which gives them an indisputable advantage by being immediately available for clinical use (off-the-shelf therapy).

[0015] Brief description of the figures.

[0016] Figure 1. Glycostem-derived EVs (Glyco-EVs) present a microRNA signature that is significantly different from their native sEVs counterpart in lyophilized samples. Using the EdgeR tool, 178 genes were identified with significant differences with respect to the native sEVs condition. Of the total, 175 were found to be increased with respect to native sEVs (Log2 fold change >0.5 and <-0.5; Adjusted p-value <0.05) and 3 were decreased with respect to the native sEVs condition (Log2 fold change >0.5 and <-0.5; Adjusted p-value <0.05). N= 4 biological samples.

[0017] Figure 2. Diagram of the experimental design of the murine osteoarthritis model. C57 BL6 mice were injected intra-articularly (IA) with 5 μL of collagenase type V1 or saline (SHAM). 7 and 14 days post-induction, animals were treated with 5 μL of saline or naive sEVs or glyco sEVs. On day 42, mice were euthanized, and joints were removed for microCT or histological imaging analysis. Clinical scores for microCT and histology were analyzed in different areas of the joint, as shown on the left, to obtain bone density and histological joint damage scores.

[0018] Figure 3. EVs according to the invention decrease bone mineralization, increasing the therapeutic potential of UC-MSC-EVs in a collagenase-induced murine model of osteoarthritis (OA). C57 BL6 mice were injected with 5pL of collagenase type V or saline (SHAM) via the intra-articular (IA) route. After 7 and 14 days post-induction, the animals were treated with 5pL of physiological saline or with UC-MSCs or UC-MSCs-oligomycin cells and their respective extracellular vesicles (sEVs). Graphs represent the mean + / - SD of 3 independent experiments using at least 5 mice per experimental group. p values ​​associated with a significant difference p < 0.05 are indicated.

[0019] Figure 4 shows the degree of joint degeneration in the analyzed areas associated with OA according to histological score. All analyses were performed in a double-blind study. The data show that Glycostem-sEVs significantly reduce OA progression, highlighting an increase in hyaline cartilage in the glycostem-sEV group, presenting greater therapeutic efficacy associated with regeneration of the damaged cartilage area in OA. Graphs represent the mean + / - SD of 3 independent experiments using at least 8 mice per experimental group. P values ​​were calculated with respect to the experimental OA group or when indicated between groups. P values ​​associated with a significant difference p < 0.05 are indicated in red. Figure 5: Lyophilized GlycoStem-derived EVs (glyco sEVs) preserve their therapeutic potential in a murine model of osteoarthritis induced by collagenase (OA).(A) Graph showing bone density values ​​in the different analysis areas. The data show that lyophilized glycostem-sEVs present the same significant improvement in chondral mineralization as their frozen counterpart, suggesting that they preserve their therapeutic capacity. (B) Graph showing the histological score values ​​according to the modified Pritzker OARSI scale in the different analysis areas. The data show that both frozen and lyophilized glycostem-sEVs present the same therapeutic activity associated with regeneration of the area, with no significant differences between the two groups. Graphs represent the mean + / - SD of 1 experiment, using 5-8 mice per experimental group. P values ​​were calculated with respect to the experimental OA group or when indicated between groups. * p < 0.05; ** p < 0.01; *** p < 0.005; ****p < 0.001.

[0020] Description of the Invention

[0021] The invention aims at a solution to the problem of osteoarthritis, with an acellular bioproduct based on extracellular nanovesicles derived from MSCs, UC-MSC-sEVs, which overexpress the proteins Keratin, type II cuticular Hb5 or P78386 (KRT85 gene); Agrin or A0A494 CO G5 (AGRN gene); and Basement membrane-specific heparan sulfate proteoglycan core protein or P98160 (HSPG2 gene). These specific nanovesicles, sEVsglyco or Glycostem-sEVs, are experimentally obtained in a greater proportion from MSCs induced towards a glycolytic metabolism with Oligomycin. These nanovesicles are useful for the treatment of OA, where the inventors have validated their efficacy and safety profile in normal and OA cartilage and in synovial tissues in vitro and in vivo.

[0022] Additionally, this product, being acellular, maintains its functional efficacy both in its frozen format at -80 and lyophilized for 48 hours, which gives the product great versatility, which can be transported at room temperature conditions while maintaining its integrity and therefore its efficacy.

[0023] The inventors have evaluated the biodistribution of the product of the invention administered locally (intra-articularly) in the knee of healthy and OA mice. It is worth mentioning that one of the main challenges of acellular therapy is the residence time of the product in the area of ​​interest. To address this question, the inventors also conducted a preliminary study using the in vivo model of murine collagenase-induced osteoarthritis (CIOA). The results showed that UC-MSC-sEVs injected intra-articularly are able to remain at the injection site for more than 96 hours, sufficient time to be internalized by the different resident acceptor cells.Additionally, preliminary results indicated that sEV of the invention significantly decreases mineralization and histological score in the joints of treated animals, as well as fibrosis in the joint, compared to UC-MSC-sEV, demonstrating a better therapeutic and regenerative effect in this animal model. In terms of protein signature and miRNA, we were able to identify specific proteins associated with cartilage regeneration such as Agrin and HSPG2; these together with KTR85 are overexpressed in the condition of sEVs derived from MSCs pretreated with oligomycin (Glycostem). Similarly, we describe a microRNA signature that was found increased only in the condition of sEVs derived from MSCs pretreated with oligomycin. Thus, the invention relates to a composition enriched in extracellular nanovesicles (sEVs) of umbilical cord mesenchymal cells, where the composition consists of.

[0024] - in a set of sEVs that increased expression of AGRN and HSPG2 proteins,

[0025] - and a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Additionally, the nanovesicles contain at least the following miRNAs: m¡R365a-5p, m¡R4257 and m¡R4535 overexpressed. Optionally, the overexpression of KRT85 can also be evaluated. In this composition, the nanovesicles are at a concentration between 5x107 to 1x1013 total particles per mL and have a size between 30 to 300 nm.

[0026] Conveniently, the AGRN and HSPG2 proteins are elevated between 1.5 and 8 times compared to the concentration in the native condition, i.e., in sEVs from UC-MSCs without oligomycin treatment. Normally, the concentration is elevated between 1.8 and 4 times. Optionally, the same occurs with the expression of KRT85.

[0027] In a second embodiment, the invention relates to a method for obtaining the composition, which comprises the following steps: a) culturing umbilical cord blood mesenchymal cells (UC-MSCs) in an induction medium supplemented with 0.01 to 100 pg / ml of Oligomycin for 2 to 48 hours, and subsequently b) isolating the nanovesicles secreted by the MSCs into the medium; c) verifying the increased expression of the AGRN and HSPG2 proteins; and d) resuspending the nanovesicles in a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH of between 6.0 and 8.0.Where, in step b) the nano vesicles are isolated from the culture supernatant by ultrafiltration, ultracentrifugation and / or centrifugation, washed at least twice with a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0, and finally resuspended in a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0. Specifically in step c) the overexpression of the proteins is evaluated with respect to the native condition, UC-MSCs nano vesicles without incubation with oligomycin. Additionally, the nanovesicles contain at least the following miRNAs: m¡R365a-5p, m¡R4257, and m¡R4535, overexpressed relative to a control of nanovesicles obtained from UC-MSCs without incubation with oligomycin. Optionally, KRT85 overexpression is evaluated.Where the composition obtained in step d) is subjected to lyophilization or freezing at -80°C. In another embodiment, the invention relates to a pharmaceutical composition comprising the composition of claim 1 and pharmaceutically acceptable vehicles for intra-articular injections; which is conveniently lyophilized.

[0028] Additionally, the invention relates to the use of the pharmaceutical composition for preparing a medicament useful in the treatment of osteoarticular diseases or osteoarticular trauma. Specifically, it is used for the regeneration of damaged cartilage, prevention of cartilage degradation, or the generation of new cartilage. The pharmaceutical composition is especially useful for the treatment of osteoarthritis, arthritis, and osteoarthritis.

[0029] Finally, the invention relates to a method for the regeneration of damaged cartilage, prevention of cartilage degradation, or for the generation of new cartilage, which comprises the administration of a therapeutically effective amount of the pharmaceutical composition of the invention, which contains the composition enriched in nanovesicles as described.

[0030] Examples

[0031] Example 1. Obtaining and characterizing nanovesicles.

[0032] Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were cultured in DMEM medium supplemented with 10% FBS, Pen / Strept, and glutamine (DMEM-10) until 80% confluence was reached, after which the culture medium was removed and washed once with buffered saline (PBS). Glycolytic stimulation was then prepared by adding fresh DMEM-10 medium supplemented with oligomycin solution containing isomers A, B, and C at a final concentration of 1pg / ml of oligomycin to the UC-MSCs. Cells were incubated with the glycolytic stimulation for 24 h. The oligomycin-containing medium was then removed, and cells were washed with buffered saline (PBS). S-glyco UC-MSCs were obtained (by glycolytic metabolism).These cells were cultured for 48 h in a medium free of animal components, specifically in high-glucose DMEM medium supplemented with only 1% L-Glut, for 48 h, after which the culture supernatant was collected.

[0033] In parallel, the same procedure was performed as a control, without the oligomycin supplementation of the culture, to observe the initial or native condition. The collected supernatant was centrifuged at 600 g at 4°C for 10 minutes to remove impurities, which precipitate.

[0034] The supernatants from the previous centrifugation were processed in an ultracentrifuge at 100,000 g for 1 hour and 10 minutes at 4°C. This step was repeated until the total volume of the supernatant was processed, where the nanovesicles or exosomes were obtained in the pellet. The supernatant was removed and the pellets were released using a vortex mixer. Subsequently, the pellets were subjected to two washes to eliminate possible contaminants. To do this, the pellets were resuspended in filtered PBS reaching a final volume of 10 ml, and centrifuged at 100,000 g at 4°C overnight. The final wash was then performed, for which the supernatants were removed and the pellets were resuspended in a final volume of 10 ml Lactated Ringer (pH 6.5, 273 mOsm / L) and processed for 1 hour and 10 minutes at 4 ° C in an ultracentrifuge at 100,000 g. Finally, the supernatant was removed, the pellets were vortexed and aliquoted for storage.

[0035] The obtained nanovesicles were evaluated, evaluating the overexpressed proteins in the nanovesicles of glycolytic cells, especially the results of the proteins of interest, KRT85, AGRN and HSPG2 with respect to the control condition, without the addition of oligomycin, the results are shown in Table 1. Where AGRN and HSPG2 are proteins associated with cartilage regeneration.

[0036] Based on these results, the inventors decided to determine the concentration of agrin and HSP G2, as a validation signature for these nanovesicles, and also the concentration of KRT85.

[0037] Table 1. Overexpressed proteins in MSC-oligomycin-sEVs condition. N= 4 biological samples. On the other hand, sEVs from glycostem present a microRNA signature that is significantly different from their sEVS-Native counterpart in lyophilized samples. 2067 miRNAs from lyophilized samples of sEVS glycostem and UC-MSC-sEVs were analyzed, of which 990 were found in the 4 biological replicates analyzed. Using the EdgeR tool, 178 genes with significant differences with respect to the native sEVs condition were obtained. Of the total, 175 were found to have increased sEVs relative to the native condition (Log2 fold change >0.5 and <-0.5; Adjusted p-value < 0.05) and 3 were found to have decreased sEVs relative to the native condition (Log2 fold change >0.5 and <-0.5; Adjusted p-value < 0.05). These results are shown in Figure 1.

[0038] For the sEVs-Glyco signature validation, the m¡R with the highest fold change with respect to the native sEVs condition were selected, which correspond to am¡R365a-5p, m¡R4257 and m¡R4535.

[0039] Example 2. In vivo studies.

[0040] To establish the effect of the GlycoStem-sEV nanovesicles of the invention, the evolution of an osteoarthritis model was evaluated when treated according to the invention versus the vehicle-only control and the cell nanovesicle control without pretreatment with oligimycin.

[0041] Healthy C57BL6 / j mice and those in the collagenase-induced OA model, of both sexes, 10-12 weeks old, appropriately randomized into the different study groups: untreated, cells and UC-MSC-sEV nanovesicles in native state and pretreated with oligomycin, GlycoStem-sEV and single administration of the vehicle (of the nanovesicular product formulation) (5 pl in PBS). The experimental design diagram is shown in Figure 2.

[0042] The monitored parameters included: mortality (with cause of mortality determined if possible), clinical observation, body weight, physical examination, and clinical pathology parameters (blood chemistry [liver, kidney, pancreas function, glycemia, various electrolytes], hematology, coagulation, urinalysis), organ weights and their histopathology, and cellular and humoral immune response.

[0043] Specifically, mice were injected intra-articularly (IA) with 5 μL of collagenase type V1 or saline (SHAM). After 7 and 14 days post-induction, animals were treated with 5 μL of saline or native sEVs or glyco sEVs. On day 42, mice were euthanized, and joints were removed for microCT or histological imaging analysis. Clinical scores for microCT and histology were analyzed in different areas of the joint to obtain bone density values ​​and histological joint damage scores.

[0044] The results are shown in Figures 3 and 4. Figure 3 shows the bone density values ​​in the different analysis areas. The data show that UC-MSCs-Glycostem-sEVs significantly improve chondral mineralization compared to native UC-MSCs-sEVs.

[0045] Figure 4 shows the degree of joint degeneration in the analyzed areas associated with OA according to histological scoring. All analyses were performed in a double-blind manner. The data show that Glycostem-sEV significantly reduces OA progression, with a notable increase in hyaline cartilage in the glycostem-sEV group, presenting greater therapeutic efficacy associated with regeneration of the damaged cartilage area in OA.

[0046] These results demonstrate that the nanovesicles of the invention have a direct effect on osteoarthritis (OA) both at the level of chondral mineralization and by reducing joint degeneration.

[0047] Example 3. Post-iophilization stability.

[0048] For this last experimental design, glycostem-derived sEVs were frozen in PBS for at least 48 hours and then divided into two samples, one that was kept at -80° for later use and the other that was lyophilized and kept at 4°C for 24 hours. For lyophilization, the samples were placed in a lyophilizer that operates at an average temperature between -80 and -90°C using a pressure of 200 mmtor. Then, the sample (which was completely frozen from -80) was placed in the equipment. A vacuum was created and the sample was left inside the equipment for two hours.

[0049] Prior to intra-articular injection, lyophilized glycostem-sEVs were resuspended in sterile miliQ water and the equivalent of 2xl0 8 Particles of both frozen and lyophilized glycostem-sEVs were injected into the animals after resuspension.

[0050] Specifically, an intra-articular injection was performed in the joint of mice induced with OA, following the experimental design shown in Figure 2. Interestingly, as shown in Figure 5, the therapeutic capacity is maintained with the lyophilized product I. Figure 5 A shows the bone density values ​​in the different analysis areas. The data show that lyophilized glycostem-sEVs I present the same significant improvement in chondral mineralization as their frozen counterpart, suggesting that they preserve their therapeutic capacity. Likewise, Figure 5 B shows the histological score values ​​according to the modified Pritzker OARSI scale in the different analysis areas. The data show that both frozen and lyophilized glycostem-sEVs I present the same therapeutic activity associated with regeneration of the area, with no significant differences between both groups.

[0051] Therefore, the nanovesicles of the invention maintain their therapeutic activity even after being lyophilized and reconstituted.

Claims

Claims. 1.- Composition enriched in nano extracellular vesicles (sEV) of umbilical cord mesenchymal cells, CHARACTERIZED because the composition consists - in a set of sEVs that have increased expression of the AGRN and HSPG2 proteins, - and a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.

0. 2.- Composition according to claim 1 CHARACTERIZED in that the nanovesicles contain at least the following miRNA: m¡R365a-5p, m¡R4257 and m¡R4535 overexpressed. 3.- Composition according to claim 1 CHARACTERIZED in that the nanovesicles have an increased expression of the KRT 85 protein. 4.- Composition according to claim 1 CHARACTERIZED in that the nanovesicles are in a concentration of between 5xl0 7 to lxlO 13 total particles per mL. 5.- Composition according to claim 3 CHARACTERIZED in that the nanovesicles have a size between 30 to 300 nm. 6.- Method for obtaining the composition of claim 1 CHARACTERIZED in that it comprises a) culturing umbilical cord mesenchymal cells (UC-MSCs), in an induction medium supplemented with 0.01 to 100 pg / ml of Oligomycin, for 2 to 48 hours, and subsequently b) isolating the nanovesicles secreted by the MSCs into the medium, c) checking the increased expression of the AGRN and HSPG2 proteins; and d) resuspending the nanovesicles in a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.

0. 7.- Method according to claim 6 CHARACTERIZED in that in step b) the nanovesicles are isolated from the culture supernatant by ultrafiltration, filtration, ultracentrifugation and / or centrifugation, they are washed at least twice with a medium or vehicle free of animal components and proteins, with an osmolality of between 250 and 310 mOsmol / L and a pH between 6.0 and 8.0, and finally they are resuspended in a medium or vehicle free of animal components and proteins, with an osmolality between 250 and 310 mOsmol / L and a pH between 6.0 and 8.

0. 8.- Method according to claim 6 CHARACTERIZED in that in step c) the overexpression of the proteins is evaluated with respect to the native condition, UC-MSCs nanovesicles without incubation with oligomycin. 9.- Method according to claim 8 CHARACTERIZED in that the nanovesicles have an increased expression of the KRT 85 protein. 10.- Method according to claim 6 CHARACTERIZED in that the nanovesicles contain at least the following miRNA: m¡R365a-5p, m¡R4257 and m¡R4535 overexpressed with respect to a control of nanovesicles obtained from UC-MSCs without incubation with oligomycin. 11.- Method according to claim 6 CHARACTERIZED in that the composition obtained in step d) is subjected to lyophilization or freezing at -80°C. 12.- Pharmaceutical composition CHARACTERIZED in that it comprises the composition of claim 1 and pharmaceutically acceptable vehicles for intra-articular injections. 13.- Pharmaceutical composition of claim 12 CHARACTERIZED in that it is lyophilized. 14.- Use of the pharmaceutical composition of claim 10 CHARACTERIZED in that it serves to prepare a medicine useful in the treatment of osteoarticular diseases or traumas. 15.- Use, according to claim 12, CHARACTERIZED in that the composition is useful in the regeneration of damaged cartilage, prevention of cartilage degradation or in the generation of new cartilage. 16.- Use, according to claim 12, CHARACTERIZED in that the composition is useful for the treatment of osteoarthritis, arthritis, osteoarthritis.

Images