Extracellular vesicles for clinical use thereof in the treatment of synucleinopathies

Abstract

The present invention relates to extracellular vesicles (EVs) derived from human dendritic cells (hDCs) comprising a RVG-hLAMP peptide construct, having sequence SEQ ID NO. 1, expressed on the surface thereof, wherein the extracellular vesicles are loaded with anti-α-synuclein shRNA minicircles (ShRNA-MCs). Likewise, the invention relates to the clinical use of said vesicles in the treatment of synucleinopathies. Lastly, the present invention relates to a method for producing the extracellular vesicles from human dendritic cells (hDCs).

Description

[0001] DESCRIPTION

[0002] Extracellular vesicles for clinical use in the treatment of synucleinopathies

[0003] Field of the invention

[0004] The present invention belongs to the technical field of medicine, in particular it relates to extracellular vesicles derived from human dendritic cells and their clinical use in the treatment of synucleinopathies such as Parkinson's disease.

[0005] Background of the invention

[0006] Synucleinopathies (also called α-synucleinopathies) are neurodegenerative diseases characterized by the abnormal accumulation of α-synuclein protein aggregates in neurons, nerve fibers, or glial cells. There are three main types of synucleinopathies: Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).

[0007] Alpha-synuclein is a neuronal protein abundantly expressed in the brain, specifically in presynaptic nerve endings. It constitutes more than 1% of the total protein in the cytosol of brain cells. In synucleinopathies, such as PD, it accumulates excessively within these cells. These deposits are traces of cellular alterations that indicate neuronal dysfunction and eventually lead to neuronal death, a phenomenon responsible for the motor symptoms of the disease.

[0008] PD is the second most common neurodegenerative disorder, affecting an estimated 10 million people worldwide (L. Hirsch, et al. The incidence of Parkinson's disease: A systematic review and meta-analysis. Neuroepidemiology 46, 292-300 (2016)) and its prevalence is estimated to double by 2040 (E.R. Dorsey, et al. The emerging evidence of the Parkinson pandemic. J Parkinsons Dis. 8, S3-S8 (2018). PD is clinically characterized by the asymmetric and progressive onset of motor symptoms including tremor, rigidity, and bradykinesia, as well as non-motor symptoms in the form of cognition, depression, and autonomic dysfunction (C. Váradi. Clinical features of Parkinson's disease: The evolution of critical symptoms. Biology (Basel). 9, 103 (2020). In PD, intracellular accumulation of α-synuclein, leading to the loss of dopaminergic neurons, takes place in the substantia nigra pars compacta (SNc) (JL Eriksen, et al. Molecular pathogenesis of Parkinson disease. Arch Neurol.62, 353-7 (2005).

[0009] Currently, there is no cure for PD, and drug treatment is primarily symptomatic and does not prevent PD progression. Furthermore, as the disease progresses, drugs become less effective and are associated with significant side effects. Therefore, there is a clear need to develop new disease-modifying strategies for this disease.

[0010] Alpha-synuclein has been linked to both sporadic and familial forms of PD, and multiple evidence supports a central role for this protein in the onset and progression of the pathogenesis of this disease (J. Simón-Sánchez, et al. Genome-wide association study reveals genetic risk underlying Parkinson's disease. Nat. Genet. 41, 1308-1312 (2009); V.M. Lee, et al. Mechanisms of Parkinson's disease linked to pathological alpha-synuclein: new targets for drug discovery. Neuron 52, 33-38 (2006); J.Y. Li, E., et al. Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation. Nat. Med. 14, 501-503 (2008); J.H. Kordower, et al. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease. Nat. Med. 14, 504–506 (2008). Consequently, therapeutic strategies targeting alpha-synuclein are a powerful approach to the prevention and treatment of PD (AT Rodger, et al.Are therapies that target a-synuclein effective at halting Parkinson's disease progression? A systematic review. Int J Mol Sci. 24, 11022 (2023); S. Menon, et al. Alpha-synuclein targeting therapeutics for Parkinson's disease and related synucleinopathies. Front Neurol. 13, 852003 (2022).

[0011] Gene therapy has emerged as a promising therapeutic option for the treatment of PD, and there are currently several clinical trials registered on ClinicalTrials.gov for PD, although none are designed to modulate α-synuclein expression.

[0012] In the state of the art, downregulation of α-synuclein levels has been carried out using small interfering RNA (siRNA) (JY Li, et al (2008)) or short hairpin RNA (shRNA) delivered by adeno-associated virus (AAV) (JH Kordower, et al. (2008)) in the SNc, observing a decrease in α-synuclein aggregates and motor deficit in transgenic or toxin-based PD models.

[0013] However, one of the main challenges for gene therapy against α-synuclein is to design a delivery system or vehicle that crosses the blood-brain barrier, to easily release molecules into the brain after peripheral administration, and modify the disease, altering genetic expression for prolonged periods, given the chronic condition of PD, preferably using vehicles that do not trigger the activation of the immune system, such as AAVs (AT Rodger, et al. 2023).

[0014] The present inventors developed a gene therapy vehicle based on extracellular vesicles (EVs) targeted to the brain by expressing the rabies virus glycoprotein (RVG) peptide on the outer surface of the EVs (RVG-EVs), capable of delivering siRNA and shRNA minicircles (MCs) into the brain after intravenous administration, resulting in gene silencing (L. Alvarez-Erviti, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341-345 (2011); J.M. Cooper, et al. Systemic exosomal siRNA delivery reduced alpha-synuclein aggregates in brains of transgenic mice. Mov. Disord. 29, 1476-1485 (2014); M. Izco, et al. Systemic exosomal delivery of shRNA minicircles prevents Parkinsonian pathology. Mol. Ther. 27, 2111-2122 (2019).

[0015] While successful, the half-life of siRNA in vivo is relatively short, and the treatment of chronic diseases requires longer-term gene silencing. Plasmids expressing shRNA are better for long-term gene silencing; however, conventional plasmids are relatively large, which prevents their loading into EVs. Minicircles (MCs), however, are double-stranded DNA vectors that contain the transgene expression cassette without additional bacterial sequences (M. Izco et al. 2019), so they are smaller in size. Furthermore, they promote greater transgene expression over longer periods and therefore have the potential to modulate target gene expression for longer periods.

[0016] Thus, α-synuclein shRNA MCs loaded into RVG-EVs were tested in a progressive mouse model of PD, observing their potential to reduce long-term brain α-synuclein expression, decrease α-synuclein aggregates, as well as prevent the loss of dopaminergic neurons in the SNc, avoiding motor deficits ( / zco et al. 2019). However, these results are not directly applicable to clinical use in humans, since the application of this technology to clinical trials requires the development of a source of modified human EVs compatible with personalized therapy, since being a chronic and progressive disease the patient will require long-term treatment.

[0017] Based on the urgent needs of the state of the art in relation to PD and other synucleinopathies, the authors of the present invention have carried out significant research work that has allowed the therapy developed in animal models to be translated into clinical practice in humans, through the development of a source of immunologically modified inert human EVs compatible with personalized therapy. Adaptation to clinical applications has required overcoming technical obstacles through the use of alternative technologies and novel approaches to guarantee the safety and efficacy of this type of long-term treatment.

[0018] The results obtained from these investigations have unexpectedly demonstrated that therapy using RVG-EVs obtained from modified human dendritic cells (hDCs), differentiated in vitro from circulating monocytes in the blood, more effectively prevents motor disorders in PD, associated with a significantly greater decrease in α-synuclein mRNA levels than in animals treated with therapy using EVs isolated from modified mouse dendritic cells (Ms-DCs).

[0019] These findings represent a highly relevant contribution to the state of the art, due to their important clinical implications for the treatment of Parkinson's and other synucleinopathies. For the first time, a therapy has been developed that allows the production of modified EVs that cross the blood-brain barrier and are produced by the patient's own cells, without activating the immune response or producing off-target effects. This enables the administration of numerous doses of EVs loaded with shRNA-MCs against α-synuclein. Description of figures

[0020] Figure 1. Loading optimization and validation of hRVG-EVs loaded with anti-α-synuclein shRNA-MC. (a and b) NTA characterization of EVs isolated from control hDCs (a) and hDCs electroporated with hLamp2b-RVG mRNA (b). (c) Western blot images to assess the expression of the markers, LAMP-2, hsc70 and Flotillin-1, in control EVs and RVG-EVs. (d) Representative TEM images of EVs isolated from control hDCs (left) and hDCs electroporated with hLamp2b-RVG mRNA (right). Scale bar represents 200 nm. (e and f) SH-SY5Y cells overexpressing α-synuclein were treated with anti-α-synuclein shRNA-MC via a transfection reaction (TR) or loaded into control hEVs (EV) and hRVG-EVs (RVG-EV). α-synuclein mRNA (e) and protein (f) levels were quantified and normalized. Typical Western blot images (g) are shown. Data are expressed as mean ± SEM (n = 4).*p < 0.05, non-parametric Kruskal-Wallis test, statistical analysis compared to untreated control cells.

[0021] Figure 2. Downregulation of α-synuclein in various brain regions of mice treated with anti-α-synuclein shRNA-MC delivered by hRVG-EVs. Analysis at 90 days post-treatment of α-synuclein mRNA expression (a, c, e) and protein levels (b, d, f) normalized against actin in ipsilateral midbrain (a, b), striatum (c, d), and cerebral cortex (e, f) of mice that received an intrastriatal injection of PBS (control) or α-synuclein PFF and 2 intravenous injections of 5% glucose (PFF), hRVG-EVs loaded with anti-α-synuclein shRNA-MC (H-EV Syn) or GFP (H-EVs GF). A group of mice was treated with anti-α-synuclein shRNA-MC delivered in Ms-RVG-EVs (Ms-EV Syn). Typical Western blot images for each structure (midbrain, striatum, and cortex) are shown. Data are expressed as mean ± SEM (n = 6–8). *p < 0.05, one-way ANOVA, statistical analysis compared to control mice.

[0022] Figure 3. Effect of anti-α-synuclein shRNA-MC-loaded hRVG-EVs on α-synuclein pathology in the SNc. (a) Immunofluorescent images of midbrain sections stained with antibodies against phospho-α-synuclein S129 (green) and TH (red), (b) Magnified image of a stained midbrain section showing the localization of α-synuclein aggregates (green) in dopaminergic neurons (red). Scale bar, 100 pm. (c) Quantification of the number of α-synuclein-positive aggregates per section in the ipsilateral SNc of PFF, H-EV Syn, H-EV GF, and Ms-EV Syn mice. Data are expressed as mean ± SEM (n = 6–10). *p < 0.05, one-way ANOVA, statistical analyses compared to PFF mice. Figure 4. α-synuclein pathology in striatum, cerebral cortex, and amygdala after treatment with hRVG-EVs loaded with anti-α-synuclein shRNA-MC.Quantification of the number of phospho-α-synuclein S129 aggregates in ipsilateral striatum (a), cerebral cortex (b), and amygdala (c) of PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice. Representative immunohistochemical images of intraneuronal phospho-α-synuclein-positive aggregates in ipsilateral striatum, cerebral cortex, and amygdala are shown. Scale bar, 100 µm. Data are expressed as mean ± SEM (n = 6–10), *p < 0.05, one-way ANOVA, statistical analyses compared to PFF mice.

[0023] Figure 5. Anti-α-synuclein hRVG-EVs shRNA-MC therapy prevents dopaminergic dysfunction and motor impairments induced by intrastriatal α-synuclein injections in PFF mice. (a) TH staining of dopaminergic neurons in coronal midbrain sections from control, PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice. Arrows indicate decreased numbers of dopaminergic neurons. The number of dopaminergic neurons was quantified by stereology in each cerebral hemisphere (I, ipsilateral; C, contralateral). (b) Dopaminergic innervation in the striatum was quantified by optical density in ipsilateral striatal sections normalized to the contralateral striatum. Representative TH staining of striatal sections is shown.(c) Quantitative analysis of mouse hindlimb position scoring when suspended by the tail, (d) Time spent hanging on a grid, (e) Time taken for the mouse to turn around and orient itself upwards after being placed in the middle of a grid tilted at an angle of 45° to the surface with its head pointing downwards. Data are expressed as mean ± SEM (n = 6–10). *p < 0.05, ***p < 0.001, one-way ANOVA, statistical analyses compared to control mice.

[0024] Figure 6. Effect of anti-α-synuclein hRVG-EV shRNA-MC therapy on α-synuclein mRNA and protein levels in the spinal cord and intestine. Quantification of α-synuclein mRNA (a and c) and protein (b and d) levels in the spinal cord (a and b) and intestine (c and d) of control, PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice. Typical Western blot images are shown. Data are expressed as mean ± SEM (n = 6–8). *p < 0.05, one-way ANOVA, statistical analyses compared to control mice.

[0025] Figure 7. Proteomic analysis of EVs produced by DCs differentiated in vitro from blood monocytes isolated from control patients, early PD and advanced PD.

[0026] Heat map and hierarchical clustering analysis of differentially expressed proteins between patients with early PD (a) or advanced PD (b) versus control individuals, (c) Venn diagram of differentially expressed proteins detected in EVs derived from early and advanced PD.

[0027] Figure 8. Regulation of α-synuclein expression in contralateral brain regions of mice treated with anti-α-synuclein shRNA-MC delivered by hRVG-EV. Analysis of α-synuclein mRNA (a) and protein (b) expression levels normalized to actin in the contralateral midbrain of control, PFF, H-EV Syn, kyiv GF, or Ms-EV Syn mice. A typical Western blot image is shown. Analysis of α-synuclein mRNA expression levels normalized to actin in the contralateral striatum (c) and cerebral cortex (d) of control, PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice. Data are expressed as mean ± SEM (n = 6). *p < 0.05, one-way ANOVA, statistical analyses compared to control mice.

[0028] Figure 9. Quantification of TH protein levels in the midbrain. Quantification of TH levels normalized to beta-actin in the ipsilateral midbrain of control, PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice. A typical Western blot image is shown. Data are expressed as mean ± SEM (n = 6).

[0029] Figure 10. Immunofluorescence images of total α-synuclein in spinal cord (a) and intestine (b) sections from control, PFF, H-EV Syn, H-EV GF, or Ms-EV Syn mice.

[0030] Description of the invention

[0031] In order to respond to the therapeutic and clinical needs existing in the state of the art, the authors of the invention have developed an effective and personalized therapy for Parkinson's Disease (PD) and other synucleinopathies, based on the use of modified EVs obtained from human dendritic cells (hDCs).

[0032] Thus, in a main aspect of the invention, an EV derived from hDCs is contemplated that comprises a peptide construct, with sequence SEQ ID NO 1, formed by the fusion of the rabies virus glycoprotein peptide (RVG) and the human Lamp2b protein (hLamp2b) (hereinafter, hLamp2b-RVG) expressed on its surface, where the EV is also loaded with anti-α-synuclein shRNA minicircles (shRNA-MCs).

[0033] In the present invention, the term Extracellular Vesicle (EV) refers to both an EV and a population of EVs.

[0034] Likewise, the use of the EVs of the invention for medical purposes is contemplated, as well as pharmaceutical compositions or medicines comprising said EV(s) as an active ingredient.

[0035] In particular, the present invention relates to the use of the EVs of the invention, or of pharmaceutical compositions or medicaments comprising them, in the treatment of synucleinopathies, particularly PD, DLB and MSA.

[0036] To produce brain-targeted EVs, the hLamp2b protein was fused to the RVG peptide. The mRNA encoding the hl_amp2b fusion protein with the RVG tag was synthesized in vitro, resulting in a polynucleotide construct of SEQ ID NO 1, and hDCs were electroporated with said mRNA.

[0037] Thus, in another main aspect of the invention, a method is contemplated for obtaining the EVs of the invention comprising the following steps: a) Isolating circulating monocytes from a blood sample of an individual, b) Differentiating in vitro the monocytes isolated in a) into human dendritic cells, c) Synthesizing in vitro an mRNA sequence that codes for the peptide construct hlamp2b-RVG of SEQ ID NO 1, d) introducing by electroporation into the hDCs obtained in b) the mRNA sequence of SEQ ID NO 1 synthesized in c), e) seeding the hDCs obtained in d), f) isolating the EVs from the hDCs seeded in e), and g) loading the isolated EVs in f) with anti-α-synuclein shRNA-MCs by electroporation.

[0038] In particular embodiments, the synthesis of the mRNA in c) is carried out using primers of sequence SEQ ID NO 2 and SEQ ID NO 3.

[0039] Changes in motor behavior in synucleinopathies are associated with a loss of dopaminergic innervation in the medial and posterior striatum. As shown in the examples provided in this application, treatment with anti-α-synuclein shRNA-MCs loaded into mouse RVG-EVs partially prevents the loss of dopaminergic innervation, while treatment with anti-α-synuclein shRNA-MCs loaded into human RVG-EVs of the present invention completely prevents the loss of dopaminergic innervation in the striatum.

[0040] Regarding α-synuclein mRNA and protein levels in the SNc, striatum and frontal cortex, mice treated with anti-α-synuclein shRNA-MCs loaded in human RVG-EVs showed a significant decrease in both mRNA and protein in all 3 regions, while mice treated with α-synuclein shRNA-MCs loaded in Ms-RVG-EVs showed a significant decrease in mRNA only in the SNc and cortex, accompanied by a slight decrease in protein levels.

[0041] Treatment with anti-α-synuclein shRNA-MCs loaded into human RVG-EVs significantly reduced the number of aggregates in all regions studied: SNc, striatum, cerebral cortex, and amygdala. Treatment with mouse RVG-EVs resulted in a significant reduction in aggregates only in the SNc and striatum.

[0042] Multidose administration of anti-α-synuclein shRNA-MC loaded into human RVG-EVs does not produce activation of the immune response, implying that this treatment can be administered repeatedly without decreasing its efficacy.

[0043] EXAMPLES

[0044] Below are some specific examples of embodiments of the invention that serve to illustrate the invention.

[0045] MATERIALS AND METHODS

[0046] Study design

[0047] The overall aim of this study was to develop a source of engineered EVs compatible with personalized therapy and to evaluate their therapeutic potential in a progressive murine model of PD. First, we optimized the production and transfection of immature DCs differentiated from monocytes recovered from leukocyte-depleting filters, and characterized their production of targeted EVs. In vitro studies using SH-SY5Y cells were designed to assess whether shRNA-MC-loaded human RVG-EVs would be able to specifically release their cargo and downregulate α-synuclein expression. For in vivo experiments, mice were treated with shRNA-MC-loaded human RVG-EVs after intrastriatal injection of preformed α-synuclein fibrils (PFFs) to evaluate therapeutic efficacy. Studies using blood samples from PD patients aimed to evaluate the viability of PD patient-derived EVs.

[0048] All studies involving human samples were approved by the Institutional Ethics Committee (CEImLar, protocol number: P.1.374). All animal studies followed the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the animal welfare ethics committee of our institution (Centro de Investigaciones Biomédicas de La Rioja, ref. LAE-03). To ensure reproducibility, a person not involved in the study randomly assigned all mice to experimental groups. Sample sizes are specified in the legends of each of the figures along with details of the statistical analysis and were estimated by power analysis using G*Power software. All analyses performed to evaluate behavioral tests and experimental procedures, including immunohistochemistry, Western blot, or qPCR, were performed blindly.

[0049] Cell culture

[0050] A clone of the human neuroblastoma cell line SH-SY5Y (American Type Culture Collection) expressing human WT α-synuclein with a C-terminal hemagglutinin (HA) tag was cultured using standard conditions (Alvarez-Erviti L, Seow Y, Schapira AH, Rodriguez-Oroz MC, Obeso JA, Cooper JM. Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease. Cell Death Dis. 2013 Mar 14;4(3):e545).

[0051] Construct synthesis

[0052] For the in vitro synthesis of mRNA encoding the LAMP2b-RVG fusion protein, the Invitrogen™ mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit was used. This kit uses T7 RNA polymerase to synthesize mRNA in vitro, for which it requires a linear DNA template containing the T7 RNA polymerase promoter. A plasmid encoding this LAMP2b-RVG sequence with a CMV promoter was used as a template. To generate the DNA template, a forward primer containing the T7 promoter sequence (SEQ ID NO 2) and a reverse primer (SEQ ID NO 3) were used. The following protocol was used for the ORC: 95°C 0:12; 56°C 0:12; 68°C 1:30 25x. The mRNA encoding the LAMP2b-RVG protein was then synthesized using the Invitrogen™ mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit following the instructions.

[0053] Dendritic cell culture and EV isolation

[0054] Peripheral blood mononuclear cells (PBMCs) were isolated from discarded blood donation fractions, 50 ml of PBS was added, and PBMCs were separated using a density gradient (Ficoll-Paque PREMIUN 1.073, Cytiva). Alternatively, PBMCs were isolated from blood samples of control individuals and PD patients (15 ml), using a density gradient (Ficoll-Paque PREMIUN 1.073, Cytiva). Monocytes were isolated, and PBMCs (5 x 10 7 cells per plate, 10 cm plate) in RPMI-1640 with GlutaMAX (GIBCO-BRL), 10% fetal bovine serum (FCS) without EVs, by centrifugation at 120,000 xg for 60 min and penicillin / streptomycin. 2 hours later, the medium was removed and replaced with fresh medium supplemented with 100 ng / ml human granulocyte-monocyte colony-stimulating factor (GM-CSF) (Preprotech) and 20 ng / ml human IL-4 (Preprotech). 7 days later, 10 6cells with 50 pg of hLamp2b-RVG mRNA (300 V-150 mA). After 24 h, the medium was collected and EVs were collected by centrifugation at 12,000 x g for 30 min to remove cell debris and the supernatant was centrifuged again at 120,000 x g for 1 h, to pellet the EVs. The isolated EVs were resuspended in 0.1 M ammonium acetate using a 27G needle. The size distribution and concentration of EVs were assessed by nanoparticle tracking analysis using an NS500 instrument (Nanosight). For in vivo injection, 10 10EVs and 450 pg of shRNA-MC in 10 ml of electroporation buffer (1.15 mM potassium phosphate [pH 7.2], 25 mM KCl, 21% OptiPrep) were electroporated (450 V, 100 mA) in a 4-mm cuvette using a Bio-Rad Gene Pulser Xcell electroporator. Samples were treated with 150 U of DNase (Promega) for 30 min at 37°C and purified by ultracentrifugation at 120,000 × g for 1 h. EVs were resuspended in 5% glucose. Scanning electron microscope (SEM)

[0055] EVs were resuspended in PBS and fixed in 2.5% glutaraldehyde solution at 4°C overnight. Samples were dehydrated using a graded ethanol series (30, 50, 70, 80, 90, and 100% * 3) for 10 minutes per step. Samples were coated with gold-palladium by sputter coating, and images were collected using a SEM (model EM-30, COXEM).

[0056] Loading RVG-EV with shRNA-MC

[0057] To determine the optimal conditions for loading EVs with shRNA-MC constructs, 1 pg of shRNA-MC (containing the shRNA sequence against α-synuclein with the H1 promoter, SEQ ID NO 4) and 3 pg of RVG-EVs were mixed in 100 pL of electroporation buffer (1.15 mM potassium phosphate [pH 7.2], 25 mM KCl, 21% OptiPrep), and electroporated at 450 V, 100 mA in a 4 mm cuvette using a Bio-Rad Gene Pulser Xcell electroporator. EV samples were treated with 1 U of DNase (Promega) for 30 min at 37°C and purified by ultracentrifugation at 120,000 × g for 1 h. EVs were resuspended in RPMI medium.

[0058] Animals

[0059] Adult male C57BL6 / C3H F1 mice, 8–9 weeks old, were purchased from Charles River Laboratories. Mice were housed under standard environmentally controlled conditions with a 12-h light / dark cycle and maintained on an ad libitum food and water diet. Every effort was made to minimize the number of animals used and their suffering.

[0060] Mice received an injection of sonicated murine α-synuclein preformed fibrils (PFFs) into the dorsal striatum and 2 and 45 days later received two intravenous injections of hRVG-EVs containing anti-α-synuclein shRNA-MCs or anti-GFP shRNA-MCs or two intravenous injections of vehicle (5% glucose). Another group of animals injected with PFF α-synuclein received intravenous injections of mouse RVG-EVs containing α-synuclein shRNA-MCs. Control animals were injected into the striatum with an equal volume of sterile PBS and received two intravenous injections of vehicle. Mice were sacrificed 90 days post-intrastriatal injection (dpi). Preparation of mouse α-synuclein PFFs

[0061] Purified mouse α-synuclein monomer was assembled into filaments by incubation at 37°C at 5 mg / ml in sterile PBS (pH 7.4) with continuous shaking at 250 rpm for 7 days. Fibrils were pelleted by centrifugation at 10,600 xg for 15 minutes and resuspended in sterile PBS at a concentration of 1 mg / ml. Fibril formation was confirmed by Congo red staining. To generate α-synuclein seeds, α-synuclein PFFs were sonicated for two 6-s cycles at 50% power (10 micron amplitude) using a probe sonicator (Soniprep 150, MSE) immediately prior to surgical injections. A fresh aliquot of sonicated α-synuclein PFFs was prepared each day of surgery.

[0062] Stereotactic surgery

[0063] Mice were deeply anesthetized with isoflurane and placed in a stereotaxic frame. Animals were unilaterally injected with 5 pl (two 2.5 pl injections) of freshly sonicated g-synuclein PFF (1 mg / ml) or PBS into the right striatum at a rate of 0.25 pl / min. Striatal coordinates were calculated relative to bregma using the atlas of Paxinos and Watson (Paxinos & Franklin, 2001): AP +0.2 mm, ML -2.0 mm, and DV -3.4 and -2.6 mm. After administration, the needle was left at the injection site for 5 min.

[0064] RVG-EV treatment of mice.

[0065] For the preparation of RVG-EV containing shRNA-MC, 10 10EVs and 450 pg of shRNA-MC in 10 ml of electroporation buffer (1.15 mM potassium phosphate [pH 7.2], 25 mM KCl, 21% OptiPrep) were electroporated (450 V, 100 mA) in a 4-mm cuvette using a Bio-Rad Gene Pulser Xcell electroporator. Samples were treated with 150 U at 37°C for 30 min. EVs were recovered by ultracentrifugation (120,000 × g for 1 h) and resuspended in 300 μl of 5% glucose immediately before intravenous injection into the mouse tail.

[0066] Behavioral tests

[0067] Mice motor function was assessed using the wire suspension, negative geotaxis, and limb-hold tests. The wire suspension test was performed before treatment, at 30-day intervals throughout the study, and before sacrifice, while the limb-hold test was performed on the day of sacrifice. Mice were habituated to the testing room for at least 30 minutes before each test, and behavioral tests were performed between 9:00 and 12:00 during the light cycle and assessed blindly by a trained observer.

[0068] - Wire suspension test

[0069] Each mouse was placed on the wire lid of a conventional cage. The lid was gently shaken three times to ensure the mouse's grip on the bars, and then turned upside down approximately 25 cm above a surface with bedding. The mice's latency to fall off the wire grid was recorded for up to 15 minutes and averaged across two trials (15 minutes apart).

[0070] - Negative geotaxis test

[0071] Each mouse was placed head down on a wire rack positioned at a 45° angle to the plane. The animal's behavior was observed for 30 s and scored as follows: 0 = turns and climbs, 1 = turns and freezes, 2 = moves but does not turn, 3 = does not move (Susick LL, Lowing JL, Bosse KE, Hildebrandt CC, Chrumka AC, Conti AC. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure. Behav Brain Res. 2014 Aug 1;269:66-74). The latency to turn 180° to an upright position and initiate climbing was recorded for all animals that received a score of 0. If the mouse was unable to turn, the default value of 30 s was taken as the maximum severity of impairment.

[0072] - Limb restraint test

[0073] Mice were held by the midsection of the tail for 10 s. During tail suspension, hindlimb position was scored from 0 to 4 according to the following criteria: 0, no limb grasp and normal escape extension; 1, assigned to mice with one hindlimb interlaced and toes showing normal extension; 2, mice with both hindlimbs interlaced inward and toes showing normal extension; 3, both hindlimbs exhibiting a curled-toe grasp and immobility; and 4, mice with the forelimbs and hindlimbs interlaced and crossed with curled toes and immobility (Guyenet SJ, Furrer SA, Damian VM, Baughan TD, La Spada AR, Garden GA. A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J Vis Exp. 2010 May 21;(39):1787).

[0074] Enzyme-linked immunosorbent assay

[0075] On the day of sacrifice, whole blood was collected from the mouse by cardiac puncture. After standing for 60 minutes, the supernatant containing serum was isolated from the whole blood by centrifugation at 2000 xg for 10 minutes. Serum proinflammatory cytokines, including TNF-α, IFN-γ, IL-1β, and IL-6, were measured using the corresponding Proteintech ELISA immunoassay kit according to the manufacturer's protocols.

[0076] Western Blot Analysis

[0077] Cell, brain, and spinal cord samples were homogenized in lysis buffer containing 10 mM Tris / HCl (pH 7.4), 0.1% SDS, protease inhibitor cocktail (Thermo Scientific), phosphatase inhibitor cocktail (Thermo Scientific), and DNAase (Promega) and incubated for 1 h at 37°C. The cells were centrifuged at 13,000 rpm for 10 min, and the supernatants were collected. Protein concentrations were determined by the BCA method. Distal intestine samples were homogenized in the same buffer, but containing 8 M urea. Protein samples were solubilized in LDS buffer and reducing agent, separated on 4%-12% Bis-Tris NuPAGE Novex gels (Invitrogen), and transferred to a PVDF membrane.Membranes were blocked with a 10% nonfat dry milk solution prepared in PBS and then incubated with the following primary antibodies: anti-α-synuclein (Abeam, ref# ab1903), anti-TH (Abeam, ref# ab112), and anti-β-actin (Abeam, ref# ab6276) antibodies. After washing steps, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies, and immunoreactivity was detected by luminol-based chemiluminescence (ECL) using the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Densitometry analysis was performed using ImageJ software and normalized to β-actin levels.

[0078] Immunohistochemistry and immunofluorescence.

[0079] For immunohistochemistry, brain, spinal cord, and intestine samples were collected after perfusion with 4% PFA in PBS, followed by overnight postfixation, cryoprotection in 30% sucrose, and freezing. Brains and spinal cords were sectioned into 30 pm coronal sections using a freezing microtome, while intestines were sectioned into 12 pm transverse sections using a cryostat. Sections were washed with TBS, and endogenous peroxidase activity was inactivated by incubation with 3% hydrogen peroxide. After washing with TBS, sections were blocked in 5% normal goat serum / 0.2% Triton X-100 in TBS followed by overnight incubation at 4°C with primary antibodies: anti-α-synuclein (Abeam, rei# ab1903, 1:2000 dilution), anti-phospho S129-α-synuclein (Abeam, rei# ab51253, 1:2000 dilution), and anti-TH (Abeam, rei# ab112, 1:2000 dilution).For colorimetric immunostaining, sections were washed and incubated with biotinylated secondary antibodies of the appropriate species, followed by incubation with peroxidase-conjugated avidin. After the washing steps, sections were incubated with diaminobenzidine. For immunofluorescence staining, sections were incubated with fluorescent secondary antibodies. All samples were processed simultaneously to allow comparison.

[0080] Image analysis

[0081] The total number of DA neurons in the SNc was assessed by stereology in a series of sections (120 pm intervals between sections) spanning the entire SNc using StereoInvestigator software (MBF Bioscience). The optical density (OD) of TH immunostaining in the striatum was used as an index of the density of striatal dopaminergic innervation. Briefly, nine representative sections at three coronal levels of the striatum (AP bregma coordinates: +1, 3, +0.5 and -0.4) from each animal were examined and the pixel densities of TH-positive fiber innervation in striatal areas were estimated using ImageJ. The obtained values ​​were normalized by subtracting the OD from the background staining measured in the white matter, where TH-positive innervation is negligible.

[0082] For the assessment of α-synuclein inclusions in SNc dopaminergic neurons, coronal sections (120 μm intervals between sections) from each animal were double-labeled using phosphoS129 and TH antibodies against α-synuclein. The number of phospho-α-synuclein aggregates was manually quantified at 20x magnification from regularly spaced sections along the rostrocaudal axis of the SNc. Data represent the mean number of aggregates per section. The number of phospho-α-synuclein aggregates in the cortex and striatum were manually counted on one image per section, with 3 representative sections (AP bregma coordinates: +1, 3, +0.5, and -0.4) from each animal. The number of phospho-α-synuclein aggregates in the amygdala was manually counted on one image for each section, with 2 representative sections (AP bregma coordinates: -1,9 and -2,2) from each animal.

[0083] Quantitative PCR

[0084] Total RNA was isolated from frozen cell and tissue samples using the RNeasy kit (Qiagen) according to the manufacturer's protocol, followed by reverse transcription with the qSCRIP Reverse Transcriptase kit (Primer Design, Southampton, England) to generate cDNA. qPCR experiments were performed using NZY Supreme qPCR Green Mastermix (Nzytech, Lisbon, Portugal) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). The primer sequences were as follows: for α-synuclein, forward: 5-GCCAAGGAGGGAGTTGTGGCTGC-3' (SEQ ID NO 5); reverse: 5- CTGTTGCCACACCATGCACCACTCC-3' (SEQ ID NO 6); for β-actin, forward: 5'- TCTACAATGAGCTGCGTGTG-3' (SEQ ID NO 7); reverse: 5'-

[0085] GGTGAGGATCTTCATGAGGT-3' (SEQ ID NO 8). α-synuclein expression was normalized to the beta-actin gene. For relative quantification, values ​​were calculated using the comparative CT (AACt) method.

[0086] Statistical analysis

[0087] All statistical analyses were performed using SPSS version 25.0 software. Data are expressed as means ± standard error of the mean (SEM). Data normality was examined using the Shapiro-Wilk test. One-way analysis of variance (ANOVA) was used for comparisons between multiple groups under the assumption of normality. Tukey's post hoc test was used for post hoc multiple comparisons to assess significance between groups after one-way ANOVA. For data that did not follow a normal distribution, statistical differences were analyzed using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test. P values ​​less than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. RESULTS

[0088] Optimization and validation of hRVG-EV loaded with shRNA-MC

[0089] To produce brain-targeted EVs, the hLamp2b protein was fused to the RVG peptide.

[0090] In previous studies using mouse DCs as a source of RVG-EVs, DCs were transfected with plasmids encoding the Lamp2b-RVG construct; however, hDCs are extremely difficult to transfect with plasmids. Alternatively, hLamp2b-RVG mRNA was synthesized in vitro, and hDCs were electroporated with the mRNA; electroporation conditions were optimized using EGFP mRNA. EVs isolated from control hDCs and hDCs electroporated with hLamp2b-RVG mRNA were characterized by NTA (Fig. 1a, 1b) and Western blot (Fig. 1c), and transmission electron microscopy (TEM) (Fig. 1d). Analysis demonstrated that control EVs and RVG-EVs have similar size and protein composition, except for the Lamp2b protein. hRVG-EVs (3 mg) were loaded with anti-α-synuclein shRNA-MCs (1 mg) using previously optimized conditions in Ms-RVG-EVs.SH-SY5Y cells overexpressing WT α-synuclein were treated with anti-α-synuclein shRNA-MC using a transfection reagent, or loaded into unmodified hEVs and hRVG-EVs. At 72 h posttreatment, decreased levels of α-synuclein mRNA (41% decrease, p = 0.037) (Fig. 1e) and protein (37% decrease, p = 0.046) (Fig. 1f) were observed. Levels of α-synuclein mRNA (39% decrease, p = 0.037) (Fig. 1e) and protein mRNA (28% decrease, p = 0.049) (Fig. 1f) were decreased to a similar extent using a transfection reagent. However, in SH-SY5Y treated with anti-α-synuclein shRNA-MC loaded with unmodified EVs, α-synuclein mRNA and protein levels were not affected (Fig. 1e, f). shRNA-MC RVG-EV decreased α-synuclein levels and pathology in the brain.

[0091] To evaluate the therapeutic effect of the treatment, hRVG-EVs loaded with anti-α-synuclein shRNA-MCs were intravenously injected into the progressive α-synuclein PFF mouse model. To generate the PD model, C57BL6 / C3H mice received unilateral intrastinal injections of murine α-synuclein PFFs. After 2 and 45 days, mice received two intravenous injections of hRVG-EVs loaded with anti-α-synuclein shRNA-MCs (n = 18) or hRVG-EVs loaded with anti-GFP shRNA-MCs (n = 18), or intravenous injections of vehicle (5% glucose; n = 18). An additional group of α-synuclein PFF-injected mice received two intravenous injections of anti-α-synuclein shRNA-MCs-RVG-EVs (n = 18). Control animals were injected into the striatum with sterile PBS and received two intravenous injections of vehicle (n = 18). Mice were sacrificed 90 days after intrastriatal injection.

[0092] After 90 days, α-synuclein mRNA levels remained downregulated in both the ipsilateral (36% decrease, p = 0.027) and contralateral midbrains (40% decrease, p = 0.024) of mice treated with hRVG-EVs containing anti-α-synuclein shRNA-MCs (Fig. 2a, Fig. 8a). The prolonged decrease in α-synuclein mRNA levels in the midbrain was associated with lower α-synuclein protein levels in this region (ipsilateral: decreased by 35%, p = 0.045; contralateral: decreased by 40%, p = 0.0039) (Fig. 2b, Fig. 8b).Likewise, α-synuclein mRNA levels were also decreased in the ipsilateral striatum (decreased by 32%, p = 0.0021) and cerebral cortex (decreased by 35%, p = 0.017) of mice treated with anti-α-synuclein shRNA-MC-loaded hRVG-EVs therapy, associated with a concomitant decrease in α-synuclein protein levels in both brain regions (striatum: decreased by 20%, p = 0.049; cortex: decreased by 25%, not significant) (Fig. 2c-f). However, in animals treated with hRVG-EVs loaded with anti-GFP shRNA-MCs, α-synuclein mRNA and protein levels were similar to those in controls and vehicle-treated PFF mice, confirming the specificity of downregulation of α-synuclein shRNA MCs (Fig. 2a-f, Fig. 9).Injection of anti-α-synuclein shRNA-MC loaded into Ms-RVG-EVs decreased α-synuclein mRNA levels in all 3 brain regions analyzed, although the reduction was only statistically significant in ipsilateral (33% decrease, p = 0.049) and contralateral midbrain (34% decrease, p = 0.037) (Fig. 2a-f, Fig. 8). α-synuclein protein levels were reduced in ipsilateral (20% decrease, not significant) and contralateral midbrain (23% decrease, not significant) but were unaffected in the striatum and cerebral cortex of mice treated with Ms-RVG-EVs anti-α-synuclein shRNA-MC (Fig. 2a-f, Fig. 8).

[0093] Furthermore, the impact of hRVG-EVs loaded with anti-α-synuclein shRNA-MCs on α-synuclein pathology was evaluated. Ninety days after PFF α-synuclein injection, immunohistological staining with phospho-α-synuclein revealed abundant phospho-α-synuclein inclusions in the ipsilateral SNc of PFF α-synuclein-injected mice and PFF-injected mice treated with hRVG-EVs loaded with anti-GFP shRNA-MCs (Fig. 3a, b). The majority of the inclusions were localized to dopaminergic neurons of the SNc (Fig. 3c). Treatment with anti-α-synuclein shRNA-MC hRVG-EV significantly reduced the number of α-synuclein aggregates in the SNc (decreased by 29%, p = 0.039), similar to the reduction observed in mice treated with Ms-RVG-EV (decreased by 37%, p = 0.027) (Fig. 3a, b).Similar results with decreased numbers of α-synuclein aggregates were observed in mice treated with anti-α-synuclein shRNA-MC loaded into hRVG-EVs or Ms-RVG-EVs in other brain regions tested, including the striatum (hRVG-EVs decreased 43%, p = 0.039; Ms-RVG-EVs decreased 49%, p = 0.038), cerebral cortex (hRVG-EVs decreased 46%, p = 0.033; Ms-RVG-EVs decreased 35%, p = 0.047), and amygdala (hRVG-EVs decreased 37%, p = 0.043; Ms-RVG-EVs decreased 36%, p = 0.041) (Fig. 4a-c).

[0094] Treatment with RVG-EVs shRNA-MC prevents neuronal degeneration and behavioral deficits

[0095] The presence of α-synuclein aggregates in the SNc is associated with decreased survival of dopaminergic neurons. Stereological evaluation of TH-positive neurons in mice injected with PFF α-synuclein revealed a unilateral 16% loss of dopaminergic neurons in the ipsilateral SNc 90 days after PFF α-synuclein injection (p = 0.047) (Fig. 5a). Western blot analysis of TH protein levels in midbrain samples confirmed these data (25% decrease, not significant) (Fig. 9). Dopaminergic degeneration was associated with a unilateral loss of dopaminergic innervation reflected by a decrease in TH staining in the anterior (10% decrease compared to control, not significant), medial (17% decrease compared to control, p = 0.016) and posterior (25% decrease compared to control, p = 0.000) striatum (Fig. 5b).A similar loss of dopaminergic neurons in the SNc (14% decrease, p = 0.049) (Fig. 5a) was detected in α-synuclein injected PFF mice treated with hRVG-EV loaded with anti-GFP shRNA-MC, this group also showed a decrease in TH staining in the anterior, medial and posterior striatum (7%, not significant; 17%, p = 0.015; 23%, p = 0.001 respectively).

[0096] Furthermore, treatment with anti-α-synuclein shRNA-MC hRVG-EVs prevented dopaminergic neuronal degeneration associated with PFF α-synuclein injection (Fig. 5a). This protective effect was associated with a significant preservation of dopaminergic innervation in the striatum (Fig. 5b). Similar results were observed in mice injected with PFF α-synuclein and treated with anti-α-synuclein shRNA-MC-loaded Ms-RVG-EVs (Fig. 5a-b).

[0097] Dopaminergic dysfunction significantly impaired the motor performance of α-synuclein-injected PFF mice in the wire suspension (p = 0.039) and limb-hold tests (p = 0.000). Similar results were obtained in the PPF group of mice treated with anti-GFP shRNA-MC delivered by hRVG-EVs (Fig. 5c, d). However, prevention of α-synuclein pathology mediated by hRVG-EV anti-α-synuclein shRNA-MC therapy was associated with improved motor performance, indistinguishable from control mice (Fig. 5c, d). Similar results were observed in the limb-hold test (Fig. 5d) in PFF-injected mice treated with Ms-RVG-EVs; however, this group exhibited abnormalities in the wire suspension test (Fig. 5c). There were no significant differences between the 5 groups in performance on the negative geotaxis test (Fig. 5e).

[0098] These results demonstrate that treatment with anti-α-synuclein hRVG-EV shRNA-MC can prevent dopaminergic neuronal degeneration and motor impairments in the α-synuclein PFF mouse model of PD.

[0099] Effects of shRNA-MC RVG-EV on the spinal cord and intestine

[0100] To investigate whether hRVG-EVs loaded with anti-α-synuclein shRNA-MC are able to downregulate α-synuclein expression in organs other than the affected brain at the early stage of the disease, α-synuclein mRNA and protein levels were analyzed in spinal cord and intestine samples. After 90 days, treatment with hRVG-EVs shRNA-MC anti-α-synuclein significantly reduced α-synuclein mRNA (decreased by 35% compared to controls, p = 0.021) (Figure 6a) and protein levels in the spinal cord (decreased by 32% compared to controls, p = 0.021) (Figure 6b). Intrastriatal injection of α-synuclein PFF was associated with phospho-α-synuclein inclusions in the spinal cord, immunohistochemical analyses confirmed that treatment with hRVG-EV loaded with anti-α-synuclein shRNA-MC prevented the formation of phospho-α-synuclein aggregates in those tissues (Fig. 10a).However, in PFF-injected mice, injected with Ms-RVG-EVs loaded with anti-α-synuclein shRNA-MCs or hRVG-EVs loaded with anti-GFP shRNA-MCs, mRNA and protein levels were similar to those in controls and vehicle-treated PFF-treated mice (Fig. 6a, b). A similar decrease in α-synuclein mRNA (decreased by 33% compared to controls, p = 0.021), protein (decreased by 15% compared to controls, not significant) and pathology was found in the distal intestine of PFF mice treated with hRVG-EVs loaded with anti-α-synuclein shRNA-MCs (Fig. 6c, d, Fig. 10b).

[0101] Finally, to exclude any detrimental effects of hRVG-EV loaded with anti-α-synuclein shRNA-MC treatment, transcriptomic analysis of contralateral cortical samples was performed, the results demonstrated no changes in mRNA expression in PFF mice treated with vehicle, hRVG-EV loaded with anti-α-synuclein shRNA-MC, or hRVG-EV loaded with anti-GFP shRNA-MC compared to controls (Table 1).

[0102] Table 1. Results of transcriptomic analysis of contralateral cortical samples

[0103] Testvs Ref # DOWN # UP # TOTAL

[0104] Furthermore, the presence of an inflammatory response was ruled out since the levels of TNF-α, IFN-γ, IL-4 or IL-1 were not affected (Table 2). Table 2. Analysis of the inflammatory response after treatment with hRVG-EV shRNA-MC anti-α-synuclein

[0105] Proteomic analysis of RVG-EVs from donors of control and PD patients.

[0106] Implementing the therapy requires the development of a source of specific gene therapy vehicles compatible with personalized therapy; this implies the development of a source of one's own cells to obtain immunologically inert vehicles.

[0107] To confirm the suitability of EVs derived from PD patients as therapeutic vehicles, proteomic analysis was performed on EVs derived from control individuals, early PD patients, and advanced PD patients. The proteomic analysis confirmed that EVs produced by DCs differentiated in vitro from blood monocytes isolated from PD patients were suitable as therapeutic vehicles. A total of 756 proteins were identified, and differential expression analysis showed minimal changes in protein abundance (Fig. 7a, b). The expression of 43 proteins was significantly different in EVs from early PD patients compared to controls, and 20 proteins were significantly different in advanced PD compared to controls. The analysis revealed 5 altered proteins in EVs derived from DCs from early and advanced PD patients, notably one of the proteins was lysosomal acid glucosylceramidase-1 (GBA-1), a protein associated with familial PD and altered in sporadic PD (Fig. 7c).

Claims

CLAIMS 1. Extracellular vesicle (EV) derived from human dendritic cells (hDCs) comprising a RVG-hLAMP peptide construct, with sequence SEQ ID NO 1, expressed on its surface, where the extracellular vesicle is loaded with anti-α-synuclein shRNA minicircles (ShRNA-MCs).

2. EV, according to claim 1, for use in medicine.

3. Pharmaceutical composition, comprising one or more EVs, according to claim 1, as an active ingredient.

4. Extracellular vesicle according to claim 1, or pharmaceutical composition according to claim 3, for clinical use in the treatment of synucleinopathies.

5. Extracellular vesicle for use according to claim 4, wherein the synucleinopathy is selected from Parkinson's disease, dementia with Lewy bodies and multiple system atrophy.

6. Polynucleotide construction of SEQ ID NO 1 encoding a recombinant protein formed by the fusion of hLAMP and RVG.

7. Method for obtaining an extracellular vesicle according to claim 1, characterized in that it comprises: a) Isolating circulating monocytes from a blood sample of an individual, b) Differentiating in vitro the monocytes isolated in a) into hDCs, c) Synthesizing in vitro an mRNA sequence that codes for the peptide construct hLAMP-RVG of SEQ ID NO 1, d) introducing by electroporation into the hDCs obtained in b) the mRNA sequence synthesized in c), e) seeding the hDCs obtained in d), f) isolating the EVs from the hDCs seeded in e), and g) loading the isolated EVs in f) with anti-α-synuclein shRNA-MCs by electroporation.

8. Method according to claim 7, wherein the synthesis of the mRNA in c) is carried out using primers of sequence SEQ ID NO 2 and SEQ ID NO 3.

Images