Rbcs-derived extracellular vesicles loaded with cargo molecules for therapeutic applications and method of production thereof

Abstract

The present invention relates to a method for preparing Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo and the RBCEVs obtainable by such method. In particular, the invention relates to such method and product, wherein the loaded cargo is a therapeutic or diagnostic compound and their use as medicament in a method of treatment.

Description

[0001] RBCs-derived Extracellular Vesicles Loaded with Cargo Molecules for Therapeutic Applications and Method of Production Thereof

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to a method for preparing Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo and the RBCEVs obtainable by such method. In particular, the invention relates to such method and product, wherein the loaded cargo is a therapeutic or diagnostic compound and their use as medicament in a method of treatment.

[0005] State of the art

[0006] Extracellular vesicles (EVs) are lipid bilayer-delimited particles naturally released from almost all types of cells (donor cell) and that can be internalized by recipient cells. EVs have been proposed as a next generation drug delivery system starting from the observations that EV can contribute to cell-to-cell communication and can deliver molecules of biological relevant interest from the cell of origin to the target cell that incorporate the EV. Based on these observations, many researchers tried to isolate, in vitro or ex vivo, native EVs from primary or cultured cells for therapeutic purposes confirming the original observation. Unfortunately, the amount of EVs needed for therapeutic applications is significantly high and the procedure must be reproducible to move toward clinical investigations. In addition, most of the methods used are not compliant with these key requirements and significant purification steps are necessary, which are time-consuming and provide limited yields of the final desired product. The gold standard method for EVs production uses antibiotics, such as ionomycin, and does not allow translation to the clinics. Moreover, in the chemical vesiculation method there is the exposure of phosphatidylserine (PS), as a consequence of calcium entry and membrane rearrangement, favors the recognition of the EVs by the reticuloendothelial system shortening their lifespan in the circulation. Moreover, the yield of EVs production and the loading efficiency remain challenging. Red Blood Cells (RBCs) have long been used in transfusion medicine and more recently as drug delivery systems. A large number of preclinical and clinical applications rely on their unique biological features that: i. permits easily to obtain these cells from any donor or patients, including children, in large amounts, repeatedly and without arming the donor and, ii. following the discovery that RBCs can be modified by the encapsulation of active agents for a number of biomedical applications already in late stages of clinical development, they can be considered one of the most useful sources for EVs development. Regarding Red Blood Cell-derived Extracellular Vesicles (RBCEVs), these can be naturally produced during the life of the cell but their production can also be artificially induced. Unfortunately, none of the proposed methods or approaches is a solution to the problem of generating RBCEVs in large amounts and in a reproducible way with reasonable efforts and able to deliver a multitude of agents.

[0007] Accordingly, there is still an urgent need in the field for the development of effective methods for preparing loaded RBCEVs and providing effective RBCEVs loaded with a cargo molecule.

[0008] Summary of the Invention

[0009] As will be further detailed in the experimental section of the present specification, the inventors have surprisingly found a method for preparing RBCEVs loaded with cargoes that overcomes the use of chemical agents to induce the production of EVs, as well as the poor yield and reproducibility. Unexpectedly, the inventors demonstrated that the method of the present invention permits to obtain an extremely high yield of RBCEVs when performing the extrusion step at low haematocrits, in contrast with the general opinion that a high number should be expected starting with a high number of cells. Furthermore, the inventors found that the loading efficiency is higher if the RBCEVs production follows the encapsulation of the cargo in the mother cell. Moreover, the inventors found that the produced RBCEVs loaded with a cargo molecule of interest are very effective in delivering the same to target cells and exerting a biological effect.

[0010] In certain aspects, the invention thus provides a method for preparing a population of Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo comprising a step of extruding a composition comprising or consisting of Red Blood Cells (RBCs) loaded with said cargo and optionally comprising a further step of isolating and / or purifying said RBCEVs obtained by such extrusion.

[0011] In certain aspects, the invention provides RBCEVs loaded with a cargo obtainable by the method herein disclosed.

[0012] In certain aspects, the invention provides a population of RBCEVs, loaded with a cargo, characterized by a size distribution with a mode between 70 and 220 nm, more preferably between 90 and 150 nm.

[0013] In certain aspects, the invention provides a population of RBCEVs, loaded with a cargo, characterized by a membrane architecture and surface antigens that mirrors those of mother RBCs,

[0014] In certain aspects, the invention provides a population of RBCEVs, loaded with a cargo, characterized by a percentage of positivity to glycophorin A at least 60%, preferably at least 70%, the CD47 expression of at least 60%, preferably at least 70% and the PS exposure lower than 30%, preferably lower than 20%.

[0015] In certain aspects, the invention provides a population of RBCEVs, loaded with a cargo, characterized by resealed nanovesicles, each of them delimited by their cell membrane, as showed by TEM analyses, in particular they are not nanoparticles belonging from the aggregation of cell debris and / or disorganized membrane components.

[0016] In certain aspects, the invention provides a pharmaceutical composition comprising the RBCEVs herein disclosed and one or more pharmaceutically acceptable excipients.

[0017] In certain aspects, the invention provides pharmaceutical composition comprising the RBCEVs herein disclosed for use in a method of treatment.

[0018] Brief description of the drawings

[0019] Fig. 1. Schematic representation of some steps of the method according to an embodiment of the invention. The method ensures the formation of EVs in a soft way that, on one hand, can preserve membrane integrity by low pressure-induced membrane budding and, on the other hand, ensure the maintenance of the cargo inside the newly formed RBCEVs thanks to the resealing of membrane after budding.

[0020] Fig. 2 - Evaluation of the loading efficiency of FITC-dextran into RBCs by Flow Cytometry . Fig. 2A) Mean fluorescence intensity found in L samples compared to UL samples. Fig 2B) Ratio calculated between L and UL samples. Histograms clearly show the higher loading efficiency of dextran into RBCs.

[0021] Fig. 3. NTA characterization of the RBCEVs. Fig. 3 A) Representative distribution plots of the UL and L RBCEVs diameter, respectively. Fig. 3B) Histograms of the physical parameters of the distribution. Bar graphs showing inter-experiments variability over several replicates (n=9) in terms of mode and mean of the RBCEVs size (particle diameter) and yield (particle number). As shown, no significant difference can be observed between UL and L samples for all the considered parameters, confirming the possibility of encapsulating cargoes inside RBCEVs (T-test (n=9), P>0.05).

[0022] Fig. 4. Transmission electron microscopy analysis of negatively stained RBCEVs. These representative pictures were obtained at 20-50, OOOx magnification and represent the size distribution of the obtained RBCEVs (50-200 nm) that correlates quite well with the NTA characterization shown in Fig. 3. Scale bar = 100 nm.

[0023] Fig. 5. Dot plots of LCD positivity. (A) and bar graph of MFI values for both UL and L RBCEVs (B) The black arrow in panel A indicates the shift in fluorescence, highlighting the specific signal of the probe. In blue, enclosed by gate P3, Dako Cytocount beads are traceable. No statistical difference can be observed between UL and L positivity in graph B. Fig. 6. Flow Cytometry characterization of RBCEVs: GYPA positivity. In (A) setting up of the marker to evaluate glycophorin A positivity. In (B) Overall statistical evaluations for PE fluorescence- GYPA RBCEVs percentages. In (C ) UL and L RBCEVs glycophorin A profiles.

[0024] Fig. 7. Flow cytometry characterization of RBCEVs: CD47 positivity. In (A) setting up of the marker to evaluate CD47 positivity. In (B) Overall statistical evaluations for PE fluorescence- CD47 RBCEVs percentages. In (C) UL and L RBCEVs CD47 profiles.

[0025] Fig. 8. Flow cytometry characterization of RBCEVs: Annexin V positivity. Bar graphs show the low mean percentages of Annexin V positivity for both UL and L RBCEVs. No statistical difference can be found between L and UL samples.

[0026] Fig. 9 - NTA evaluation of Cascade Blue-Dextran loading into RBCEVs. Dot plots showing vesicle number and size distribution using scattering (A) and fluorescence (B) in L samples compared to UL as control. Representative snapshots of the Cascade Blue positive particles in UL and L samples (C). Data prove that loading of the selected cargo was efficiently achieved.

[0027] Fig. 10- Bar graph of MFI Cascade blue in UL and L RBCEVs. An extremely significant higher value is shown for L samples compared to UL ones.

[0028] Fig. 11. Evaluation by FC of FITC-dextran loading into RBCEVs. A) Dot plot showing FITC positivity of LCD positive events in L samples compared to UL controls. B) Representative histogram overlay for UL green fluorescent RBCEVs (grey histogram) and for L green fluorescent-FITC dextran (red histogram) RBCEVs evaluation. C) Bar graph reporting MFI values from the same, representative experiment. D) Statistical evaluations for green fluorescence MFI in UL and L RBCEVs. E) MFI ratio (L / UL RBCEVs) calculated from all the dextran-conjugates loading.

[0029] Fig. 12. RBCEVs uptake in HUVEC at 24 h-incubation and evaluation of cell vitality.

[0030] A) Dot plots of 7’ AAD to assess eventual cell death in HUVEC treated with RBCEVs or not. B) Histograms of PHK26 positivity in HUVEC treated with labelled RBCEVs or not. C) Confocal images of PHK26-RBCEVs (red) into HUVEC cells (green). D) Bar graphs of PHK26 MFI in HUVEC treated with labelled RBCEVs compared to untreated ones. E) Bar graphs of 7’ AAD percentage of positivity in HUVEC treated or not. Cytometry and confocal analyses both demonstrate high uptake of RBCEVs after 24 h and no toxicity in HUVEC.

[0031] Fig. 13 RBCEVs uptake into PBMCs at 24 h-incubation and evaluation of cell vitality.

[0032] A) Dot plots of FSC and SSC to identify cell subpopulations (i.e., lymphocytes and monocytes) into the isolated PBMCs. B) Histograms of PHK26 positivity into lymphocytes subpopulation treated or not with labelled RBCEVs. C) Histograms of PHK26 positivity into monocytes subpopulation treated or not with labelled RBCEVs. D) Bar graphs of PHK26 MFI in treated PBMCs compared to untreated ones. E) Bar graphs of 7’ AAD-positive percentages into control or treated PBMCs. F) Confocal images of PBMCs treated or not with PHK26-RBCEVs. Cytometry and confocal analyses both demonstrate high uptake of RBCEVs after 24 h and no toxicity in PBMCs.

[0033] Fig. 14. RBCEVs uptake at 4 h-incubation in HUVEC and PBMCs. A) Histograms of PHK26 positivity in HUVEC treated or not with labelled RBCEVs for 4 h. B) Histograms of PHK26 positivity into lymphocyte subpopulation treated or not with labelled RBCEVs for 4 h. C) Histograms of PHK26 positivity into monocytes subpopulation treated or not with labelled RBCEVs for 4 h. D) Time-course of PHK26 MFI at 0, 4 and 24 hours of incubation into the selected cell lines.

[0034] Fig. 15. Absolute quantification of miR-210 into RBCs and RBCEVs by qPCR. Absolute miR-210 concentrations have been calculated by a standard curve set up with synthetic RNA standards at 20-0.0002 nM. miR-210 concentration into loaded RBCEVs are compared to UL RBCEVs and to the starting concentration obtained into the mother RBCs (UL and L) before vesiculation. High loading efficiency can be appreciated. The amount of miR-210 found in UL RBCs and RBCEVs are due to the endogenous miRNA. Data are mean and SEM (n=4).

[0035] Fig. 16. Relative quantification of miR-210 into HUVEC. Relative quantification has been performed using U6 snRNA as reference gene and UL sample as control. miR-210 concentration found in HUVEC treated with L RBCEVs are compared to cells treated with UL RBCEVs and to cells transfected at different miRNA concentrations. Data are mean and SEM, n=4 (Unpaired t-test; *two-tailed p-values<0.05).

[0036] Fig. 17. Evaluation of the effect of miR210-loaded RBCEVs at the mRNA level. Relative quantification of PTBIB mRNA has been performed using ATCB as reference gene and UL sample as control. PTP1B mRNA found in HUVEC treated with L RBCEVs is compared to cells treated with UL RBCEVs and to cells transfected at different miRNA concentrations. Data are mean and SEM, n=4 (Unpaired t-test; *two-tailed p-values<0.05).

[0037] Fig. 18. Evaluation of the effect of miR210-loaded RBCEVs at the protein level. A) Western blot of PTP1B in protein extracts from HUVEC treated with RBCEVs UL or L and transfected with different amounts of miR-210. B) Quantification of PTP1B band normalised to total proteins. Data are the mean and SEM, n=4 (Unpaired t-test; p-values *<0.05, ***<0.001, ****<0.0001).

[0038] Detailed description of the invention

[0039] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), incorporated herein by reference. The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0040] The term “extracellular vesicle” (EV) as used herein refers to a small plasma membrane derived extracellular vesicles structure released from a cell into the extracellular environment. In particularly preferred aspects disclosed herein, the extracellular vesicles are derived from red blood cells (RBCEVs).

[0041] EVs are substantially spherical lipid-based nanoparticles with a composition similar to plasma membrane between 50 and 1000 nm in diameter.. Extracellular vesicles have a membrane.

[0042] The membrane may be a double layer membrane (i.e., a lipid bilayer). The membrane may originate from plasma membranes. Accordingly, the membrane of the extracellular vesicle may have a similar composition to the cell from which it is derived.

[0043] Extracellular vesicles may be classified as exosomes, microvesicles or apoptotic bodies, based on their size and origin of formation. Microvesicles are a particularly preferred class of extracellular vesicles according to the invention disclosed herein. Preferably, the extracellular vesicles of the invention have been shed from the plasma membrane, and do not originate from the endosomal system. In preferred aspects described herein, the extracellular vesicles are red blood cell derived extracellular vesicles, derived from the plasma membrane of a red blood cell through outward budding and fission of the plasma membrane.

[0044] In this description, the expression "loaded erythrocytes with a cargo" means erythrocytes (also referred to as red blood cells or RBCs) that encapsulate variable amounts of one or more compounds of interest, in particular exogenous compounds of interest, wherein exogenous compound means a compound that are not naturally present in the erythrocytes. In this description, the expression "loaded Red Blood Cell-derived Extracellular Vesicles with a cargo " means Red Blood Cell-derived Extracellular Vesicles (also referred to as RBCEVs) that encapsulate variable amounts of one or more compounds of interest, in particular exogenous compounds of interest. A population of RBCEVs may be used to indicate a plurality of RBCEVs.

[0045] In this description, the term “cargo” is used interchangeably with “load” herein.

[0046] In this specification the term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). The term "polypeptide" encompasses native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence.

[0047] For the purposes of the present invention, and in the specific technical field, the terms "lysis" or "hemolysis" or "partial lysis" mean the reversible opening of the pores on the cell membrane with consequent free passage in both directions of intra- and extra-cellular materials. Therefore, lysis is a phenomenon of temporary and reversible permeabilization and does not involve a complete and irreversible rupture of the cell membrane.

[0048] It follows that the term "lysed erythrocyte" refers to an erythrocyte whose plasma membrane features pores that can be reclosed in such a way that the integrity of the cell membrane is restored.

[0049] For the purposes of the present description, the expression "(re)sealing solution" means a solution used that is able to close the pores in the plasma membrane of the erythrocytes. This solution allows to encapsulate the substance(s) of pharmaceutical interest within the erythrocytes thanks to the opening of said pores.

[0050] For the purposes of the present invention, the expression "sealed erythrocytes" refers to red blood cells which, unlike the lysed erythrocyte, feature a plasma membrane permeability comparable to (overlapping with) that of untreated red blood cells.

[0051] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0052] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0053] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0054] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0055] A first object of the present invention is a method in vitro or ex vivo for preparing a population of Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo comprising a step a) of extruding a composition comprising or consisting of a Red Blood Cells (RBCs) loaded with said cargo, across a membrane and optionally a further step b) of isolating and / or purifying said extruded RBCEVs by the step a).

[0056] The composition of a Red Blood Cells (RBCs) used in the method of the invention comprising or consisting of a population of loaded RBCs with one or more cargoes. The composition of RBCs is preferably a blood sample or a derivate of a blood sample, obtained from a human subject. In some embodiments, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, at least about 97% or 100% of the RBCs encapsulate at least one cargo. In some embodiments, the red cells or red cell precursors may be modified or engineered the cargo. In some embodiments, the loaded RBCs comprise on average at least about 5,000, 50,000, 100,000, 150,000, 200,000, 1,000,000 or more molecules of cargo per each red cell. Preferably, the composition of a blood sample used in the extrusion step has a haematocrit (cell percentage volume / volume) between 4 and 8%, preferably between 5 and 7%, more preferably about 6%. In one embodiment the blood sample is characterized by depletion of leukocytes, in particular of granulocytes, preferably at least 80%, 85%, 90%, 95%, at least about 97% of said depletion. The cargo may be encapsulated in the RBCs according to any methods known in the art, preferably according to the procedures disclosed in Magnani, Panzani et al. 1998 or in EP2994117B 1 (both documents herein incorporated by references). According to one embodiment the composition of loaded RBCs is prepared from a blood sample by the following process: a) swelling the erythrocytes using a first hypotonic solution, wherein said first solution, brings the erythrocytes to an osmolality between 250-200 mOsm / Kg; b) further swelling the erythrocytes obtained in step a), without reaching the lysis, using a second hypotonic solution more hypotonic than the first hypotonic solution, wherein said second hypotonic solution brings the erythrocytes to an osmolality between 200 and 170 mOsm / Kg; c) concentrating the erythrocytes obtained in step b); d) placing the concentrated erythrocytes in contact with a solution comprising one or more cargoes, and subsequently e) adding a resealing solution for the purpose of obtaining a population of erythrocytes loaded with said cargo. The cargo loaded in the RBCs and consequentially in the derivative RBCEVs may be any therapeutic or diagnostic compounds, any active ingredients, in particular any active ingredient with a specific therapeutic effect. For example peptides, oligopeptides, proteins, polypeptide, DNA, mRNA, circRNA, miRNA, siRNA, CRISPR / CAS9 and gRNA complexes, CRISPR / deaminase (base editor) and gRNA complexes, oligonucleotides, antisense oligonucleotides, nucleotide analogs, nucleosides, nucleoside analogs, hormones, immunosuppressant, inhibitors of malignant cell growth, corticosteroids, glucocorticoids, anti-retroviral and non-steroidal anti-inflammatory agents, cytokines, toxins, immunogens, contrast media for diagnostics; particles or nanoparticles selected from nanoparticles containing a metal, magnetic nanoparticles, super-paramagnetic nanoparticles (SPIO), and nanoparticle-active molecule complexes. Specific examples of cargo as therapeutic compound may be 6-mercaptopurine, fludarabine phosphate, phosphorylated azidothymidine, dideoxycytosine, dideoxyinosine, glutathione, bisphosphonates, prednisolone, prednisolone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, betamethasone, betamethasone sodium phosphate, thymidine phosphorylase, phenylalanine ammonia lyase, indocyanine green, and super-paramagnetic particles, dexamethasone and beta dexamethasone, also in form of phosphate, and deflazacort, prodrugs, namely precursors of bioactive ingredients. In one embodiment such cargo is the miR- 210. The RBCEVs loaded with the miR-210 may be used for the treatment of diabetes, cardiovascular and neurodegenerative diseases.

[0057] Following the extrusion step, the method may comprise a step of isolating and / or purifying said extruded RBCEVs. RBCEVs disclosed herein may be isolated and / or purified by standard techniques, including but not limited to centrifugation, ultracentrifugation, chromatography and combinations thereof. In one embodiment the purification step to obtain a purer preparation of loaded RBCEVs. may comprise one or more centrifugation and / or an ultracentrifugation step. In one preferred embodiment the purification step comprises or consisting of an ultracentrifugation step performed once at 50,000 x g, preferably for at least about 1 hr, more preferably at about 4° C. In one embodiment the purification step comprises also a centrifugation step at 3,000 x g, preferably for 5-10 mins at room temperature, more preferably followed by a filtering step through a 0.45 pm filter. The centrifugation step is advantageously performed before an ultracentrifugation step.

[0058] In one embodiment the method comprises a reannealing step before the purification step, wherein the RBCEVs product extracted in step a) is incubated at 37°C at least for about 5 min.

[0059] The method of the invention comprises a step a) of extruding loaded Red Blood Cells (RBCs) across a membrane, preferably said membrane is a polycarbonate membrane. According to one preferred embodiment the composition of loaded Red Blood Cells is extruded across a first membrane of 5-pm and a second membrane 1-pm membrane. Such extrusion step a) consist of at least 1, 2, 3 or 4 cycles or more cycles, preferably at least 3 or 4 cycles.

[0060] A further object of the present invention is a population of RBCEVs obtainable by the method herein disclosed, in particular wherein said cargo is a therapeutic or diagnostic compound.

[0061] In one embodiment the invention provides a population of RBCEVs, loaded with a cargo, characterized by a size distribution with a mode between 70 and 220 nm, more preferably between 90 and 150 nm. Mode of RBCEV distribution is the particle diameter value that appears most often in the EV population. Mean of distribution is the point in the size distribution where 90% of the sample is contained. In one embodiment, the invention provides a population of RBCEVs, loaded with a cargo, characterized by a membrane architecture and surface antigens that mirrors those of mother RBCs, and by a percentage of positivity to glycophorin A at least 60%, preferably at least 70%, the CD47 expression of at least 60%, preferably at least 70% and the PS exposure lower than 30%, preferably lower than 20%.

[0062] In some embodiments, the invention provides a population of RBCEVs wherein each RBCEV of the population encapsulated at least at least about 500, 5,000, 10,000, 15,000, 20,000, 100,000 or more molecules. In some embodiments, the invention provides a population of RBCEVs wherein at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, at least about 97% or substantially 100% RBCEVs of the population encapsulate at least one cargo.

[0063] According to one embodiment, said population of loaded RBCEVs are derived from a human or animal blood sample or red blood cells derived from primary cells or immobilized red blood cell lines. The blood cells may be type matched to the patient to be treated, and thus the blood cells may be Group A, Group B, Group AB, Group O or Blood Group Oh. For example, the blood is Group O. The blood may be rhesus positive or rhesus negative. In some cases, the blood is Group O and / or rhesus negative, such as Type O-. The blood may be free from disease or disorder, such as free from HIV, sickle cell anaemia, malaria. However, any blood type may be used. In some cases, the RBCEVs are autologous and derived from a blood sample obtained from the patient to be treated. In some cases, the loaded RBCEVs are allogenic and not derived from a blood sample obtained from the patient to be treated.

[0064] A further object of the present invention is a pharmaceutical composition comprising loaded Red Blood Cell-derived Extracellular Vesicles obtainable according to the method of the invention and a pharmacologically acceptable excipient and its use as a medicament in a therapeutic treatment. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient or stabilizer.

[0065] In another aspect of the present disclosure a method of treating a subject in need of treatment is provided, the method comprising administering to the subject a therapeutically effective amount of loaded RBCEV, as described herein or a pharmaceutical composition comprising a population of loaded RBCEVs, as described herein, thereby treating the subject. Extracellular vesicles and pharmaceutical compositions described herein may be administered, or formulated for administration, by a number of routes, including but not limited to systemic, intratumoral, intraperitoneal, parenteral, intravenous, intra-arterial, intradermal, subcutaneous, intramuscular, intravitreal, sub-retinal, oral and nasal. The medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body to treat.

[0066] A population of RBCEVs, as described herein or a pharmaceutical composition comprising said population of loaded RBCEVs may be used in the treatment of treating diseases including, but not limiting to cancers, liver, diseases (hemochromatosis, chronic hepatitis, hepatic tumor), thrombosis, excess iron, parasitic diseases, viral and / or bacterial infection, diabetes, cardiovascular disease, neurodegenerative diseases, osteoporosis, anemia, lung infection, respiratory diseases, coagulopathies, and immune disorders. In some embodiments, RBCEVs may also be loaded with mRNAs encoding a protein suitable for use in enzyme replacement therapy (ERT) or gene therapy applications.

[0067] The following experimental section is provided solely by way of illustration and not limitation and does not intend to restrict the scope of the invention as defined in the appended claims. The claims are an integral part of the description.

[0068] EXAMPLES AND EXPERIMENTAL SECTIONS

[0069] 1. MATERIALS AND METHODS

[0070] Loading of cargoes into human RBCs

[0071] Red Blood Cells (RBCs) have been purified from fresh whole blood samples collected in EDTA from healthy donors afferent to the Transfusion Center of the Presidio Ospedaliero Unico "Santa Maria della Misericordia" Urbino (PU). Whole blood samples have been centrifuged at 1,800 x g at 4°C for 10 min to allow separation of cells from plasma, then RBCs were purified through two washes in Hepes solution (HEPES lOmM, NaCl 154 mM, glucose 5mM) at 300 mOsm or IX Phosphate-Buffered Saline (PBS, 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HPO4, and 1.8 mM KH2PO4) at pH 7.4 to remove white blood cells. Total depletion of the remaining leukocytes (mainly granulocytes) from the RBCs sample was carried out by filtration with White Blood Cell Acrodisc® Syringe Filter (PALL). At the end of purification, removal efficiency has been assessed and the haematocrit (Het) of the purified RBCs was evaluated by ABX Micros automated haematology analyzer (Horiba). The purified RBCs were resuspended in 70% Hepes for the next step. The loading process was accomplished with the “hypotonic dialysis and isotonic resealing” method as already reported (Magnani et al., 1989). Briefly, RBCs were put inside a cellulose dialysis bag and the bag was placed in 50 mL of hypotonic solution at 60 mOsm (NaH2PO4 lOmM, NaHCO3 lOmM, glucose 20mM, ATP 2mM, GSH 3mM) or 0.2-0.3X PBS and let incubate on a rotating plate at 4°C for 60-90 min. At the end of the dialysis step, dialysed RBCs were collected from the bag and subjected to osmolarity measurement using the Osmomat 3000 osmometer (Gonotec) to assess the opening. Then, resealing of the membrane pores was performed by the addition of 0.1 volumes of PIGPA (a hypertonic solution at 3,000 mOsm containing inosine lOOmM, ATP 20mM, glucose lOmM, sodium pyruvate lOOmM, MgC12 4mM, NaCl 190mM, KC1 1.666mM, NaH2PO4 33mM). The reannealing of membrane segments was achieved by incubation of the same for 25 min at 37°C. Finally, the washing of unincorporated cargoes was carried out by two washes in Hepes and subsequent centrifugation at 1,500 x g for 10 min at 4°C. Encapsulation of dextran-conjugates (Fluorescein isothiocyanate-dextran average Mw 70,000 Da, Sigma-Aldrich and Dextran- Cascade Blue average Mw 10,000 Da, ThermoFisher Scientific) has been carried out by adding the conjugates into the dialysis bag (14.4 and 3.5 kDa MWCO cellulose tube, respectively) during the dialysis step. Whereas, for the encapsulation of synthetic miR-210 purchased by, Merk), this was resuspended at a concentration of 200 pM in RNase-free water and added to the swelled (opened) RBCs for 30 minutes at 37°C, before the addition of PIGPA. All the procedures were performed under sterile conditions. For miR-210 loading, all the reagents were treated with Diethyl Pyrocarbonate (DEPC) 0.1% and the used plasticware was RNase-free. In each experiment, an "un-loaded" (UL) control sample, obtained from RBCs subjected to the same dialysis procedure but without the addition of exogenous molecules, was prepared.

[0072] Production of RBCEVs by physical vesiculation method

[0073] At the end of the loading procedure, unloaded and loaded RBCs were diluted at different haematocrit (cell percentage volume / volume) pre-warmed PB S and then extruded for 4 times across polycarbonate (PC) membranes (Avanti Polar Lipids) of different size pores using a Mini-Extruder kit (Avanti Polar Lipids). The process was conducted at room temperature or at 37°C but not higher temperature to not denature macromolecules. The final optimized protocol envisaged resuspension of RBCs at 6% in PBS, serial extrusion across 5-pm and 1- pm membrane for 4 cycles each and temperature maintenance at 37°C during the whole procedure. To allow perfect reannealing of the EV membranes, a step was included in which the samples were incubated at 37°C for 5 min before purification. In Fig. 1 a schematic representation of the process.

[0074] The process herein disclosed has been demonstrated by using a lab-scale mini-extruder but can easily be scaled-up by the use of automated extruders already used in the clinical settings for the production of liposomes.

[0075] Characterization by DLS

[0076] The products obtained at the end of the extrusion processes were initially characterised in terms of average particle size and poly dispersity index (PDI) using a Malvern Zetasizer Nano S instrument (Malvern Instrument ltd, UK). Before the measurements, the formulations were diluted at 1 :20 in distilled water. The measurements were performed at the end of all the extrusion steps to evaluate the influence of the extrusion process on the physical parameters evaluated and to have a confirmation of the reproducibility of the process between the produced batches. The final physical characteristics of the produced vesicles were studied after the purification process using the Nanoparticle Tracking Assay technique as reported below.

[0077] Purification of RBCEVs

[0078] After the extrusion, EV suspension, containing the produced vesicles but also contaminants, was subjected to an initial centrifugation at 3,000 x g for 10 min at room temperature to remove cell debris, and then the supernatant was filtered through a 0.45 pm PES filter (Pall). Purification of the RBECVs was first optimised through serial ultracentrifugation at increasing speeds (20,000 x g, 50,000 x g, and 110,000 x g) to select the best performing one and finally performed through a single ultracentrifugation step at 50,000 x g at 4°C for 1 h with Beckman Rotor type 90Ti (Beckman). At the various steps, the samples were collected and analysed by different techniques to monitor the process and characterise the final products.

[0079] Characterization by Nanoparticle Tracking Assay (NTA)

[0080] NTA measurements were performed with a NanoSight LM10 (NanoSight, Amesbury, United Kingdom) and three videos of either 30 or 60 s were recorded of each sample. All measurements were performed at room temperature, never above 25 °C. The software used for capturing and analysing the data was the NTA 3.1 (Nanosight). Data are presented as the mean ± SD of the three video recordings. Samples containing high particle numbers were diluted before analysis and the relative concentration was then calculated according to the dilution factor. 100 and 400 nm beads, supplied by Malvern Instruments Ltd. (Malvern, UK), were used as control.

[0081] Characterization by Flow Cytometry (FC)

[0082] Pre-analytic conditions: As recommended by Welsh and Van der Pol (Welsh, Van Der Pol et al. 2020) we adopted several measures to ensure a reliable EVs detection by Conventional Flow Cytometry. The Flow cytometer instrument is a FACScanto II, equipped with three lasers (488 nm, 633 nm, and 405 nm, BD Biosciences). Firstly, to minimise the “swarm effect” we diluted EVs from each sample, at least 1 :40 based on the EVs absolute counts obtained by NTA (van der Pol, van Gemert et al. 2012). Each buffer and solution for diluting, labelling (i.e., binding buffer for Annexin V staining), and preparing samples for Cytometry acquisition were filtered by 0.22 pm membrane filters. Indeed, different reagents were employed to detect: i., Glycophorin A (anti-GYPA PE mAb, Clone:GA-R2 (HIR2) BD Biosciences), ii., CD47 as “don’t eat me signal” (anti CD47 PE mAb, clone 07, Sino biological) and iii., Annexin V PE (abl4155, Abeam) for extruded phosphatidylserine and iv., anti-CD44 PE as irrelevant mAb (clone J.173, Beckman Coulter) As yet proposed, we stained EVs with a lipophilic cationic dye (LCD, kit. Cat. 626267, BD Biosciences, Custom) recently identified as a generic EV tracer, highly useful when samples containing heterogeneous EV population need to be analysed (Brocco, Lanuti et al. 2019) or when EVs are artificially produced and do not express the classical tetraspanin markers (CD9, CD81, CD63), as in our study. All reagents were spun at 21000 g, following general guidelines, as in (Simeone, Celia et al. 2020). The Flow cytometer cleaning is vital and involves the use of detergent, bleach, and DI water. We run FACSrinse for 10 minutes on HIGH, followed by 10% bleach for 10 minutes, and 0.2um filtered DI water for 10 minutes to sufficiently clean the sample line.

[0083] Sample composition and acquisition: EV size distribution and concentration were evaluated before analysing the samples by flow cytometry (FC), following FC guidelines. 50 microliter of UL and L EV samples (diluted at least 1 :40 from the initial NTA counts) were placed at the bottom of the tube and 1 microliter of LCD was placed in each tube. The antiCD47 PE- conjugated mAb (1,5 microliter), anti-glycophorin PE-conjugated mAb (4,5 microliter), and Annexin V PE-conjugated (6,5 microliters in 200 microliters Binding Buffer) and anti-CD44 PE as irrelevant mAb) (2 microliters) were added. To select EVs, we set the trigger threshold on SSC, following Megamix plus (Biocytex) and Rosetta beads (Exometry) calibration System and contemporary we selected the true EVs events from the channel in which the LCD emits (APC -Allophycocyanin). FSC and SSC parameters were set on a log scale.

[0084] Each tube was collected further gating the EV dimensions (EVs 100-900 nm): in particular, datasets were acquired from the following tubes:

[0085] 1) unlabelled UL and L;

[0086] 2) LCD-labelled UL and L;

[0087] 3) LCD and anti-GYPA PE mAb labelled UL and L;

[0088] 4) LCD and anti-CD47 PE mAb labelled UL and L;

[0089] 5) LCD and Annexin V-labelled UL and L.

[0090] Finally, other tubes have to be added to the above-cited list:

[0091] 6) Buffer only;

[0092] 7) Buffer plus reagents (e.g. antibodies); same concentration of reagents used with EVs;

[0093] 8) Irrelevant mAb, to be employed as Isotype controls, besides RBCEVs autofluorescence.

[0094] All the tubes were gently mixed and incubated in the dark at RT for 40 minutes. 400 microliter of PBS (or Annexin V buffer) was added to each tube, and the contents were analysed by FACS within 2 hours. Finally, in starting experiments, Triton-X 100 was added to a final 2% concentration to the RBCEV samples. This is based on the fact that lipid membrane enclosed vesicles are more sensitive to detergent lysis than protein aggregates.

[0095] At the end of each run, a fresh tube of filtered buffer in between EV samples was inserted, since carryover from a previously run sample into the next sample should be negligible. An acceptable background level was obtained using 0.22um filtered PBS or DI water at the same flow rate as the samples. A low flow rate is recommended (Inglis, Danesh et al. 2015). Acceptable background levels (expressed as events per second) were considered <100 events / sec with triggering on triggering scatter. Our protocol takes into account the FMO (Fluorescence Minus One) controls. This control may help determine if FRET or quenching is occurring from the use of multiple fluorescent reagents.

[0096] Final analysis: Analyses were conducted gating on the FSC log vs SSC log plots, then combining LCD positivity (Boolean gating) and finally showing RBCEVs events in the FL2 -PE histograms, to evaluate glycophorin A, CD47, and Annexin V positivity, as well as in the FL1-FITC histogram to evaluate the presence of loading.

[0097] TEM

[0098] For transmission electron microscopy analysis, specimen drops were deposited on formvar- carbon-coated 300 mesh grids. They were immediately fixed with 2.5% glutaraldehyde for 1 min and then negatively stained with 2% (wt / vol) Na-phosphotungstate for 1 min. The observations were carried out employing a Philips CM10 transmission electron microscope at 80 kV.

[0099] RBCEVs uptake into HUVEC and PBMC

[0100] For uptake studies, obtained RBCEVs were labelled by the PKH26 fluorophore and administered to different cell kinds (i.e., HUVEC and PBMC). Briefly, PKH26 stock solution (Red Fluorescent Cell linker for General Cell Membrane, SigmaAldrich) (1 pM) was added to diluent C solution and incubated at 37° C for 15 minutes. Then, 1-2 pL of RBCEVs were added to PKH26 in diluent C, resulting in a sample with 1011 parti cles / mL of RBCEVs and 4 pM of PKH26. EV compositions from UL and L samples, after PKH26 labelling procedure, were added in approximately 1.5-2.0 x 1011 on 4 x 105 cells, and fluorescence detection was performed both by Flow Cytometry and Confocal Microscopy ((Leica TCS SP5 II confocal microscope (Leica Microsystem) with 488, 543, and 633 nm lasers). After 24h, HUVEC cells were detached by Trypsin 0,25% solution, then washed with PBS and prepared for Flow Cytometry PKH26 fluorescence quantification. Indeed, by confocal microscopy the same cells detached and acquired through a cytometric analysis were analysed, together with other samples of HUVEC adherent cells, directly grown on MatTEK plates (MatTek Corporation). Indeed, the same culture conditions were set up for PBMCs, isolated from donor blood peripheral blood by density gradient separation (Ficoll- PaqueTM Plus solution, GE Healthcare, Little Chalfont, UK) in which the uptake was evaluated after 24h of EVs treatment. To evaluate EVs’ possible induction of cell toxicity, cells were labelled by the viability dye, 7-amino-actinomycin (7-AAD, Beckman Coulter, USA).

[0101] Production of miR-210-loaded RBCEVs and HUVEC treatment

[0102] RBCEVs loaded with synthetic miR-210 and obtained via physical vesiculation were used to evaluate the biological effect on human umbilical vein endothelial cells (HUVECs). To do that RBEVs loaded with miR-210 (L) and control ones (UL) were prepared as already described in the previous paragraphs.

[0103] HUVECs were cultured in EndoGro™ basal medium (Merck) with the addition of endothelial growth supplements (such as EndoGro-LS Supplement 0. 2%, rh EGF 5ng / mL, ascorbic acid 50pg / mL, L-glutamine 10 mM, hydrocortisone hemisuccinate Ipg / mL, heparan sulphate 0.75 U / mL and FBS 2%) and maintained at 37°C in a humidified incubator and 5% CO2. Cells were seeded at the concentration of 100,000 / pz in 6-well plates and, after 48h of growth, treated with 1.5-2.0 x 1011 RBCEVs. For the uptake experiments, UL and L RBCEVs were added and let incubate for 4 to 24h. For the evaluation of the biological effect, positive controls were set up by adding increasing concentrations of “nude” miRNA in the presence of a transfection reagent (Trans-IT 2X, ThermoFisher Scientific). At the end of the incubation time, HUVECs were collected for imaging, and flow cytometry or total RNA and protein extractions for the evaluation of the biological effect at the mRNA and protein levels, respectively.

[0104] TotRNA extraction

[0105] Total RNA extraction from RBCs, RBCEVs, and HUVEC was performed with the miRNeasy Mini Kit (QIAGEN) following the manufacturer's protocol. This kit allows the isolation of total RNA including also miRNAs. First, 700 pL of QIAzol Lysis Reagent (QIAGEN) was added to the cell or vesicle samples and incubated for 5 minutes at room temperature. As per protocol, 140 pL of chloroform was added, vortexed, and incubated for 3 minutes at room temperature. After that, the samples were centrifuged at 12,000 x g at 4°C for 15 minutes to allow the separation of the aqueous phase containing nucleic acids, an interphase containing denatured proteins and an organic phase. The aqueous phase was recovered and added with 1.5 volumes of 100% ethanol to allow precipitation of the nucleic acids. Then, the RNA samples were loaded into RNeasy® Mini columns placed in 2mL collection tubes and centrifuged at 8,000 x g for 15 seconds at room temperature. At the end of the centrifugation, the flow-through was removed, the remaining sample volume was transferred to the column, and the centrifugation and removal of the flow-through were repeated. The column then underwent a wash with 700 pL of RWT buffer and a centrifugation under the same conditions described above followed by two washes with 500 pl of RPE buffer and two centrifugations at 8,000 x g at room temperature for 15 seconds and 2 minutes, respectively. When the collection tube was changed, the column was centrifuged at 12,000 x g for 1 minute to allow the membrane to dry completely. Elution was performed by adding 30 pL of RNase-free water followed by centrifugation at 8,000 x g for 1 minute. At the end of RNA extraction, the purity and concentration of the samples were evaluated by NanoDrop ND- 1000 spectrophotometer (Thermo Fisher).

[0106] Small RNA Assay: cDNA synthesis and qPCR

[0107] For small RNAs analyses, complementary DNA (cDNA) synthesis was performed by using the TaqMan® Small RNA Assays (Applied Biosystems). The reaction for RT with singlestranded miRNA was prepared with 7 pL of RT Reaction Mix and 10 ng of extracted RNA with the addition of 3 pL of specific looped primer (see Table SI for assays ID) and nuclease- free water to obtain 15 pL as final volume. The reaction was then placed in a GeneAMP PCR SYSTEM 2700 thermal cycler and subjected to the thermal profile required by the protocol. This involves a first step of annealing between the primer and the target miRNA that takes place at 16°C for 30 min, as the complementarity between primer and miRNA is mild. The second step is meant to activate the reverse transcriptase enzyme, responsible for the double strand formation, which requires another 30 minutes and 42°C. The third and final step involves enzyme inactivation and thus stopping cDNA synthesis.

[0108] The cDNA sample was then subjected to amplification and quantitative analysis by Real Time PCR using the TaqMan® Small RNA Assays. The reaction was prepared by adding 1 pL of cDNA to 10 pL of TaqMan™ Universal Master Mix II, no UNG (ThermoFisher Scientific), and 1 pL of specific assay (see table SI for assays ID). The reactions were brought to a final volume of 20 pl with nuclease-free water. Reactions were conducted in triplicate and a No Template Control (NTC) was provided for each amplification. The strips were put into the 7500 Real Time PCR System (Applied Biosystems) and ran with a two- steps thermal profile: denaturation at 95°C for 15 seconds and annealing / extension at 60°C for 1 minute. The results obtained were analysed by 7500 System Software (Applied Biosystem). Relative quantification was performed by using U6 snRNA as endogenous control, whereas absolute quantification was done by setting up a standard curve at decreasing concentrations (20, 2, 0.2, 0.02, 0.002, and 0.0002 nM) of the same miRNA. RNA standards were subjected to cDNA synthesis and PCR amplification, as for the samples, and the relative Cts plotted against the concentration to obtain the standard curve according to (Simmonds 2019).

[0109] Long RNAs: cDNA synthesis and qPCR For long RNAs (PTP1B, ACTB, etc.), cDNA synthesis was performed using the PrimeScript™ RT Master Mix and kit (Takara) starting from 500 ng of total RNA, following the manufacturer's instructions. The reverse transcription reaction was performed on a final volume of 10 pL at 37°C for 15 minutes, at the end of which enzyme inactivation was performed at high temperatures (85°C for 5 seconds). Finally, 40 pl of RNase-free water (1 :5 dilution) was added to bring the sample to a theoretical 10 ng / pL, and the final cDNA was used for subsequent gene expression study by Real Time PCR. For the PTP1B mRNA expression assay, the reaction was done with 1 pL of diluted cDNA, 10 pL of the same master mix as small RNAs, and 1 pL of the specific TaqMan® Gene Expression Assay (Hs00942477_ml, Catalog #4331182, ThermoFisher Scientific). Reactions were brought to 20 pL as final volume and ran with the same protocol described above. The results obtained were analysed by 7500 System Software (Applied Biosystem) and relative expression calculated by the AACt method using ACTB (Hs01060665_gl, Catalog #4331182, ThermoFisher Scientific) as reference gene, ran in the same conditions as for the target gene.

[0110] Protein extraction and western blotting

[0111] Protein extraction from HUVEC was performed by adding 100 pL of RIP A buffer (150 mM NaCl, 50 mM Tris, 1% Triton, 0.1% NaDeoxycholate, 0.1% SDS at pH 8.0) directly in the well. The extracted proteins were quantified by the Bradford assay for preliminary quantification before the western blotting. 5-10 pg of total proteins were loaded onto a 10% SDS-PAGE and ran at 10A and 20A until the end of running according to the Laemmli method (Laemmli 1970). The electrophoresis was followed by the transfer onto Polyvinylidene Difluoride (PVDF) membrane using the TransBlot apparatus (BioRad). The transfer was done in the Towbin’s buffer at 100V for 70 minutes. Transferring efficiency and loading control were done by No-Stain labelling (ThermoFisher Scientific) and Imager acquisition. Then, blocking with 5% of nonfat dry milk was performed for Ih at room temperature. Incubation with primary antibodies (PTP1B #5311, GAPDH #2118, Cell Signal Technology #) was performed overnight at 4°C at the recommended concentration and conditions. Incubation with HRP-conjugated secondary antibody was performed in a blocking solution for Ih at room temperature. At the end of the washings, a chemiluminescent reaction was carried out with the WestemBright ECL western blot detection kit (Advansta). ChemiDoc Imager and Image Lab software (Bio Rad) were used for imaging and quantification, respectively. 2. RESULTS

[0112] Loading of tracking and therapeutic molecules into human RBCs

[0113] RBCs have been selected as donor cells for the production of EVs because they are easily obtainable in large quantities and can be conveniently loaded with several molecules. As mentioned in the method section, human RBCs were purified from 5 ml of fresh whole blood by means of two washes in Hepes followed by filtration with a leukodepletion filter to completely remove white blood cells. The removal of granulocytes was really effective, as demonstrated by the haemocytometer analysis. Regarding the loading of cargoes into RBCs, this was extremely efficient, showing that both low and high molecular weight can be effectively loaded thanks to the versatile protocol that can be opportunely optimised on the basis of the kind of molecule (e.g., the choice of the MWCO of the dialysis tube, dialysis times, and addition of miRNA after the dialysis step). With respect to dextran-conjugates, the loading was assessed by flow cytometry and the efficiency calculated by the ratio of Mean Fluorescence Intensity (MFI) of loaded RBCs versus the UL ones (Fig. 2). While for miR210 mimic, evaluation of the loading was assessed by Real Time PCR and the loading efficiency calculated by comparing the obtained final concentration into loaded RBCs (1.96 ± 0.67 pM) and the starting concentration put in the swelled RBCs (10 pM). A mean efficiency of 19.58 ± 7.98 % was achieved. Moreover, the cell recovery was high (60.07% and 60.63% for RBCs UL and L, respectively) demonstrating that the procedure is independent of the presence of the cargo.

[0114] Production of RBCEVs by the newly developed physical vesiculation method

[0115] Setting up of the physical vesiculation method for RBCEVs production from loaded RBCs was the most critical step and required a lot of several changes during the experiments, proving that the extrusion is not banal at all. We started from standard extrusion of loaded RBCs at physiological haematocrit (i.e. 40% V / V) across a 1-pm PC membrane at room temperature, but unfortunately the first results were not good and we encountered a lot of troubles (i.e., difficult extrusion, breakage of the filter, breaking of the syringe, leaking of the content, etc.). Thus, we needed to add several additional and inventive steps during the procedure. The first one was to completely remove granulocytes (mainly neutrophils) that in our hypothesis could have affected the extrusion process owing to the bigger dimensions. Nevertheless, the extrusion process was once again detrimental and led to poor recovery of intact RBCEVs. So, we decided to work on RBCs concentration (i.e., hematocrit) and extrusion temperature (i.e., room temperature and 37°C). Regarding RBCs concentration, we tested several haematocrits (ranging from 20 to 4 %) and we unexpectedly found that the yield of production was higher at lower percentages. Whereas, an optimal yield was achieved when haematocrit was maintained between 5 and 7%; thus, we selected 6% for further steps. Concerning temperature, we found that performing the procedure at 37°C produced a higher yield As the last optimization step, we decided to perform a preliminary passage onto a 5- micron membrane. Unexpectedly, we found that vesiculation occurred already using this porous membrane, even if the dimensions of pores are comparable with the cell dimensions (5 pm against the 7-8 pm of diameter of RBCs). This preliminary step was followed by a second extrusion through a 1 -micron membrane. This was likely the most important step that allowed us to obtain a highly reproducible process with high homogeneity and a higher yield of recovered RBCEVs with an increased amount of cargo inside the vesicles. All these inventive steps made the process as soft as possible to not damage cells and lose their content and deeply transformed the standard extrusion process. For this reason, we called the newly developed method “soft extrusion”. Results reported below and in the next paragraphs have been obtained with the newly developed protocol that envisaged complete leukocyte removal, 6% haematocrit of loaded RBCs, controlled temperature (i.e., 37°C) and two serial extrusions that was composed of 4 cycles across a 5-pm PC membrane followed by 4 cycles across a 1-pm PC membrane.

[0116] The extrusion process has been monitored using DLS technique to evaluate the vesiculation and the reproducibility of the steps from batch to batch by physically characterising the raw product. Table 1 shows the dimensions and the poly dispersity index of the obtained product at the end of each step of extrusion. Results obtained starting from both UL and L RBCs are also compared. As reported, after the first passage at 5 pm we obtained a product with a mean Z-average of 312.4 and 311.1, respectively for UL and L. This demonstrates that vesiculation actually occurred already at the first extrusion; however, after the second extrusion we can observe a decrease in particle size for both samples (202.9 and 207.1, respectively). The decrease of the dimensions after the second extrusion was in accordance with the lower size of the membrane’s pores. In addition, the difference in terms of size when the vesicles were loaded (L) compared to the unloaded (UL) was not significant after each cycle of extrusion, indicating that the payload was not affecting the physical characteristics of the vesicles. Regarding the Pdl values, which is a measure of the heterogeneity of a sample based on size, we had high values (about 0.5-0.6) owing to the presence of a small peak in all the samples, with a size of 6-7 nm that has been associated with the haemoglobin released by some cells that inevitably broke. The raw product was then subjected to purification by ultracentrifugation and the final product physically and biologically characterised by NT A, TEM and FC.

[0117] TABLE 1 - DLS characterization of the raw products

[0118] Characterization by NTA

[0119] NTA can often provide higher resolution, while DLS may be a faster assessment of the mean size and polydispersity. Thus, we moved to this kind analysis to monitor the purification and to characterize the final product. Purification has been optimized starting from serial centrifugations and according to NTA results (data not shown). The final protocol envisaged a first low speed centrifugation to remove cell debris, filtration across a 0.45 PES filter and finally a single UC step at 50,000 x g. NTA characterization of the final product (Fig. 3) showed that UL and L RBCEVs have similar morphological characteristics, with a hydrodynamic diameter ranging from 100 to 300 nm. Furthermore, the proposed methodology resulted in the formation of very reproducible vesicles as shown by the mode and mean of vesicle diameter distribution corresponding to about 130 and 200 nm, respectively (n=9). In terms of yield, we obtained from 4 to 5x1012 particles / ml starting from 1 mL of RBCs at 6% Het (that corresponds to about 5.70x108). Theoretically, each RBC should give rise to 500-600 EVs (considering the ratio between the RBC area = 15 pm2 and the RBCEV area = 0.015-0.020 pm2). We obtained about 4.5x10x103 RBCEVs per RBC, a ratio very close to the theoretical yield. This demonstrates that the soft extrusion actually allowed the vesiculation of almost all RBCs and that the breakage of cells was minimal.

[0120] Characterization by TEM

[0121] Purified products were further analysed by transmission electron microscopy (TEM) using negative staining. The obtained RBCEVs appeared as membrane closed rounded vesicles delimited by a well-defined thin bilayer with a measured diameter ranging from 50 to 200 nm (Fig. 4). Of note, the little discrepancy between the vesicle dimensions resulting from TEM and NTA could be due to sample fixation commonly leading to sample dehydration. This a tangible demonstration that our products are indeed extracellular vesicles delimited by intact membranes, confirming once again that the developed process was suitable and very efficient for vesiculation.

[0122] Characterization by FC

[0123] RBCEVs have been finally characterised from a biological point of view using a cytofluorimetric-based approach, in an attempt to demonstrate the presence of the RBCs typical proteins onto the membrane of the obtained vesicles and that these proteins are correctly orientated. As previously mentioned, the threshold was mainly placed on SSC, and the correct area of analysis was established using Rosetta beads (Fig. SI A, B). Indeed, we also run Megamix plus beads for the general setting of the instrument. Rosetta beads allow to define an area that ranges from P2 to P6 (Fig. SI A). In the dot plot (Fig. SIB), FSC vs SSC can check and confirm the SSC values of the acquisition threshold or a back-check of the physical characteristics, in case of thresholding on fluorescence.

[0124] In addition, the general EV tracer LCD was employed to support the acquisition and the analysis of the EVs. LCD is an APC-emitting Lipophilic Cationic Dye (red), which diffuses into double layer structures assisted by membrane potential, thus staining both particles and cells (Cappellano, Raineri et al. 2021). Indeed, LCD use enables the application of a simplified protocol, combined with the possibility of applying a fluorescence triggering, representing a substantial step forward in the process of unequivocally identifying and enumerating EVs by FC. LCD mean fluorescence intensity (MFI) data are shown in Fig.5, highlighting similar profiles for UL and L samples.

[0125] The UL and L compositions were then labelled with the reagents indicated in the Materials and Methods Section, highlighting the following results. First of all, we wanted to analyse the presence of one of the most expressed RBCs antigen, that is Glycophorin A (GYP A). Most of the EVs appeared Glycophorin A (GYP A) positive (Fig. 6A-D), indicating their unequivocal origin from RBCs. GYPA positivity also means that the membrane of the vesicles was correctly oriented, since eventual inside-out phenomena would have internalised this membrane antigen. As observable, percentages of GYPA positivity reached the 85% in the last experiments that are the most optimised for both EV production process and EV characterization protocols. Finally, percentages do not present a significant difference in UL and L samples revealing that the cargo encapsulation does not alter the expression of this transmembrane protein.

[0126] Next, we checked for the presence of another very important antigen, the CD47, which has been associated with the “don’t eat me” signal, indicating that its presence can avoid RBCs, or in this case RBCEVs, removal by RES. The labelling by anti-CD47 mAb gave similar results (Fig. 7A-D), namely high positivity for both UL and L samples, confirming the absence of relevant and significant differences in UL and L composition and highlighting that RBCEVs mirror the mother cells (Stewart, Urbaniak et al. 2005, Tripisciano, Weiss et al. 2020). As mentioned, in RBCs this molecule inhibits phagocytosis through interaction with the inhibitory immunoreceptor SIRPa expressed by macrophages.

[0127] Natively produced and chemically produced RBCEVs usually exposed phosphatidylserine (PS) in their outer phospholipid bilayer, thus favouring their removal from the circulation, as well. Actually, the uptake of EVs by phagocytes has been associated with the presence of Annexin V on their surface, while the presence of ANNEXIN V on the surface of the phagocytic cells themselves did not contribute to this process. Furthermore, the EVs induced exposure of PS on the phagocyte membranes, thereby explaining the mechanism by which Annexin V-coated EVs would bind to phagocytes, as PS is the phospholipid ligand preferred by annexins (Lizarbe, Barrasa et al. 2013), particularly Annexin V (Kohler, Hering et al. 1997, Tontanahal, Arvidsson et al. 2021). For these reasons and for the reported biochemical composition of physiologically released-EVs (CD63+ / CD81+-EVs, Annexin V+ EVs) (Thangaraju, Neerukonda et al. 2020), to complete the EV characterization, Annexin V positivity was investigated. Results reported in Fig. 8 revealed very low percentages of positivity, which goes in the same sense of CD47 data and are in agreement with the literature on mother cells (Ensinck, Brajovich et al. 2019) and physiologically-produced RBCEVs (Gamonet, Desmarets et al. 2020). Finally, the autofluorescence of RBCEVs and the fluorescence of an irrelevant antibody have been checked as control. Representative histograms are reported in Fig. S2.

[0128] Taken together, the data reported so far demonstrate the feasibility and the reproducibility of the process and the homogeneity in terms of physical and biological parameters of the produced population of RBCEVs. A list of examples of application are next reported to prove that the process is able to produce RBCEVs loaded with different cargoes starting from preloaded RBCs, that loaded RBCEVs can be efficiently up taken by different cell kinds and that the cargo is conveniently released inside cells to produce a biological effect.

[0129] Example 1 - Production of Cascade Blue-Dextran-loaded RBCEVs

[0130] As a first example, we produced RBCEVs starting from RBCs preloaded with a low molecular weight dextran-conjugate (10,000 Da), where the fluorophore was Cascade Blue. RBCEVs have been produced according to the newly developed procedure and the final products were analysed by scattering- and fluorescence-based NTA. Nanoparticle quantification and characterization under scattering conditions revealed the presence of abundant quantities of RBCEVs in both UL and L samples, with dimensions typical of extracellular vesicles (Fig 9 A, B). When dextran-loaded RBCEVs were analysed in fluorescence mode, particles were detectable only in L samples clearly demonstrating that Cascade Blue-Dextran was encapsulated into RBCs and retained in RBCEVs after the procedure of vesicle formation (Fig. 9 C, D). Of note, the strong difference between particle number revealed under light scattering and fluorescence mode in L samples is due to the lower sensitivity of NTA fluorescence-based detection. See supplementary materials for the video of CB-Dextran-loaded RBCEVs recorded at the NTA.

[0131] The loading of Cascade-Blue dextran into RBCEVs was assessed and confirmed also by flow cytometry. Fig. 10 shows that L samples presented a significantly higher MFI with respect to control (UL) RBCEVs.

[0132] Example 2 - Production of FITC-dextran-loaded RBCEVs and characterization

[0133] As a second example, we produced RBCEVs starting from RBCs loaded with high molecular weight dextran (70,000 Da) to demonstrate that also high molecular weight molecules can be efficiently loaded both in RBCs and RBCEVs, as well. This kind of dextran was conjugated with FITC so the presence of the cargo in the final product was analysed by flow cytometry in the respective channel. In Fig.11, Cytometry (A-C) and statistical histograms (D) demonstrate the presence of the cargo in L RBCEVs and an extremely significantly higher MFI. While the bar graph in E displays the ratio between the overall MFI signals obtained in RBEVs loaded with both dextran-conjugates, which is even more significant.

[0134] Taken together, data from examples 1 and 2 clearly prove that cargo loading could be assessed by NTA and / or FC and that loading efficiency was high for both low and high molecular weight polymers.

[0135] Example 3 - Uptake of PHK26-labelled RBCEVs in HUVEC and PBMC

[0136] As a third example, we evaluated the ability of RBCEVs to be internalised by different kinds of cells (i.e., HUVEC, lymphocytes and monocytes isolated from PBMC). To this end, the produced RBCEVs were labelled with PHK26, a dye that is commonly used for these experiments. After labelling, 1.5x1011 RBCEVs were administered to the cells and let incubate for 4 and / or 24 h.

[0137] After the incubation-times of EVs with the specific cell lines employed for uptake evaluation, confocal analyses, and flow cytometry quantitation were carried out. Results are reported in Figg. 12-14.

[0138] Fig. 12 displays data from HUVEC at 24h. In (A), the frequencies of dead cells were revealed by means of 7-AAD positivity, and the relative statistical report is shown in (E). This preliminary analysis ensured that, at the used concentrations, RBCEVs were not toxic and did not cause cell death. In panels B and D, flow cytometry quantification of PKH26 fluorescence in HUVEC cells showed an extremely high uptake of labelled RBCEVs. These results were confirmed by the confocal images (panel C) that highlighted red dots (PKH26- labelled RBCEVs) internalised into HUVEC cells (stained by green fluorescent probes).

[0139] In Fig. 13, data from 24 h-incubation with PBMCs are reported. In (A) physical characteristics from the flow cytometry parameters FSC vs SSC are given, underlining the presence of both lymphocytes (blue-coloured) and monocytes (magenta-coloured). In (B) and (C), flow cytometry histograms revealed a small but appreciable uptake in lymphocytes, while a strong internalisation was revealed in monocytes. The relative quantitation is shown in (D) demonstrating a highly significant uptake of labelled RBCEVs with respect to control. As for HUVEC cells, PBMCs did not reveal any necrotic / apoptotic process induced by RBCEVs, at least for the time investigated (E). Confocal images of (F) highlight magenta dots (PKH-26+EVs) internalised into monocytes, confirming the FC results. Finally, Fig. 14 shows PKH26 fluorescence in the different cell lineages investigated, after 4h. In all the tested cell lines, uptake of RBCEVs is already appreciable after 4-h incubation, however is markedly increased after 24h. For this reason, these incubation conditions have been selected for the following experiments.

[0140] Example 4 - Production of miR-210-loaded RBCEVs and evaluation of the biological effects

[0141] After having proven the feasibility of the procedure to produce loaded RBCEVs and their uptake thanks to the use of tracking molecules, we moved to the demonstration of their biological effect by loading a pharmacologically active cargo. Indeed, as a fourth example, we produced miR-210-loaded RBCEVs thanks to the same invented methodology. Here, we report the loading efficiency and the biological effect of the obtained vesicles loaded with the miRNA mimic. For the calculation of the loading efficiency, we evaluated the amount of miRNA loaded into the purified RBCEVs by totRNA extraction, cDNA synthesis, and absolute quantification via qPCR. In Fig. 15, we report the mean concentration of miRNA achieved in the starting RBCs (1370.61 ± 336.09 pmol / ml) and the one obtained in the final RBCEVs (44.65 ± 2.12 pmol / ml). If we express the concentration on the number of vesicles, we obtained a mean concentration of about 10 pmoles per 1.0x1012 EVs. For each experiment, we have also calculated the loading efficiency into the RBCEVs; the mean value is 3.26 ± 0.90 %. Therefore, an efficient loading and high efficiency into RBCEVs were demonstrated also for active pharmaceutical compounds.RNA samples were finally used to evaluate the effect of the delivered miRNA on the mRNA of a known target of miR-210, that is PTP1B (Zhou, Collado et al. 2022). According to the literature, the effect of mR-210 is not exerted at the RNA level (Hu, Huang et al. 2010). Actually, we have seen an increase in the mRNA levels both in the samples treated with the loaded RBCEVs and in the transfected samples (Fig. 17). This can be a feedback effect due to the block of protein translation and was observed also by (Hu, Huang et al. 2010). Thus, we moved to evaluate the effect at the protein level. To do this, we used protein extracts from the same experiments and performed SDS-PAGE, western blotting, and immunoblotting. After each loading, run, and transferring, we proceeded to band quantification and total protein normalisation. Fig. 18A shows a representative blot and the respective total proteins, while Fig. 18B the quantification. As can be seen, we were able to detect a significant downregulation in the cells treated with RBCEVs loaded with miR-210 (L) compared with UL. The effect was a little bit lower than that obtained in the cells transfected with 1 nM of miRNA.

[0142] Our data confirm that the effect of the miR-210 is exerted at the protein level as reported by other authors (Barile, Lionetti et al. 2014). What is most important, in this example of application we were able to demonstrate that miR-210-loaded RBCEVs can exert their biological effect at least in vitro and can be considered a proof-of-concept for further applications.

[0143] Final observations

[0144] In conclusion, the invention herein proposed can represent a new platform for the production of a next generation nanocarriers based on RBCs that can be loaded with several kinds of therapeutic molecules. In the reported example a small RNA has been used; however, long RNAs can be used as well. This new tool may thus allow many therapeutic applications, ranging from silencing of unwanted RNAs / proteins to gene editing or to RNA-based therapies for genetic diseases, thanks to the loading of siRNAs / miRNAs, Crispr / cas9 plus gRNA complexes and functional mRNAs, respectively. The main points of strength are the higher yield and reproducibility, with respect to the methods up to now proposed, and the relatively easy translatability to the clinics.

[0145] Declaration according to Art. 170bis

[0146] In compliance with Art. 170bis of the Italian code of industrial property, the applicant of the present patent application declares that: For the biological material, containing microorganisms or genetically modified organisms, object or used in the aforementioned patent application, the obligations deriving from national or Community regulations, and in particular, from the provisions referred to in paragraph 6 of the Legislative Decree of 12 April 2001 n.206 and 8 July 2003 n. 224, concerning these modifications, have been respected; -The donors of the human blood samples used in the present patent application gave their informed written consent. The study was approved by local ethics committees.

Claims

CLAIMS1. A method for preparing a population of Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo comprising the following steps: a) extruding a composition comprising or consisting of a plurality of Red Blood Cells (RBCs) loaded with said cargo across a membrane, wherein said extrusion is performed in an isotonic buffer; b) isolating and / or purifying the extruded RBCEVs by the step a); wherein the method further comprises a reannealing step before the purification step b) in which said RBCEVs sample obtained from said extrusion step a) is incubated at 37°C at least for 5 min.

2. The method according to claim 1, wherein said membrane in step a) is a polycarbonate membrane.3.. The method according to claims 1 or 2, wherein said composition is a blood sample, in particular a human blood sample, characterized by a hematocrit (cell percentage volume / volume) between 4 and 8%, preferably between 5 and 7%, more preferably 6%.

4. The method according to any one of claims from 1 to 3, wherein said extrusion step a) consists of at least 2, 3 or 4 cycles, preferably 4 cycles.

5. The method according to any one of claims from 1 to 4, wherein in the extrusion step a) said composition is extruded across a first membrane of 5-pm and a second membrane 1- pm membrane.

6. The method according to any one of claims from 1 to 5, wherein said purification step b) comprising one or more centrifugation of the RBCEVs sample obtained from said extrusion step a).

7. The method according to claim 6, wherein said purification step comprises a centrifugation and / or an ultracentrifugation step.

8. The method according to claim 7, wherein said ultracentrifugation step is performed once at 50,000 x g, preferably for at least about 1 hr, more preferably at 4° C.

9. The method according to claims 7 or 8, wherein said centrifugation step is performed at 3,000 x g, preferably for 5-10 mins at room temperature, more preferably followed by a filtering step through a 0.45 pm filter.

10. The method according to any one of claims from 1 to 9, wherein said cargo is a therapeutic or diagnostic compound.

11. The method according to any one of claims from 1 to 10, wherein said cargo is selected from peptides, oligopeptides, proteins, polypeptide, DNA, mRNA, circRNA, miRNA, siRNA, CRISPR / CAS9 and gRNA complexes, CRISPR / deaminase (base editor) and gRNA complexes, oligonucleotides, antisense oligonucleotides, nucleotide analogs, nucleosides, nucleoside analogs, hormones, immunosuppressant, inhibitors of malignant cell growth, corticosteroids, glucocorticoids, anti-retroviral and non-steroidal anti-inflammatory agents, cytokines, toxins, immunogens, contrast media for diagnostics; particles or nanoparticles selected from nanoparticles containing a metal, magnetic nanoparticles, super-paramagnetic nanoparticles (SPIO), and nanoparticle-active molecule complexes.

12. The method according to any one of claims from 1 to 11, wherein said composition is a blood sample, in particular a human blood sample, preferably a blood sample characterized by a depletion of leukocytes, in particular granulocytes.

13. The method according to any one of claims from 1 to 12, comprising the following steps: i) preparing a composition of Red Blood Cells (RBCs) loaded with a cargo from a blood sample of a human subject, preferably leukocytes are not present in said composition; ii) reducing the hematocrit of said composition to a value between 5 and 7%, preferably 6%; iii) extruding said composition across a first membrane of 5-pm and a second membrane 1- pm membrane, preferably for more than 1 cycle, more preferably for 4 cycles.

14. The method according to any one of claims from 1 to 13, wherein one or more steps of said method are conducted at a temperature not higher than 37°C.

15. A population of Red Blood Cell-derived Extracellular Vesicles (RBCEVs) loaded with a cargo obtainable by the method according to any one of the claims from 1 to 14.

16. A population of Red Blood Cell-derived Extracellular Vesicles (RBCEVs), loaded with a cargo characterized by a size distribution with a mode between 70 and 220 nm, more preferably between 90 and 150 nm, characterized by a percentage of positivity to glycophorin A at least 60%, preferably at least 70%, the CD47 expression of at least 60%, preferably at least 70% and the PS exposure lower than 30%, preferably lower than 20%.

17. The population of RBCEVs according to claim 15 or 16 characterized by a membrane architecture and surface antigens that mirrors those of mother RBCs.

18. The population of RBCEVs according to any one of the claims from 15 to 17, characterized being resealed nanovesicles, each of them delimited by their cell membrane, as showed by TEM analyses, in particular they are not nanoparticles belonging from the aggregation of cell debris and / or disorganized membrane components.

19. The population of RBCEVs according to any one of the claims from 15 to 18, wherein each RBCEV of the population encapsulated at least at least about 500, 5,000, 10,000, 15,000, 20,000, 100,000 or more molecules.

20. The population of RBCEVs according to any one of the claims from 15 to 19, wherein said population of RBCEVs wherein at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% of the population encapsulate at least one cargo.

21. The population of RBCEVs according to any one of the claims between 15 to 20 wherein said cargo is a therapeutic or diagnostic compound.

22. The population of RBCEVs according to any one of the claims between 15 to 21 wherein said cargo is selected from peptides, oligopeptides, proteins, polypeptide, DNA, mRNA, circRNA, miRNA, siRNA, CRISPR / CAS9 and gRNA complexes, CRISPR / deaminase (base editor) and gRNA complexes, oligonucleotides, antisense oligonucleotides, nucleotide analogs, nucleosides, nucleoside analogs, hormones, immunosuppressant, inhibitors of malignant cell growth, corticosteroids, glucocorticoids, anti-retroviral and non-steroidal anti-inflammatory agents, cytokines, toxins, immunogens, contrast media for diagnostics; particles or nanoparticles selected from nanoparticles containing a metal, magnetic nanoparticles, super-paramagnetic nanoparticles (SPIO), and nanoparticle-active molecule complexes.

23. A pharmaceutical composition comprising the population of RBCEVs according to any one of claims from 15 to 22 and one or more pharmaceutically acceptable excipients.

24. The population of RBCEVs according to any one of claims from 15 to 22 or the composition according to claim 23 for use as a medicament in a therapeutic treatment of a disease.

25. The population of RBCEVs or the composition for use according to claim 24 wherein said disease is selected from cancers, liver diseases, thrombosis, excess iron, parasitic diseases, viral and / or bacterial infection, diabetes, cardiovascular disease, osteoporosis, anemia, lung infection, respiratory diseases, coagulopathies, immune disorders, neurodegenerative diseases.

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