process

Abstract

The present invention relates to methods of production, isolation and application of extracellular vesicles (EV)s derived from stem cells from human exfoliated deciduous teeth. The invention also relates to the use of said EVs in therapy, such as in the treatment of Parkinson's disease.

Description

[0001] PROCESS

[0002] FIELD OF THE INVENTION

[0003] The disclosure relates to methods of production, isolation and application of extracellular vesicles (EV)s derived from stem cells from human exfoliated deciduous teeth (SHEDs). The disclosure also relates to the use of said EVs in therapy, such as in the treatment of Parkinson’s disease (PD).

[0004] BACKGROUND TO THE INVENTION

[0005] Parkinson’s disease (PD) is the second leading progressive neurological disorder affecting mostly elderly people. It has been estimated that today from 7 to 10 million of patients are suffering from the PD and these numbers are expected to double by 2030 [1,2],

[0006] PD is defined by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta located in the midbrain and formation of Lewy bodies that represent cytoplasmic inclusions of insoluble alpha-synuclein aggregates. At present, there is no effective cure for PD, treatments (Levodopa and analogs) are symptomatic and do not halt progression of neurodegeneration [1], At the same time patient healthcare and social security require enormous investments. Consequently, in order to prolong the quality life expectancy for PD patients and to reduce economic and social burden it is crucial to develop innovative and effective treatment methods for PD. It is now widely accepted that new effective PD therapies should promote regeneration and (or) neuroprotection delaying progression of the disease and mitigating disability in PD patients [1],

[0007] The potential use of extracellular vesicles (EVs) for therapeutic purposes attracted a great deal of interest in recent years [2], In a first proof-of concept study therapeutic efficacy of intranasal administration of EVs derived from stem cells from human exfoliated deciduous teeth (SHEDs) was demonstrated on a unilateral 6-hydroxydopamine (6-OHDA) medial forebrain bundle (MFB) rat model of PD [3], Intranasal therapy significantly improved gait and cognitive functions of PD affected rats, and normalized the expression of tyrosine hydroxylase (TH) in the SN and striatum [3,4],

[0008] However, there are major challenges that need to be solved before these findings can be exploited for the development of an improved, minimally invasive clinical therapy against PD.

[0009] SUMMARY OF THE INVENTION

[0010] In the proof-of concept studies EVs isolated by laboratory scale methods were used. SHEDs were cultivated in standard cultivation flasks, EVs were purified from the cell culture supernatants using differential ultracentrifugation protocol [3,4], However, therapeutic use requires a large amount of EVs, that cannot be produced by laboratory scale methods, these amounts of EVs can only be obtained by scaling up production and isolation.

[0011] Furthermore, the composition and therapeutic properties of EVs depends on the cell type and its physiological state. Even slight alterations in the culture conditions may dramatically affect composition and therapeutic properties of EVs [5,6], therefore scale up of EV production needs to be optimized to maintain maximum therapeutic efficacy.

[0012] To overcome these challenges, the present inventors have developed and demonstrated therapeutic efficacy of a new technology for production, isolation and application of EVs in the 6-OHDA rat model of PD.

[0013] Therefore, the present invention provides:

[0014] A method of production of extracellular vesicles (EVs) comprising:

[0015] (a) culturing human stem cells from exfoliated deciduous teeth (SHED)s on microcarriers in the presence of xeno-free medium;

[0016] (b) passaging said SHEDs at least twice to achieve a conditioned supernatant containing EVs;

[0017] (c) concentrating and diafiltrating said conditioned supernatant by tangential flow filtration (TFF); and

[0018] (d) subjecting said concentrated EV suspension to size-exclusion chromatography (SEC) to obtain a purified EV suspension. The present invention also provides:

[0019] Extracellular vesicles (EVs) obtainable by, or obtained by, the method of the invention. The present invention also provides:

[0020] Extracellular vesicles (EVs) obtainable by, or obtained by, the method of the invention, for use in therapy.

[0021] The present invention also provides:

[0022] Extracellular vesicles (EVs) obtainable by, or obtained by, the method of the invention, for use in the treatment of Parkinson’s disease.

[0023] The present invention also provides:

[0024] Extracellular vesicles (EVs) obtainable by, or obtained by the method of the invention, for use in the treatment of neurological disorders.

[0025] BRIEF DESCRIPTION OF FIGURES

[0026] Figure 1: Schematic of scaling up production of extracellular vesicles using microcarrier cultures of human immortalized dental pulp cells.

[0027] Figure 2: Principal component analysis (PCA) using RPM for normalization of miRNA reads.

[0028] Figure 3. Characterization of different RNA species enriched in different EV preparations and producing cell lines.

[0029] Figure 4A. Cluster analysis of proteomic cargo from EVs derived using standard and scaled up methods.

[0030] Figure 4B. Unique protein identifications within groups. An UpSet diagram shows information on the combinations of intersections between different groups of EVs. Figure 5. Transcriptomic and proteomic analysis to characterize the miRNA, mRNA and protein content in EVs before (2D_UC), during (3D_UC) and after scale-up (3D TFF- SEC). A, D, G: Upset plots were used to compare the complexity in miRNA, mRNA, and protein profiles between the samples. B, E, H: principal component analysis (PCA) was performed to investigate the relationship between molecule patterns and cultivation and purification methods, respectively. PCI loading exceeded 60% in all three analyses. C, F, I:

[0031] Spearman correlation heatmaps together with clustering of samples were performed to investigate the relationship between EV cargo profiles based on the subset of consistently detected miRNAs, mRNA, and proteins.

[0032] Figure 6: Experimental design of the study. APO, Apomorphine; Asc, Ascorbate; 6- OHDA, 6-hydroxydopamine D, Day; CW, CatWalk; MWM, Morris water maze.

[0033] Figure 7: Effects of extracellular vesicles (EV) on gait performance of healthy and 6- hydroxydopamine (6-OHDA)-injected rats. Data shown as mean values ± S.D. (n=9-12 animals per group). RF, right front; RH, right hind; LF, left front; LH, left hind paw. * / ?<0.05, ** / ?<0.01, *** / ?< 0.001 and **** / ?< 0.0001 vs. Control; #p<0.05, ##p<0.01, ### p < 0.001 and #### p < 0.0001 vs. 6-OHDA.

[0034] Figure 8: Effects of extracellular vesicles (EV) on spatial learning and memory of healthy and 6-hydroxydopamine (6-OHDA)-injected rats. Spatial learning was assessed during 4 training days (A), whereas spatial memory - during probe trial on day 5 as amount of target zone crossings (B) and time spent in target quadrant (C). Data shown as mean values ± S.D. (n=9-12 animals per group). ** p < 0.01 and **** p < 0.0001 vs. Control; #p< 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 vs. 6-OHDA.

[0035] Figure 9: Effects of extracellular vesicles (EVs) on the nigrostriatal optical density of tyrosine hydroxylase (TH) staining. TH density in the striatum is shown in (A) and substantia nigra - in (C) at xlO magnification and in the insert in (A) - at x400 magnification. Densitometry bars show optical density of TH in the striatum (B) and substantia nigra (D). Data shown as mean values ± S.D. (n=4 per group). *p < 0.05, **p < 0.01, ***p < 0.001 and **** / ? < 0.0001 vs. 6-OHDA. Figure 10: Changes in glial fibrillary acidic protein (GFAP) density following treatment with extracellular vesicles (EVs) in the hippocampal dentate gyrus (DG). Representative images (A) show hippocampal GFAP staining at x 100 magnification, whereas histograms (B) show the comparison of GFAP density between groups. Data shown as mean values ± S.D. (n=4 per group). *p < 0.05 and ***p < 0.001 vs. 6-OHDA.

[0036] Figure 11: Altered ionized calcium-binding adapter protein-1 (Iba-1) density after treatment with extracellular vesicles (EVs) in the hippocampal dentate gyrus (DG). Microphotographs (A) depict Iba-1 staining in the hippocampal DG at x 100 magnification, whereas histograms (B) show Iba-1 density analysis between groups. Data shown as mean values ± S.D. (n=4 per group). *p < 0.05 and ***p < 0.001 vs. 6-OHDA.

[0037] Figure 12: Changes in interleukin-1 beta (IL-1 ) count in the substantia nigra (SN) of controls, lesion group (6-OHDA) and rats treated with extracellular vesicles (EVs). Microphotographs (A) depict IL- 10- positive cells in the SN at x200 magnification, whereas histogram (B) shows the analysis between groups. Data shown as mean values ± S.D. (n=4 per group). **p<0.01 and *** / ?< 0.001 vs. 6-OHDA.

[0038] DETAILED DESCRIPTION

[0039] It is to be understood that different applications of the disclosed methods and products may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0040] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0041] General definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a celV includes “cells” and the like.

[0042] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “a method of production of extracellular vesicles (EV) comprising culturing human stem cells from exfoliated deciduous teeth (SHED)s on microcarriers in the presence of xeno serum-free medium” should be interpreted to mean the method has at least the described culturing step, but may contain other steps.

[0043] In some aspects of the disclosure, the word “comprising is replaced with the phrase “consisting of . The term “consisting of is intended to be limiting. For example, the phrase “a method of production of extracellular vesicles (EV) consisting of the following steps” should be understood to mean that the method involved only those steps and no others.

[0044] Method of production of Extracellular vesicles (EVs)

[0045] The invention encompasses a method of production of extracellular vesicles (EVs) comprising:

[0046] (a) culturing human stem cells from exfoliated deciduous teeth (SHED)s on microcarriers in the presence of xeno-free medium;

[0047] (b) passaging said SHEDs at least twice to achieve a conditioned supernatant containing EVs;

[0048] (c) concentrating and diafiltrating said conditioned supernatant by tangential flow filtration (TFF) to achieve a concentrated EV suspension; and

[0049] (d) subjecting said concentrated EV suspension to size-exclusion chromatography (SEC) to obtain a purified EV suspension.

[0050] Culturing cells Cells suitable for use in the method of the invention may include cells taken from an immortalised cell line. Cells suitable for use in the method of the invention may by human dental pulp stem cells. In a preferred embodiment the cells for use in the method of the invention are human stem cells from exfoliated deciduous teeth (SHED)s. In a preferred embodiment of the invention the SHEDs are immortalized (iSHED)s.

[0051] The basal media for the method of the invention will include minimal essential medium such as GMEM with Glutamine and a synthetic media such as KOSR. The medium can be supplemented. The culture media may be DMEM-GlutaMAX. The basal media may include a carbon source such as sodium pyruvate, essential and non-essential amino acids and one or more antibiotics such as gentamycin, penicillin and / or streptomycin. The basal medium for use in the method is free of animal-based products. The basal medium for use in the method is xeno-free medium.

[0052] In a preferred embodiment of the invention the media for use in the method are (i) an iSHED expansion medium DMEM-GlutaMAX medium containing 1 g / L D-glucose and supplemented with 10% fetal bovine serum with antibiotics; and (ii) production medium which is xeno-free medium supplemented with xeno-free supplement s).

[0053] In the method described herein a suitable number of cells are inoculated on (or seeded on) microcarriers and are then cultured in the xeno-free medium on microcarriers. The skilled person can determine what quantity of cells is sufficient to inoculate on microcarriers. For example, iSHEDs may be first expanded in T-flasks in xeno-free medium until a suitable number of cells are obtained. In an embodiment of the invention, cells are seeded on microcarriers at a density of about 10,000 cells / cm2.

[0054] A microcarrier is a support matrix that allows for the growth of adherent cells in bioreactors. The microcarriers used in the method of the invention may be liquid microcarriers or solid particles. The microcarriers used in the method of the invention may any commercially available microcarrier. The microcarriers used in the method of the invention may be alginate-based (GEM, Global Cell Solutions), dextran-based (Cytodex, GE Healthcare), collagen-based (Cultispher, Percell), or polystyrene-based (SoloHill Engineering) microcarriers. In a preferred embodiment of the method of the invention, the microcarriers are dextran-based. In a preferred embodiment of the method of the invention, the microcarriers are Cytodex microcarriers.

[0055] Cells are cultured (and expanded) in the microcarriers on a bioreactor until a suitable confluency is reached. Cell culture may continue for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, or longer. Cell culture will continue until about 80% of the surface area of the microcarriers are covered. In one embodiment, cell culture will continue until there is about 80% confluence of cells on the surface of the microcarriers. “Confluence” or “confluency” refers to the percentage of the surface of a culture vessel or well that is covered by adherent cells. The culturing and expansion of the stem cells is preferable such that there are enough cells to seed for generation of the conditioned supernatant containing EVs.

[0056] Preparation of conditioned supernatant

[0057] Cells are passaged at least twice to achieve a conditioned supernatant containing EVs. The skilled person is aware of passaging methods involving collection of cell culture supernatant and reseeding of cells. For example, about 90% volume of cell culture supernatant can be collected, and the remaining cell-microcarrier suspension collected. The microcarriers can be washed and cells proteolytically detached. The cell-microcarrier suspension can be strained to separate the cells, and the cells resuspended in fresh medium, then reseeded on prepared microcarriers. Following reseeding and subsequent culturing as set out above, supernatants containing EVs can be collected from the microcarrier cultures.

[0058] In the method of the invention cells are passaged at least two times to obtain a conditioned supernatant containing EVs. “Conditioned" can be considered to mean that the supernatant contains factors and EVs secreted from the cells being cultured. The term “conditioned" is a known term to the person skilled in the art.

[0059] Preparation of clarified supernatant The conditioned supernatant derived from the bioreactor microcarrier culture is concentrated and diafiltrated by tangential flow filtration to achieve a concentrated EV suspension.

[0060] Tangential flow filtration (TFF) is a rapid and efficient method for the separation and purification of biomolecules by utilizing ultrafiltration membranes. Unlike normal flow filtration (NFF), the feed in TFF flows parallel to the membrane instead of being pushed through the membrane. The advantage is that in TFF, there is less of a chance for filter clogging, and it is also gentler on shear-sensitive products. In an embodiment of the method of the invention about a 300 kDa cutoff filter is used in the TFF step. The supernatant is diafiltered to achieve a concentrated, or clarified, EV suspension. Concentration of the conditioned supernatant as a result of the TFF step may be 5 fold, 6 fold, 7, fold, 8 fold, 9 fold, 10 fold or more. The skilled person would be able to determine suitable TFF flow speeds and transmembrane pressures to be used in the method of the invention.

[0061] In an embodiment of the invention the conditioned supernatant is centrifuged before TFF. In an embodiment of the invention the conditioned supernatant is filtered after centrifugation and before TFF.

[0062] Preparation of purified EVs

[0063] The concentrated EV suspension is subjected to size-exclusion chromatography to obtain a purified EV suspension. Size-exclusion chromatography (SEC) separates molecules based on their size by filtration through a column. In an embodiment of the invention SEC column with about a 700 kDa cut off is used. EVs are eluted from the column to obtain a purified EV suspension. " urified' can be considered to mean substantially free of impurities.

[0064] “Substantially” can be considered to mean to a great or significant extent, such that any remaining impurities do not affect the functional properties of the EVs.

[0065] Concentration of purified EV suspension

[0066] In an embodiment of the invention, the purified EV suspension may be further concentrated after the size-exclusion chromatography step. In one embodiment, this concentration may occur by centrifugation. Extracellular vesicles obtained by the method

[0067] Also encompassed by the invention are EVs obtained by, or produced by, the methods of the invention. Also encompassed by the invention are EVs obtainable by the methods of the invention. EVs produced by, obtained by, or obtainable by, the methods of the invention have a distinct cargo profile when compared with EVs produced by known methods, which allows the EVs of the invention to be distinguished from EVs produced by known methods.

[0068] Pharmaceutical compositions

[0069] Also encompassed by the invention is a pharmaceutical composition comprising EVs obtainable by, or obtained by, a method of the invention and a pharmaceutically acceptable carrier or diluent. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. In an embodiment of the invention the pharmaceutical composition comprises EVs obtainable by, or obtained by, a method of the invention in a phosphate- buffered saline suspension.

[0070] Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0071] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0072] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0073] Methods of treatment and medical uses

[0074] The EVs obtainable by, or obtained by, the method of the invention maybe used in therapy. In therapeutic applications, EVs are administered to a subject already suffering from a disorder or condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a "therapeutically effective amount" . Effective amounts for a given purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. As used herein, the term "subject" includes any human.

[0075] The EVs thereof of the present invention, or the pharmaceutical compositions as defined herein, are particularly suited for use in the treatment or prevention of inflammatory conditions. "Inflammatory Conditions" or ‘inflammatory diseases" refers to any of a number of conditions or diseases, which are characterized by vascular changes: edema and infiltration of neutrophils (e.g., acute inflammatory reactions); infiltration of tissues by mononuclear cells; tissue destruction by inflammatory cells, connective tissue cells and their cellular products; and attempts at repair by connective tissue replacement (e.g., chronic inflammatory reactions). In a preferred embodiment the inflammatory condition is a neurodegenerative condition. In a preferred embodiment the neurodegenerative condition is Parkinson’s disease.

[0076] The EVs of the invention carry carry many proteins, miRNAs and mRNAs that can be involved in therapeutic action by suppressing oxidative stress and inflammatory response.

[0077] The invention encompasses a method of treatment comprising the administration of a therapeutically effective amount of EVs obtainable by, or obtained by, the methods described herein, or a pharmaceutical composition comprising said EVs, to a subject or patient in need thereof. The invention encompasses a method of treatment of Parkinson’s disease comprising the administration of a therapeutically effective amount of EVs obtainable by, or obtained by, the methods described herein, or a pharmaceutical composition comprising said EVs, to a subject or patient in need thereof.

[0078] The invention encompasses a method of manufacture of a medicament comprising a therapeutically effective amount of EVs obtainable by, or obtained by, the methods described herein, or a pharmaceutical composition comprising said EVs, for the treatment of disease, comprising the administration of said medicament to a subject or patient in need thereof. The invention encompasses a method of manufacture of a medicament comprising a therapeutically effective amount of EVs obtainable by, or obtained by, the methods described herein, or a pharmaceutical composition comprising said EVs, for the treatment of Parkinson’s disease, comprising the administration of said medicament to a subject or patient in need thereof.

[0079] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. The mode of administration may be via injection or infusion. The therapeutic methods of the invention also encompass use of EVs that are derived from autologous cells of an individual or patient to be treated or are allogenic to cells of an individual or patient to be treated.

[0080] The EVs of the invention carry carry many proteins, miRNAs and mRNAs that can be involved in therapeutic action by suppressing oxidative stress and inflammatory response. EXAMPLES

[0081] Example 1: Bioreactor culture

[0082] Expansion of cells before seeding on microcarriers

[0083] A human immortalized (Lenti-hTERT-2A-CDK4) SHED line (later defined as Cells) was used in the expansion method. To achieve sufficient numbers of cells for the inoculation on microcarriers, SHEDs were first expanded in T-flasks. Cells were cultured in DMEM-GlutaMAX medium containing 1 g / L D-glucose and supplemented with 10% fetal bovine serum (FBS, Gibco, 10500064, LOT 2412072H), 100 U / ml penicillin and 100 pg / ml streptomycin, in a 37 °C incubator, in a humid, 5% CO2 atmosphere. For passaging and before inoculation onto the microcarriers, cells were washed once with a phosphate buffered saline (PBS) solution, then a 0.25% trypsin-EDTA solution (Gibco, 25200-072) was added, and cells incubated at 37 °C for about 3-5 min until suspension was formed. Trypsin-EDTA was neutralized by adding double volume of culture medium, then cell suspension centrifuged at 500 x g for 5 min.

[0084] Inoculation on microcarriers

[0085] Cytodex-1 microcarriers (Cytiva, 17044802) were suspended in Cellartis MSC Xeno-Free basal medium with Cellartis MSC Xeno-Free supplement (Takara Bio, Y50200), 100 U / ml penicillin and 100 pg / ml streptomycin (later defined as Medium) and kept at 37 °C for about an hour until inoculation. The cells obtained after expansion were suspended in Medium and seeded on microcarriers at a density of 10,000 cells / cm2in Applikon 2 L bioreactor under constant control of temperature (37 °C), CO2 (5%) and pH (7.4). The density of the microcarriers was 10 cm2 / ml, the volume of the Medium in the bioreactor was 600 ml. Stirring was set at 20 rpm overnight.

[0086] Expansion in bioreactor

[0087] After inoculation the stirring speed was increased to 100 rpm. Microcarriers were inspected daily microscopically, to assess cell attachment and colonization efficiency. Cells were cultured until -80% of the surface area of the microcarriers was covered, typically for 4-5 days.

[0088] Collection of supernatants and reseeding of cells First, 90% volume of cell culture supernatant was collected, then the remaining cellmicrocarrier suspension was collected into test tubes, allowing for microcarriers to settle down. The medium was then removed and microcarriers washed 3 times with PBS. Then warm TrypLE select (Gibco, 12563-029) was added, mixed and incubated at 37 °C for 10 minutes. After incubation, the cell-microcarrier suspension was mixed with a serological pipette and placed onto the 100 pm cell strainers (Th. Geyer, 7.696 769) in the 50 ml test tubes. After washing strainers 3 times with PBS, the separated cells were counted, resuspended in a fresh Medium, and reseeded on freshly prepared (see above) microcarriers under the same conditions as before.

[0089] For in vivo experiments, supernatants containing extracellular vesicles (EVs) were collected from the cells that have been reseeded on microcarriers twice (EV isolation-III) (Figure 1). Example 2: Isolation of EVs from the cell culture supernatants

[0090] Preparation of cell culture medium

[0091] To remove detached cells and large debris, conditioned cell culture medium was clarified by the centrifugation at 500xg for 10 min 4 °C, then supernatant (500 ml) was collected and filtered using 0.22 pm vacuum filters (Corning, 430769).

[0092] Tangential flow filtration

[0093] Clarified cell culture medium was concentrated using 300 kDa cutoff hollow fiber filters (MidiKros 65 cm 300 kDa mPES, Repligen, D06-E300-05-N) in Cogent pScale TFF system (Merc Millipore). All processes were carried out at 100.5ml / min flow speed (30% max) and at 3.0 psi transmembrane pressure. Total volume of 500 ml medium was concentrated to a final volume of 50 ml at the rate 100.5ml / min. Concentration was followed by diafiltration with 500 ml PBS buffer. The final volume of concentrated and dialyzed EV suspension was 50 ml.

[0094] Size-exclusion chromatography

[0095] Concentrated and dialysed EV suspensions were subjected to the size-exclusion chromatography using HiScreen Capto Core 700 column (Cytiva, 17548115) in AKTA avant 25 (Cytiva) preparative chromatography system. EVs were eluted with PBS buffer and 40 ml fractions collected in 50 ml tubes. The elution of EVs was detected by absorption of 280 UV light. Internalized small impurities (<700kDa) were washed out from the column with 2M NaCl solution.

[0096] Concentration

[0097] Suspensions of purified EVs were concentrated 10 times in Amicon Ultra 3 kDa MWCO (Merc Millipore Cat. No. UFC9003) centrifugal filter units by centrifugation at 40007g at 4°C. The final EV samples (5 ml) were aliquoted and stored at -80°C until use.

[0098] Nanoparticle tracking analysis (NTA) revealed that in both samples, the main population of EVs peaks at around 100-130 nm. 2D-UC EVs exhibited a broader size distribution, including a greater proportion of larger particles with small peaks at 239 nm, 335 nm and 459 nm, while 3D-TFF / SEC EVs were more uniform in size. Western blot confirmed that both types of EVs expressed key EV markers such as HSP70, syntenin-1 and CD63.

[0099] The use of a 3D bioreactor system for SHED culture on microcarriers, combined with subsequent EV isolation by ultracentrifugation (3D-UC), demonstrated a remarkable 20.3-fold increase in EV yield compared to the standard 2D culture approach using ultracentrifugation (2D-UC). Moreover, further purification of EVs using TFF followed by SEC (3D-TFF-SEC) resulted in an additional 22.8-fold increase in EV yield. When combined, the optimized 3D culture system and advanced purification protocol achieved an impressive 463 -fold increase in overall EV production compared to the conventional 2D culture and ultracentrifugation method. EV yields, as determined by NTA, were normalized to the growth surface area to ensure accurate comparisons across culture conditions.

[0100] Nanoparticle tracking assays showed that when compared to laboratory scale method employing cell culture on the T flasks and EV isolation using ultracentrifugation, the new protocol increased the scale of EV production by 463 fold.

[0101] Example 3: Culture conditions (3D versus 2D) and purification methods (UC versus TFF / SEC) impacts molecular cargo of Evs RNA extraction

[0102] Total RNA was extracted from EV samples and cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) with an on-column DNase I digest. For the EV samples, 7 pL glycogen (5 mg / mL) was added for enhanced precipitation. RNA was eluted in 30 pL nuclease-free water and stored at -80°C until further analysis. For cell samples, the quality of extracted total RNA was assessed using the Agilent RNA 6000 Nano kit (5067-1511, Agilent Technologies, Waldbronn, Germany) for 2100 Bioanalyzer systems.

[0103] Small RNA library preparation

[0104] 8.5 pL (EVs) or 100 ng (cells) total RNA were used as input for the generation of small RNA sequencing libraries. Libraries were generated with the RealSeq Biofluids library preparation kit (RealSeq Biosciences, CA, USA) according to the manufacturer’s instructions. To each sample, 1 pL miND® spike-in standards (TAmiRNA, Vienna, Austria) were added during the first step as described previously (Khamina K, Diendorfer AB, Skalicky S, Weigl M, et al.

[0105] 2022. A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers. IntJ Mol Sci 23.). Adapter-ligated libraries were amplified (20 cycles for EV samples; 18 cycles for cells) using barcoded Illumina reverse primers in combination with the Illumina forward primer. Library quality control was performed using DNA 1000 chips (Agilent Technologies, Waldbronn, Germany). All samples were pooled equimolarly and processed with the Blue Pippin system (Sage Science, MA, USA) using 3% agarose size selection cassettes, following the manufacturer’s instructions (size range: 130-160 bp). Sequencing was performed on an Illumina NovaSeq SP Flowcell in SR100 mode.

[0106] Whole-transcriptome sequencing library preparation

[0107] 6 pl total RNA from EVs and 10 ng total RNA from cells were used as input for the generation of whole-transcriptome sequencing libraries using the SMART er stranded total RNA sequencing kit v3.0 pico (TakaraBio) according to the manufacturer's instructions. Preamplification was performed using 10 PCR cycles in steps 1 and 2, respectively. Library yield was assessed on the Bioanalyzer DNA high-sensitivity assay between 200 and 2000 bp. Libraries were pooled equimolarly and sequenced on a NovaSeq S4 in paired-end 150 mode (Illumina).

[0108] Bioinformatics analysis of transcriptomics data

[0109] Small RNA-sequencing data were analyzed using the miND® analysis pipeline and evaluated with fastQC vO.11.9 and multiQC vl.14. Reads were adapter-trimmed, and quality filtered using cutadapt v3.3. Mapping steps were performed with Bowtie vl.3.0 and miRDeep2 v2.0.1.2. Reads were initially mapped against the genomic reference GRCh38.pl2 by Ensembl allowing two mismatches and subsequently against miRBase v22.1, filtered for microRNAs of hsa, allowing one mismatch. For a general RNA composition, non-microRNA mapped reads were mapped against RNAcentral vl9.0 and assigned to RNA species of interest.

[0110] Overall quality of the whole-transcriptomic next-generation sequencing data was evaluated automatically and manually using fastQC v0.11.8 and multiQC vl.7. Sequencing reads from all passing samples were adapter-trimmed and quality filtered with bbduk from the bbmap package v38.69, removing sequencing adapters, low-quality bases, and short reads below a predefined length threshold. Reads were then aligned to the Homo sapiens reference genome GRCh38.pl3 provided by Ensembl using STAR v2.7. Gene expression quantification was carried out using Salmon vl.l in alignment-based mode.

[0111] Statistical Analysis

[0112] Statistical analysis of NGS data (miRNA and whole-transcriptomic) and proteomics was conducted with R V4.0. Prior to unsupervised investigation and overlap analysis, whole-transcriptomic data were filtered for protein-coding genes only. Overlap analysis was conducted using the package UpSetR vl.4. Transcriptomics count data and log-transformed proteomics data were used for unsupervised exploration. Spearman correlation distances were calculated using rstatix vO.7.2. and clustered using the package pheatmap vl.0.12. Principal component analysis was performed using the pcaMethods vl.98.0 package. RNA seq revealed that samples containing EVs isolated using standard lab scale methods (EV2DUC) and the scaled up protocol of the invention (EV3DTFFSEC) form separate clusters (Figure 2). In addition, our data showed that EV purification using TFF / SEC significantly decreased complexity of RNA species (Figure 3).

[0113] A reduction of miRNA and Inc RNA levels in EV samples purified using TFF / SEC of approx.

[0114] 10 - 15 times was observed. Proteomic data showed that samples containing EVs isolated using standard lab scale (EV2DUC) and the scaled up methods of the invention (EV3DTFFSEC) form separate clusters (Figure 4A). Purification using TFF / SEC resulted in a substantial decrease of complexity of EV proteome (by approx. 25 %) (Figure 4B).

[0115] To assess the impact of purification (UC vs TFF-SEC) and cultivation (2D vs 3D), a systematic analysis of the EV-associated miRNA, mRNA, and protein cargo was performed. The resulting data sets for each molecule type were investigated for their i) complexity (number of identified proteins, mRNAs, and miRNAs), ii) patterns in molecule abundance using principal component analysis (PCA), and iii) reproducibility using Spearman correlation analysis (Figure 5).

[0116] The microRNA data (Figures 5A, 5B and 5C) showed that overall, > 700 microRNAs were detected, of which 270 were found in all samples (Figure 5A). The complexity was higher in 2D UC EV samples compared to TFF, with 276 miRNAs only detected in 2D UC but not 3D UC or 3D TFF SEC. PCA analysis showed that miRNA expression patterns in EVs harvested from 3D cultivated cells were similar, independent of the purification method, while 2D_UC EV exhibited marked differences in miRNA profiles (Figure 5B), which was confirmed by Pearson correlation analysis using the common set of 270 miRNAs (Figure 5C).

[0117] This demonstrates that miRNA changes are subject to change when cultivation parameters are changed.

[0118] The mRNA data in EVs (Figures 5D, 5E and 5F) showed high uniformity across all samples. mRNA complexity was high, with 13902 detected mRNAs of which 9503 were detected in all samples. 2D UC and 3D UC samples exhibited slightly higher complexity, and PCA analysis indicates that mRNA patterns differ between UC and TFF-SEC purified EVs, however, Pearson Correlation analysis showed highly reproducible mRNA patterns across all EV samples. This demonstrates that mRNA profiles in EV preparations are only subject to small changes during the scale-up of EV production.

[0119] The protein data in EVs (Figures 3G, 3H, and 31) showed a different pattern. Overall, 2457 proteins were detected, of which 862 were present in all 8 EV preparations. Interestingly, protein complexity was highest in 2D UC and 3D UC samples, with 1018 proteins only detected in these groups but not in 3D TFF SEC. Vice versa, no proteins could be identified that are exclusively present in 3D TFF SEC but not in 3D UC or 2D UC. PC A analysis showed that protein patterns are highly reproducible in 3D TFF SEC samples, but different from 2D UC and 3D UC samples. Pearson correlation between the set of 862 overlapping proteins confirmed this result. This indicates that the change in EV purification from UC to TFF-SEC has a strong impact on the protein content in EV preparations.

[0120] In conclusion, both culture conditions (3D versus 2D) and purification methods (UC versus TFF / SEC) dramatically impacts molecular cargo profile of EVs. The results demonstrate that culture conditions and isolation method differentially affect proteomic and miRNA cargo of EVs: when compared with UC, isolation using TFF / SEC substantially reduced the complexity of proteomic cargo of EVs, whereas culture conditions (2D versus 3D) affected miRNA, but not mRNA and proteomic content of the EVs.

[0121] Example 4: Evaluation of therapeutic efficacy of EVs in animal model of Parkinson’s disease (PD)

[0122] Animal model

[0123] A rat 6-hydroxydopamine (6-OHDA) model of PD was used in the experiment. Unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) was performed on day 0 (DO) as described previously [3,4],

[0124] Experimental design is shown in Figure 6. The rats were randomly divided into the six groups (n = 9-12). 1. Control. 0.1% ascorbic acid (Asc) intra-MFB + PBS (intranasal application, i.n.);

[0125] 2. EVI. 0.1% Asc intra-MFB + EVI (EVs produced and isolated using a new scaled up protocol) (i.n.);

[0126] 3. EV2. 0.1% Asc intra-MFB + EV2 (EVs produced and isolated using standart protocol) (i.n.); 4. 6-OHDA. 6-OHDA 20 pg in 3 pl of 0.1% Asc intra-MFB+ PBS (i.n.);

[0127] 5. 6-0HDA+ EVI. 6-OHDA intra-MFB + EVI (i n );

[0128] 6. 6-0HDA+ EV2. 6-OHDA intra-MFB + EV2 (i n ).

[0129] On D8 after the 6-OHDA injection rats received intranasal (each nostril) injection of EV suspension (Figure 6). A single dose of EVs contained 3,8xl08EVs suspended in 10 pl of PBS. In total, during the treatment course, each rat received 17 daily EV treatments (until D24). Functional gait (Catwalk, CW) and cognitive (Morris water maze, MWM) tests were performed as described elsewhere [3,4], On D31 animals were euthanized and immunohistochemical analysis performed as described [3],

[0130] Improvement of gait parameters following intranasal administration of EVs

[0131] Marked impairments in all studied gait parameters in the CatWalk test were observed. Interactions between groups were significant regarding the following parameters: stand duration ( s, 228 = 18.54, / ? < 0.0001, Figure 7A), stride length (F5, 228 = 29.02, / ? < 0.0001, Figure 7B), step cycle (F5, 228 = 23.32, / ? < 0.0001, Figure 7C), and duty cycle (F5.228 = 20.25, / ? < 0.0001, Figure 7D). Holm-Sidak’s post-hoc analysis revealed that 6-OHDA group rats had significantly longer standing time of right front ( / ? < 0.05), right hind ( / ? < 0.001) and left hind (p< 0.001) paws compared to sham-operated animals (Figure 7A). Stand time was shorter in 6-OHDA+EV1 group rats, specifically that of the right hind (p < 0.01) and left hind (p < 0.05) paws.

[0132] In 6-OHDA+EV2 group, stand time with all paws was shorter compared to that of 6-OHDA group rats (p < 0.05 for right and left front paws, p < 0.001 for right and left hind paws). Rats that received 6-OHDA injection also had significantly shorter stride length of all paws (p< 0.001 for right front and hind paws and left hind paw, / ?< 0.0001 of the left hind paw, Figure 7B) in comparison to Sham group animals. Rats from the 6-0HDA+EV1 group showed markedly longer stride length of all paws (p< 0.0001 for right front, left front and left hind paws, p < 0.001 for the right hind paw) when compared to 6-OHDA group.

[0133] Significant increase in stride length was also observed in 6-OHDA+EV2 group rats in comparison to 6-OHDA group animals in all paws (p< 0.0001 for right front and left front paws, p < 0.01 for the right hind and p < 0.001 for the left hind paw). Step cycle of 6-OHDA was markedly prolonged for all paws (p < 0.01 for right and left front paws, p < 0.05 for right and left hind paws) compared to Sham group (Figure 7C). In comparison to 6-OHDA group rats, this parameter was significantly shorter in 6-0HDA+EV1 group, namely for right front and right hind paws (p< 0.001), as well as the left hind paw ( / ?<0.05). Moreover, 6-0HDA+EV2 group animals had significantly shorter step cycle in all paws when compared to 6-OHDA group rats (p < 0.001 for the right front and left hind paw and p < 0.01 for the right hind and left front paw).

[0134] Finally, the duty cycle percentage of all paws in 6-OHDA group rate was substantially increased (p< 0.001) except for the left front paw (Figure 7D). 6-0HDA+EV1 group showed significantly lower duty cycle when compared to 6-OHDA group animals (p < 0.0001 for right front and hind paws, p < 0.01 for the left hind paw). Duty cycle of all paws was lower in 6-0HDA+EV2 group rats (p < 0.001 for the right hind paw, p < 0.01 for the right hind and left front paw and p < 0.001 for the left hind paw) in comparison to 6-OHDA group.

[0135] Conclusion: both EV preparations significantly improved all tested gait functions in the 6-OHDA rats.

[0136] Improvement of spatial learning and memory after intranasal administration ofEVs

[0137] Rat spatial learning in the Morris water maze test was significantly altered between groups (Fs, 55 = 16.3, p< 0.0001, Figure 8A). Holm-Sidak’s post-hoc analysis further revealed that 6-OHDA-injected rats had longer escape latency on days 2 ( / ?<0.01), 3 ( / ?<0.01) and 4 (p < 0.0001) compared to Control (Figure 8A), while 6-0HDA+EV1 group rats demonstrated significantly shorter latency to 6-OHDA group (p < 0.05 on day 2 and 3, / ? <0.01 on day 4). Rats from 6-OHDA+EV2 group also showed markedly shorter escape latency on these days when compared to 6-OHDA group (p < 0.01 on day 2, / ? <0.001 on day 3 and / ? <0.0001 on day 4). In the probe trial on day 5, significant differences between groups were also detected for platform zone crossings (F5, 52 = 8.94, p < 0.0001, Figure 8B) and time spent in the platform quadrant (F5, 52 = 12, p< 0.0001, Figure 8C). 6-OHDA group animals crossed the platform zone considerably less than controls (p< 0.0001, Figure 8B) and spent significantly less time in the platform quadrant (p< 0.0001, Figure 8B). Rats from 6-OHDA+EV1 and 6-OHDA+EV2 groups crossed the platform zone more times than the 6-OHDA group animsl (p < 0.05 in 6-OHDA+EV1 and ? < 0.01 in 6-OHDA+EV2 group). Meantime spent in the target quadrant was longer in 6-OHDA+EV2 (p < 0.0001), but not 6-OHDA+EV1 group compared to 6-OHDA group animals.

[0138] Conclusion: both EV preparations significantly improved spatial learning and memory of the 6-OHDA rats.

[0139] Preservation of tyrosine hydroxylase (TH) density in the striatum and SN after intranasal administration ofEVs to the 6-OHDA-treated rats

[0140] A substantial decrease in TH density was observed in the SN (p< 0.0001, Figure 9A-B) and striatum (p< 0.001 , Figure 9C-D) of rats injected with 6-OHDA compared to controls. Compared to 6-OHDA-injected rats, rats that received EVs exhibited increased TH density in the SN (p < 0.01 in 6-OHDA+EV1 and p < 0.001 in 6-OHDA+EV-2 group) and in the striatum (p < 0.05 in both 6-OHDA+EV1 and in 6-OHDA+EV-2 group).

[0141] Intranasal administration ofEVs decrease astrogliosis in the 6-OHDA-treated rats Density of astroglial protein GFAP was markedly elevated in the hippocampal dentate gyrus (DG) of 6-OHDA group rats (p< 0.001, Figure 10A-B) in comparison to controls. Those 6-OHDA-injected animals that were treated with intranasally administered EVs showed a decrease in GFAP density compared to 6-OHDA group rats (p < 0.05 in both 6-OHDA+EV-l and in 6-OHDA+EV-2 group).

[0142] Intranasal administration of EVs decrease microgliosis and interleukin-1 beta expression in the 6-OHDA-treated rats The density of microglial marker altered ionized calcium -binding adapter protein- 1 (Iba-1) was increased significantly in DG of lesion group rats (p< 0.0001 vs. Control, Figure 11A-B). In 6-OHDA+EV1 and 6-OHDA+EV2 groups, Iba-1 density was lower (p < 0.05 vs. 6-OHDA).

[0143] Similarly, inflammasome-related marker IL-ip-positive cells were counted in the SN of controls, 6-OHD A-inj ected and 6-OHDA group animals that received EVs. Marked elevation of IL-ip-positive cells was observed in SN of 6-OHDA group rats in comparison to controls (p< 0.0001 vs. Control, Figure 12A-B).

[0144] Rats treated with EVs demonstrated a substantial decrease of the IL-ip expression in the SN (p< 0.001 in 6-OHDA+EV-l and / ?<0.01 in 6-OHDA+EV-2 groups) when compared to 6-OHDA group animals.

[0145] It is proposed that EVs suppress the neurotoxic action of the 6-OHDA by reducing oxidative stress in dopaminergic neurons of the SN. Indeed, proteomic analysis identified several proteins that were abundant in the EV preparations and were previously described as potent suppressors of oxidative stress in PD, such as Apolipoprotein D (ApoD), glutathione S-transferase Pi 1 (GSTP1), and glutathione S-transferase omega 1 (GSTO-1). The EVs were also found to contain superoxide dismutase 1 (SOD1), catalase (CAT) and peroxiredoxin-2 (PRDX2), enzymes essential for neutralizing ROS and protecting dopaminergic neurons from oxidative damage in PD. Proteomic analysis also identified high levels of anti-inflammatory proteins ANXA1, ANXA2 and ANXA5 in the EV preparations.

[0146] Conclusion: both EV preparations preserved tyrosine hydroxylase density in the striatum and SN, decreased astrogliosis and suppressed neuroinflammation in the 6-OHDA rats.

[0147] General Conclusions

[0148] 1. Our newly developed protocol increases the scale of EV production from immortalized human stem cells from exfoliated deciduous teeth by 463 fold.

[0149] 2. Culture conditions (microcarrier culture versus standard) and purification methods (ultracentrifugation versus TFF / SEC) significantly impact molecular cargo of EVs. EV purification using TFF / SEC significantly decreased complexity of proteomic content and RNA species.

[0150] 3. EVs produced by the new protocol and used according to our unique treatment regimen improve gait and cognitive functions, normalize tyrosine hydroxylase expression and suppress neuroinflammation in PD rats.

[0151] 4. Our new technology can be used for the large-scale production of EVs suitable for preclinical and early phase clinical trials against PD.

[0152] REFERENCES

[0153] 1. Diaz ML (2019) Regenerative medicine: could Parkinson's be the first neurodegenerative disease to be cured? Future Sci OA 5 (9):FSO418. doi:10.2144 / fsoa-2019-0035

[0154] 2. Hermann DM, Peruzzotti-Jametti L, Giebel B, Pluchino S (2024) Extracellular vesicles set the stage for brain plasticity and recovery by multimodal signalling. Brain 147 (2):372-389. doi:10.1093 / brain / awad332

[0155] 3. Narbute K, Pilipenko V, Pupure J, Dzirkale Z, Jonavice U, Tunaitis V, Kriauciunaite K, Jarmalaviciute A, Jansone B, Klusa V, Pivoriunas A (2019) Intranasal Administration of Extracellular Vesicles Derived from Human Teeth Stem Cells Improves Motor Symptoms and Normalizes Tyrosine Hydroxylase Expression in the Substantia Nigra and Striatum of the 6-Hydroxydopamine-Treated Rats. Stem Cells Transl Med 8 (5):490-499. doi:10.1002 / sctm,18-0162

[0156] 4. Narbute K, Pilipenko V, Pupure J, Klinovics T, Auders J, Jonavice U, Kriauciunaite K, Pivoriunas A, Klusa V (2021) Time-Dependent Memory and Gait Improvement by Intranasally- Administered Extracellular Vesicles in Parkinson's Disease Model Rats. Cell Mol Neurobiol 41 (3):605-613. doi:10.1007 / sl0571-020-00865-8

[0157] 5. Jarmalaviciute A, Tunaitis V, Pivoraite U, Venalis A, Pivoriunas A (2015) Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis. Cytotherapy 17 (7):932-939. doi:10.1016 / j.jcyt.2014.07.013

[0158] 6. Tkach M, Thery C (2016) Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell 164 (6): 1226-1232. doi: 10.1016 / j .cell.2016.01.043

Claims

CLAIMS1. A method of production of extracellular vesicles (EVs) comprising:(a) culturing human stem cells from exfoliated deciduous teeth (SHED)s on microcarriers in the presence of xeno-free medium;(b) passaging said SHEDs at least twice to achieve a conditioned supernatant containing EVs;(c) concentrating and diafiltrating said conditioned supernatant by tangential flow filtration (TFF) to achieve a concentrated EV suspension; and(d) subjecting said concentrated EV suspension to size-exclusion chromatography (SEC) to obtain a purified EV suspension.

2. The method of claim 1, wherein tangential flow filtration comprises the use of a filter with a 300 kDa cut off.

3. The method of claim 1 or claim 2, wherein size-exclusion chromatography comprises use of a column with a 700 kDa cut off.

4. The method of any one of the preceding claims, wherein the purified EV suspension is subsequently concentrated.

5. Extracellular vesicles obtainable by, or obtained by, the method of any one of claims 1 to 4.

6. A pharmaceutical composition comprising the extracellular vesicles of claim 5 and a pharmaceutically acceptable carrier.

7. The extracellular vesicles of claim 5, or the pharmaceutical composition of claim 6, for use in therapy.

8. The extracellular vesicles of claim 5, or the pharmaceutical composition of claim 6, for use in the treatment of Parkinson’s disease.

269. The extracellular vesicles, or pharmaceutical composition, for use according to claim 7 or claim 8, wherein the SHEDs are autologous or allogenic to a patient being treated.

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