RU2800729C2 - Vesicles containing pten inhibitor and their use - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical compositions containing membrane vesicles, including extracellular vesicles, including so-called exosomes loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN). Methods for the treatment of neurological diseases, disorders or conditions using extracellular vesicles are provided. In addition, isolated extracellular vesicles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN) are provided.

EFFECT: invention provides an additional safe, effective and convenient method of treating spinal cord injury.

29 cl, 14 dwg, 6 ex

Description

FIELD OF TECHNOLOGY

[1] The present invention relates to membrane vesicles loaded with a PTEN inhibitor, pharmaceutical compositions containing said vesicles and their use in the treatment of neurological disorders, and more particularly to cell-derived extracellular vesicles loaded with an exogenous PTEN inhibitor, pharmaceutical compositions containing them, and their use in the treatment of spinal cord injuries.

BACKGROUND OF THE INVENTION

[2] Injury to the spinal cord can lead to autonomic dysfunction, loss of sensation, or loss of mobility. Such spinal cord injury (SCI) is caused by trauma, tumors, ischemia, developmental disorders, neurodegenerative diseases, demyelinating diseases, transverse myelitis, vascular malformations, or other causes. The consequences of SCI depend on the nature of the injury and its location in the spinal cord. In addition, because SCI is a dynamic process, the degree of damage may not be fully apparent initially in all acute spinal cord syndromes. An incomplete injury to the spinal cord may develop into a more complete injury; more often the level of damage rises one or two levels within hours or days after the initial event. This clinical deterioration is explained by a complex cascade of pathophysiological events.

[3] The psychological and social consequences of SCI often overwhelm the patient. Some of the common disabling conditions associated with SCI are permanent limb paralysis, chronic pain, muscle atrophy, loss of voluntary bladder and bowel control, sexual dysfunction, and infertility.

[4] Recent advances in neuroscience have brought significant attention to SCI research and have made significantly improved treatment and rehabilitation options available. For example, the potential of functional electrical stimulation (FES) to enhance nerve regeneration has been shown to significantly improve recovery and improve functional capacity after SCI. However, not all patients with SCI are suitable for FES (complete spinal cord involvement is required); the patient must be in a neurologically stable condition; peripheral nerves must be intact and responsive to exogenous electrical stimulation.

[5] Axonal regeneration after SCI is limited due to the extremely limited growth capacity inherent in mature neurons, as well as due to external factors, such as glial scar maturing over time, and inhibitory substances. There have been attempts to change external factors, but with limited success. For example, removal of an extracellular inhibitory molecule, delivery of a neurotrophic factor, or transplantation of a permissive substrate failed to induce sustained regeneration of the damaged corticospinal tract.

[6] Phosphatase-tensin homologue (PTEN) is a highly conserved protein tyrosine phosphatase with dual specificity. This protein dephosphorylates the lipid second messengers phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] to form phosphatidylinositol-4,5 -bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-phosphate [PI(4)P], respectively. This unique activity makes PTEN a major homeostatic regulator and tumor suppressor protein that is absent or impaired in a large number of tumors as a result of somatic changes. The important role of the PI3K/AKT/mTOR signaling pathway in cell growth, regeneration, and survival confirms the validity of the therapeutic effect on PTEN. Proposed therapeutic targets based on PTEN inhibition include nerve growth and regeneration after trauma or injury, treatment of cardiac ischemia/reperfusion and associated diseases, wound healing, and infertility (for review see Pulido, 2018, Molecules, 23, 285). Interestingly, the main paradigm of involvement of PTEN in carcinogenesis is that it is a cancer suppressor, and it has been demonstrated that PTEN inhibition can occur in brain metastases (Zhang et al., Nature, 2015, 527, 100-104).

[7] PTEN is predominantly expressed in adult brain neurons and plays an important role in controlling the regeneration of corticospinal neurons by suppressing the target of rapamycin (mTOR) activity in mammals. mTOR activity is strongly downregulated in axotomized adult neurons, limiting the synthesis of a new protein required for sustained axonal regeneration. Several publications mention the involvement of PTEN in the suppression of nerve regeneration, and others show the positive effect of PTEN depletion on conditions associated with damage or dysfunction of axons. Effective inhibition of PTEN could potentially increase mTOR, thereby promoting nerve regeneration.

[8] WO 2009/117389 describes the therapeutic use of PTEN inhibitors for the treatment of neurodegenerative disorders. WO 2015/066701 describes a method for regenerating nerves or ameliorating nerve degeneration by administering a PTEN inhibitory peptide to or near an injured nerve. WO 2011/044701 describes specific PTEN inhibitory peptides and their use in the treatment of diseases associated with cytotoxic stress, including diseases and injuries of the central nervous system.

[9] A wide variety of carriers have been proposed for the delivery of siRNA molecules, including, but not limited to, liposomes, protein particles, micelles, and lipid particles. Rungta et al. (Molecular Therapy-Nucleic Acids, 2013, 2, e136) have shown that siRNA in lipid nanoparticles (LNP) can induce efficient silencing of gene expression in neurons.

[10] Extracellular membrane vesicles (EV) are membrane vesicles secreted by various cell types. EVs are present in the bloodstream under normal physiological conditions and are elevated in various diseases such as diabetes and related vascular complications, cardiovascular disease, hematological malignancies, and solid tumors.

[11] EV can be divided into three subpopulations: (i) exosomes 30–100 nm in diameter, originating from endosomal compartments; (II) microvesicles with a diameter of 100 nm to 1 μm, released from the cell surface through "vesiculation"; and (III) apoptotic bodies 1-5 μm in diameter, which are released from apoptotic cells. EVs contain some of the elements of the original cell, including proteins, DNA fragments, microRNAs, and mRNAs.

[12] EP 2254586 deals with exosomes isolated from mesenchymal stem cells, said exosomes having at least one biological property of mesenchymal stem cells. In addition, exosomes have been proposed as carriers for various drugs, including small molecule compounds and non-coding RNAs (for example, US 2017/0247708 and Ha et al., Acta Pharmaceutica Sinica B 2016;6(4):287-296). Typically, siRNAs are introduced into exosomes by electroporation.

[13] WO 2018/033911 issued to some authors of the present application describes exosomes derived from mesenchymal stem cells for the treatment of neurological disorders.

[14] Neuronal injury in general, and spinal cord injury (SCI) in particular, involves a long and complex cascade of secondary events following the injury itself. The complexity of the cascade may affect the efficacy of proposed treatments, and there is an unmet need to develop additional safe, effective, and convenient treatments for SCI.

BRIEF DESCRIPTION OF THE INVENTION

[15] In one aspect, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN).

[16] In yet another aspect, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous phosphatase and tensin homologue inhibitor (PTEN) for use in the treatment of a neurological disease, disorder or condition.

[17] In accordance with a further aspect of the present invention, there is provided a method of treating a neurological disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of membrane particles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN), resulting in treatment neurological disease or condition. In some embodiments, the membrane particles are extracellular vesicles of cellular origin.

[18] According to another aspect, the present invention provides isolated extracellular vesicles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN).

[19] According to any of the above aspects, extracellular vesicles are selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes and exovesicles. According to additional features in the described embodiments, the particles contain exosomes. In some embodiments, extracellular vesicles are a combination of exosomes and microvesicles. In some embodiments, extracellular vesicles, such as exosomes, are derived from adherent cells expressing mesenchymal markers. In some embodiments, adherent cells expressing mesenchymal markers are selected from mesenchymal stem cells, oral mucosal stem cells, or olfactory parietal cells. In other embodiments, extracellular vesicles, such as exosomes, are derived from adherent cells expressing neural crest cell markers. In one embodiment, neural crest cells comprise cranial neural crest cells. According to additional features in the described embodiments, the cranial neural crest cells are selected from the group consisting of dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP) and dental follicle progenitor cells (DFPC). In any of the above aspects and embodiments, the extracellular vesicles are isolated extracellular vesicles. According to some aspects and implementation options, the pharmaceutical composition according to the present invention is a cell-free composition.

[20] In any of the above aspects and embodiments, a PTEN inhibitor comprises a polynucleotide or oligonucleotide inhibitor of PTEN expression. In some embodiments, the PTEN inhibitor is an RNA interference oligonucleotide. In some embodiments, the RNA interference oligonucleotide is an anti-PTEN siRNA. In some embodiments, the siRNA contains a sequence complementary to the nucleic acid encoding the human PTEN protein. In some embodiments, the oligonucleotide inhibitor contains a hydrophobic moiety. In some embodiments, the hydrophobic moiety is a sterol, ganglioside, lipid, vitamin, fatty acid, peptide, or a combination thereof. In some embodiments, the hydrophobic moiety is a sterol. In some embodiments, the sterol includes cholesterol.

[21] According to additional features of any of the described aspects or implementation options, the PTEN inhibitor includes a peptide inhibitor.

[22] According to some of the above aspects and embodiments, the neurological disease is a neurodegenerative disease. In other aspects and embodiments, the neurological condition is a spinal cord injury. According to some of the aforementioned aspects and embodiments, the treatment of spinal cord injury comprises administering a pharmaceutical composition or isolated extracellular vesicles to a subject in need thereof. In some embodiments, administration includes intranasal administration. In some embodiments, treatment further comprises administering to the subject a therapeutically effective amount of chondroitinase ABC or a polynucleotide encoding it.

[23] Unless otherwise indicated, all technical and/or scientific terms used herein have the same meaning as generally implied by a person skilled in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the implementation or testing of embodiments of the present invention, typical methods and/or materials are described below. In case of conflict, the description of the patent, including definitions, shall prevail. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

Brief description of the drawings

[24] Some embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings. With respect to the specific detailed drawings, it should be emphasized that the details are shown by way of example and for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description of the drawings makes the methods for practicing the embodiments of the present invention obvious to those skilled in the art.

[25] FIG. 1 shows MSC-Exo rendered using Cryo-TEM.

[26] FIG. 2 shows a micro-CT scan of all CNS tissues in injured (upper panel) and healthy (lower panel) rats. The figure shows that exosomes penetrate the blood-brain barrier and reach the site of spinal cord injury.

[27] FIG. 3 shows the quantification of gold nanoparticles (GNP) by intranasal administration of GNP exosomes by inductively coupled plasma (ICP) assessment in the CNS (left panel) and major organs (right panel) using FAAS in healthy and injured rats. *p < 0.05, ***p < 0.001.

[28] Figure 4 shows the quantification of mean PKH26 signal intensity in the T10 spinal segment using PKH26-labeled exosomes (PKH26-Exo) in intact and injured rats. **p < 0.01.

[29] Figure 5 shows the quantification of PKH26-Exo signals co-localized with neurons, astrocytes, and activated microglia (p < 0.01).

[30] FIG. 6 shows NanoSight analysis of Cy3-tagged self-delivering MAPK siRNA incubated with exosomes. The figures show the size distribution of non-fluorescent exosomes (grey) and Cy3 exosomes (black).

[31] FIG. 7 shows representative immunofluorescence staining images of dorsal root ganglion (DRG) neurons treated with: control (medium) ( Fig. 7A ), non-specific control siRNA ( Fig. 7B ), MSC-Exo ( Fig. 7C ), PTEN-siRNA ( Fig. 7D ) or ExoPTEN ( Fig. 7E ). Scale 100 µm.

[32] FIG. 8 shows the number of branching points in relation to the level of branching for all groups. The number of branch points at the mean branch levels, quantified by the IMARIS software, was compared across groups. At the middle level of branches, PTEN miRNA exosomes contributed to the formation of a significantly larger number of axon branch points than all other groups.

[33] Figure 9 shows total axon length ( Figure 9A ), number of axons ( Figure 9B ), number of branching points ( Figure 9C ), and maximum level of branching ( Figure 9D ) measured and compared across all groups.

[34] Figure 10 shows protein and mRNA expression in the SCI region and in the liver with intranasal administration of ExoPTEN. In FIG. 10A and B show PTEN protein expression in the spinal cord and liver, respectively, in exosome-treated (IN) and ExoPTEN-treated (IN) rats compared to untreated SCI rats. In FIG. 10C and D show RT-qPCR analysis of spinal cord and liver PTEN mRNA expression in untreated, exosome (IN) and ExoPTEN (IN) treated SCI rats, with GAPDH used as an internal control. Data are presented in the format "mean ± standard error of the mean" with multiple comparisons according to Kruskal-Wallis; IL: injection into the lesion; B: intranasally.

[35] FIG. 11 shows that treatment with intranasal administration of ExoPTEN induces recovery of motor, sensory and bladder functions. In FIG. 11A shows weekly Basso, Beatty, and Bresnahan (BBB) motor scores in SCI rats: untreated (n = 15, black) or treated with PTEN-siRNA (IL) (n = 3), ExoPTEN (IL) (n = 4 ), exosomes (IN) (n = 10) or ExoPTEN (IN) (n = 7); IL: injection into the lesion; B: intranasally. A two-way analysis of variance was performed followed by Tukey's multiple comparison test (***p < 0.001, ****p < 0.0001 between control dissection group and ExoPTEN (IN)). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 between exosomes (IN) and ExoPTEN (IN). In FIG. 11B shows the distribution of BBB motor scores in each group over 8 weeks. The highest score obtained by each rat in each group is presented. The ratio of the number of rats with a given score to the total number of rats in each group is shown. In FIG. 11C shows mean weekly body weight (±SEM) values for animals in the control dissection group or rats treated with PTEN-siRNA (IL), ExoPTEN (IL), exosomes (IN), or ExoPTEN (IN). A two-way analysis of variance was performed followed by the calculation of Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ****p < 0.0001 between control dissection group and ExoPTEN (IN). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 between exosome (IN) and ExoPTEN (IN). In FIG. 11D shows body weight versus BBB score for each group at week 8. Pearson's correlation coefficient, denoted as R ² , was calculated and showed a high correlation between BBB scores and weight values for all groups. In FIG. 11E shows percentage recovery of sensation at 0, 4, 8 weeks after treatment. In FIG. 11F shows bladder function expressed as time to spontaneous urinary reflex (days) from start of treatment. In FIG. 11G shows post-cross-sectional images of untreated (upper panel), exosome-treated (middle panel), and PTEN-miRNA exosome-treated (lower panel) rats from day 3 to week 8 post-surgery.

[36] FIG. 12 shows the measurement of MRI and electrophysiological parameters at the lesion in treated and untreated rats: FIG. 12A and FIG. 12B shows the cross-sectional area at 4 mm rostral and caudal from the epicenter of the lesion, respectively; on fis. 12C shows mean fractional anisotropy (FA) values within the lesion; in fig. 12D and FIG. 12E shows the amplitude ( FIG. 12D ) and latency ( FIG. 12E ) of potentials evoked by motor stimuli in exosome-treated (IN), ExoPTEN (IN) or healthy rats.

[37] Figure 13 shows the quantification and comparison of immunofluorescent markers in three groups of rats: untreated, exosome-treated, and ExoPTEN-treated. The markers are β-III-tubulin ( Figure 13A) , CD11b ( Figure 13B ), GFAP ( Fig. 13C) and CD31 ( Fig. 13D ). Data are presented in the format "mean ± standard error of the mean", one-way analysis of variance was performed, followed by the calculation of Tukey's multiple comparison test (*p < 0.05, **p < 0.01, ****p < 0.0001) .

[38] Onfig. 14 shows representative images of corticospinal tract axons tracked by BDA in untreated (top panel, n = 3) treated with exosomes (middle panel, n = 3) and treated with ExoPTEN (bottom panel, n = 2) rats with SCM. Scale 500 µm. The boxes show enlarged selections. White arrows indicate BDA-positive fibers caudal to the epicenter. Scale 100 µm. After intranasal administration of ExoPTEN (upper panel), significantly more axonal outgrowth was observed compared to the cut control or exosome treatment groups.

Detailed description of the invention

[39] The present invention, in some embodiments, relates to vesicles loaded with an exogenous PTEN inhibitor for the treatment of neurological disorders, and more specifically, but not exclusively, to cellular-derived vesicles loaded with a PTEN inhibitor for the treatment of spinal cord injury. Before explaining in detail at least one embodiment of the present invention, it should be understood that the present invention is not necessarily limited in its application to the detailed information set forth in the following description or illustrated by examples. The present invention may be embodied in other embodiments, or embodied or carried out in various ways.

[40] In particular, the present inventors demonstrated that exosomes can be efficiently loaded with cholesterol-conjugated siRNA ( Fig. 6 ). As shown in the examples, more than a third of the exosomes had a detectable amount of siRNA. Moreover, exosomes loaded with PTEN-miRNA promoted sustained axonal regeneration of dorsal root ganglion neurons in vitro ( Fig. 7 ). Moving on to in vivo studies, histological and microCT images of the entire central nervous system confirmed that intranasally injected exosomes crossed the blood-brain barrier, traveled to the spinal cord lesion, and were internalized by neurons at the lesion ( Fig. 2 ). Even more strikingly, intranasally injected exosomes loaded with PTEN miRNA promoted reliable axon regeneration and angiogenesis, which was accompanied by a decrease in astrogliosis and microgliosis ( Fig. 13 ). most importantly, it provided a pronounced restoration of motor functions. ( Fig. 11 ).

[41] In accordance with the idea of the present invention, exosomes loaded with agents capable of suppressing the expression and/or activity of PTEN can be used in the treatment of other neurological diseases or conditions in general and, more specifically, degenerative neurological diseases or conditions. In particular, from the present invention it is clear that the inhibition of activity and, in particular, inhibition of the expression of PTEN in the lesion allows effective treatment of spinal cord injury.

[42] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising particles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN).

[43] The terms "phosphatase-tensin homologue" "PTEN" are used interchangeably herein and refer to the human phosphatase-tensin homologue enzyme product of EntrezGene ID: 5728, which can be characterized by the amino acid sequence corresponding to UniProt Accession Number: P60484. Other non-human homologues can be readily identified using known methods such as BLAST searches and are considered to be included within the spirit of the invention. The PTEN protein acts as a phosphatase to dephosphorylate phosphatidylinositol(3,4,5)-trisphosphate (PtdIns(3,4,5)P3 or PIP3). PTEN specifically catalyzes the dephosphorylation of the 3'-phosphate of the inositol ring in PIP3, resulting in the formation of PIP2 biphosphate (PtdIns(4,5)P2). This dephosphorylation is important because it leads to inhibition of the AKT signaling pathway. An exemplary amino acid sequence of PTEN is shown in SEQ ID NO: 1.

[44] The terms "particle" and "vesicle" are used interchangeably herein and refer to the discrete entity used to deliver the active agent. The terms "active agent" and "active fragment" are used interchangeably herein and refer to an agent having biological activity, pharmacological action and/or therapeutic utility. Non-limiting examples of active agents are small molecules, RNA, DNA, peptides and proteins. In some embodiments, the active agent is a non-endogenous active agent, i. not present in a living cell in nature. In one embodiment, "non-endogenous" means that the active agent is not present in the human body and/or in human cells.

[45] The particle may contain a vesicle or a flattened sphere delimited by a lipid bilayer. The particle diameter may be 40-100 nm. Particles can be formed by budding of the endosomal membrane inwards. The particles may have a density of approximately 1.13-1.19 g/ml and may float on sucrose gradients. The particles can be enriched in cholesterol and sphingomyelin as well as lipid raft markers such as GM1, GM3, flotilin and src protein kinase Lyn. The particles may contain one or more proteins present in mesenchymal stem cells or mesenchymal stem cell conditioned medium (MSC-CM), for example, a protein characteristic or specific for MSC or MSC-CM. They may contain RNA, such as miRNA.

[46] In some embodiments, the particles are membrane vesicles. As used herein, the term "membrane vesicles" refers to any structure of vesicles containing fluid enclosed in a lipid bilayer. Thus, in some embodiments, the particles are lipid bilayer phospholipid membrane vesicles. In some embodiments, membrane vesicles are selected from extracellular vesicles, liposomes, ectosomes, and transferosomes. In some embodiments, the membrane vesicles are synthetic membrane vesicles. In other embodiments, membrane vesicles are particles of cellular origin. In some embodiments, the cells are eukaryotic cells. In one embodiment, the membrane vesicles are liposomes.

[47] In one embodiment, the membrane vesicles are extracellular vesicles. In some embodiments, the pharmaceutical composition of the present invention comprises extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN), wherein the extracellular vesicles are of cellular origin.

[48] The terms "extracellular vesicles" and "EV" are used interchangeably herein and refer to vesicles of cellular origin containing a membrane covering the interior. Usually, the diameter of extracellular vesicles is from 30 nm to 1000 nm, and they can contain various macromolecular cargo in the interior, on the outer surface of the extracellular vesicle, or on its membrane. Said cargo may contain nucleic acids, proteins, carbohydrates, lipids, low molecular weight compounds and/or combinations thereof. The term "extracellular vesicles" includes the terms "exosome" and "microvesicles". The terms "exosomes" and "nanovesicles" are used interchangeably herein and refer to EVs between 30 and 100 nm in diameter. As a rule, regardless of specific theoretical concepts, exosomes are formed inside cells and released from the cell upon fusion of the multivesicular body (MVB) with the plasma membrane. Alternatively, exosomes are released directly from the plasma membrane. As used herein, the term "microvesicles" refers to EVs between 100 and 1000 nm in diameter. The term "ectosomes" refers to vesicles of various sizes (eg, 0.1-1 mm in diameter) budding directly from the plasma membrane.

[49] In some embodiments, the extracellular vesicles are selected from exosomes, microvesicles, ectosomes, exovesicles, and combinations thereof.

[50] In one embodiment, EVs are exosomes. Exosomes may have at least one of the following properties: (a) have a size of 50 to 100 nm, according to electron microscopy; (b) contain a >100 kDa molecular weight complex containing <100 kDa proteins; (c) contain a >300 kDa molecular weight complex containing <300 kDa proteins; (d) contain a complex with a molecular weight > 1000 kDa; or (e) have a hydrodynamic radius of less than 100 nm as measured by laser diffraction or dynamic light scattering. According to one implementation variant, the diameter of the exosomes is from 50 to 100 nm.

[51] In another embodiment, the extracellular vesicles are microvesicles. In one embodiment, EVs are microvesicles. In one embodiment, the diameter of the microvesicles is 100 to 1000 nm, 120 to 800 nm, 150 to 600 nm, or 200 to 400 nm. According to another implementation variant, the size of the microvesicles is from 100 to 300 nm or from 150 to 250 nm. In some embodiments, the EV diameter is 30 to 250 nm, or 50 to 200 nm. In some embodiments, the EV diameter is 70 to 170 nm, or 80 to 150 nm.

[52] The size of the EV may be greater than 2 nm. The EV can be larger than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm. The size of the EV may be greater than 100 nm, such as greater than 150 nm. The size of the EV may be substantially 200 nm or more.

[53] The size of the EV may be within a certain range, such as 2 nm to 20 nm, 2 nm to 50 nm, 2 nm to 100 nm, 2 nm to 150 nm, or 2 nm to 200 nm. The EV size can be 20 to 50 nm, 20 to 100 nm, 20 to 150 nm, or 20 to 200 nm. The EV size can be 50 to 100 nm, 50 to 150 nm, or 50 to 200 nm. The size of the EV may be from 100 nm to 150 nm, or from 100 nm to 200 nm. The EV size can be from 150 to 200 nm. The EV size can be 100 to 600 nm, 150 to 500 nm, or 200 to 400 nm.

[54] The size can be determined in various ways. In principle, the size can be determined by size fractionation and membrane filtration with appropriate size limitation. Particle size can then be determined by monitoring the segregation of constituent proteins by SDS-PAGE or by biological analysis.

[55] The size may be the hydrodynamic radius. The hydrodynamic radius EV may be less than 100 nm. It can be from about 30 nm to about 70 nm. The hydrodynamic radius may be from about 40 nm to about 60 nm, for example, from about 45 nm to about 55 nm. The hydrodynamic radius may be approximately 50 nm. The hydrodynamic radius EV can be determined by any suitable means, such as laser diffraction or dynamic light scattering.

[56] According to another implementation variant, extracellular vesicles are a combination of exosomes and microvesicles. In some embodiments, the extracellular vesicles are isolated extracellular vesicles. In yet another embodiment, the EVs are exovesicles or ectosomes.

[57] As described above, extracellular vesicles are derived from cells. The terms "derived from" and "derived from" are used interchangeably herein and refer to vesicles that are produced within, by, or from a particular cell, cell type, or cell population. As used herein, the terms "parent cell", "producer cell" and "original cell" include any cell from which an extracellular vesicle originates and is released. The terms also include a cell that uses a protein, lipid, carbohydrate, or nucleotide component of an extracellular vesicle. For example, the terms "parent cell" or "producer cell" include the cell that is the source of the extracellular vesicle. In some embodiments, the cells are eukaryotic cells.

[58] Extracellular vesicles (EV) can be obtained from biological cells by any of several methods, for example, by secretion, budding or dispersion from biological cells. EVs can be objects that can be isolated from mesenchymal stem cells (MSC), neural crest cells (NCC), mesenchymal stem cell conditioned medium (MSC-CM), or neural crest cell conditioned medium. EVs may be responsible for the activity of the original cells, for example, MSC, NCC, NCC-CM or MSC-CM, or at least have their activity. EVs may be responsible for and perform essentially most or all of the functions of the original cells, such as MSC, NCC, NCC-CM, or MSC-CM. For example, EVs may be a replacement (or biological replacement) for MSC, NCC, NCC-CM, or MSC-CM. For example, extracellular vesicles can be produced, secreted, generated or distributed by biological cells. If the biological cell is in cell culture, the particle may be secreted into the cell culture medium.

[59] Examples of biological cells from which EVs can be obtained include adherent cells expressing mesenchymal markers, such as mesenchymal stem cells, oral mucosal or olfactory parietal cells, astrocytes, and neural crest cells. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous PTEN inhibitor, the extracellular vesicles being derived from adherent cells expressing mesenchymal markers. In one embodiment, adherent cells expressing mesenchymal markers are selected from mesenchymal stem cells (MSCs), oral mucosal stem cells, and olfactory parietal cells. In one embodiment, the cells are mesenchymal stem cells (MSCs).

[60] The term "mesenchymal stem cells" refers to multipotent stromal cells that can differentiate into various cell types well known in the art, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells). ) and adipocytes (fat cells).

[61] In their pluripotent state, mesenchymal stem cells typically express the following markers: CD105, CD166, CD29, CD90, and CD73, but do not express CD34, CD45, and CD133.

[62] Mesenchymal stem cells can be isolated from various tissues, including but not limited to bone marrow, adipose tissue, dental pulp, oral mucosa, peripheral blood, and amniotic fluid. In one embodiment, mesenchymal stem cells are isolated from bone marrow. In one embodiment, the mesenchymal stem cells are derived from a region selected from bone marrow, adipose tissue, umbilical cord, dental pulp, oral mucosa, peripheral blood, and amniotic fluid. In some embodiments, EV is derived from bone marrow-derived MSCs. According to another implementation variant, EV is obtained from MSCs derived from adipose tissue. In some of these embodiments, EVs are selected from exosomes, microvesicles, and combinations thereof. In some embodiments, cells express markers CD105, CD166, CD29, CD90, and CD73. In an additional embodiment, the cells express CD105, CD166, CD29, CD90 and CD73 and do not express CD34, CD45 and CD133. In some embodiments, the cells are selected from dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP) and dental follicle progenitor cells (DFPC).

[63] EVs may contain one or more proteins, oligonucleotides, or polynucleotides secreted by a particular type of cell, such as mesenchymal stem cells or neural crest cells. EVs may contain one or more proteins or polynucleotides present in mesenchymal stem cell conditioned medium (MSC-CM). In a particular embodiment, EVs may contain microRNAs derived from MSCs or neural crest cells.

[64] For example, EVs may contain 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more of these proteins and/or polynucleotides. EVs may contain essentially about 75% of the indicated proteins and/or polynucleotides. Proteins can be specified by reference to a list of proteins or products of a list of genes.

[65] EVs may have at least one property of mesenchymal stem cells. The particle may have a biological property, such as biological activity. The particle may have any of the biological activities of the MSC. The particle may, for example, have the therapeutic or restorative activity of the MSC.

[66] Methods for isolating, purifying, and expanding mesenchymal stem cells (MSCs) are known in the art and include, for example, those described by Caplan and Haynesworth in US Pat. No. 5,486,359 and Jones E.A. et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

[67] Cultures of mesenchymal stem cells can be obtained by diluting BM aspirates (typically 20 ml) with equal volumes of Hank's Balanced Salt Solution (HBSS; GIBCO Laboratories, Grand Island, NY, USA) and layering the diluted cells onto an approximately 10 ml column with Ficoll (Ficoll-Paque; Pharmacia, Piscataway, NJ, USA). After 30 minutes centrifugation at 2500 xg, the mononuclear cell layer is removed from the interface and suspended in HBSS. The cells are then centrifuged at 1500 xg for 15 minutes and resuspended in complete medium (MEM, α medium without deoxyribonucleotides or ribonucleotides; GIBCO); 20% fetal calf serum (FCS) from a batch selected for MSC rapid growth (Atlanta Biologicals, Norcross, GA, USA); 100 units/ml penicillin (GIBCO), 100μg/ml streptomycin (GIBCO); and 2 mM L-glutamine (GIBCO). The resuspended cells are placed in approximately 25 ml of medium in a 10 cm culture dish (Corning Glass Works, Corning, NY, USA) and incubated at 37°C in a humidified atmosphere with 5% CO ₂ . After 24 hours of culture, non-adherent cells are discarded and adherent cells are thoroughly washed twice with phosphate buffered saline (PBS). The medium is replaced with fresh complete medium every 3-4 days for approximately 14 days. Adherent cells are then harvested using 0.25% trypsin and 1 mM EDTA (trypsin/EDTA, GIBCO) for 5 minutes at 37° C., plated in a 6 cm dish, and cultured for another 14 days. Cells are then trypsinized and counted using a cell counting device such as a hemocytometer (Hausser Scientific, Horsham, PA, USA). Cultured cells are recovered by centrifugation and resuspended in 5% DMSO and 30% FCS at a concentration of 1 to 2 x 10 ⁶ cells per ml. Aliquots of 1 ml are slowly frozen and stored in liquid nitrogen.

[68] To expand the mesenchymal stem cell fraction, frozen cells are thawed at 37°C, diluted with complete medium, and reconstituted by centrifugation to remove DMSO. Cells are resuspended in complete medium and seeded at a concentration of approximately 5000 cells/cm ² . After 24 hours of culture, non-adherent cells are removed and adherent cells are harvested using trypsin/EDTA, dissociated by passage through a tapered Pasteur pipette, and preferably replated at a density of about 1.5 to about 3.0 cells/cm ² . Under these conditions, MSC cultures can grow up to about 50 population doublings and increase their numbers by about 2000 times [Colter DC., et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000].

[69] MSC cultures used in some embodiments of the present invention include three groups of cells, defined by their morphological features: small and agranular cells (hereinafter referred to as RS-1), small and granular cells (hereinafter referred to as RS- 2) and large and moderately granular cells (hereinafter referred to as mature MSCs). The presence and concentration of such cells in culture can be analyzed by detecting the presence or absence of various cell surface markers using, for example, immunofluorescence, in situ hybridization, and activity assays.

[70] In some embodiment, EVs are not derived from astrocytes. Thus, the present invention contemplates compositions containing particles loaded with a PTEN inhibitor, provided that the particles are not astrocyte-derived exosomes.

[71] In a specific embodiment, the extracellular vesicles are derived from cells expressing neural crest cell markers. In a specific embodiment, EVs are derived from neural crest cells. In another embodiment, the neural crest cells are cranial neural crest cells. In some embodiments, cranial neural crest cells include dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP), and dental follicle progenitor cells (DFPC). ), but are not limited to them. In some embodiments, such cells express mesenchymal markers as defined above.

[72] EV can be obtained or allocated in several ways. Such a method may include isolation of EV from mesenchymal stem cells (MSC) or from neural crest cells (NCC).

[73] Therefore, the EVs of the present invention are isolated EVs. Thus, the present invention provides a pharmaceutical composition comprising isolated extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN), the extracellular vesicles being separated from the cells. In some embodiments, the cells are human cells.

[74] In any of the above embodiments, the extracellular vesicles are isolated extracellular vesicles. As used herein, the terms "purify", "purified", "purification", "isolated", "isolated", and "isolation" are used interchangeably and refer to the state of a population (e.g., a plurality of known or unknown number and/or concentration) of extracellular vesicles. subjected to one or more of the purification processes, for example, the selection of the desired extracellular vesicles or, alternatively, the removal or reduction of residual biological products. In one embodiment, the ratio of EV to residual parent cells is at least 2, 3, 4, 5, 6, 8, or 10 times higher, or, in some preferred embodiments, at least 50, 100, or 1000 times higher than in the original material. In some preferred embodiments, the term "isolated" has the meaning of "substantially cell-free" or "cell-free", and can be replaced by this term. Thus, in some embodiments, the pharmaceutical composition of the present invention comprises isolated extracellular vesicles loaded with a PTEN inhibitor. In some embodiments, the pharmaceutical composition is a cell-free composition, i.e. does not contain a detectable number of cells.

[75] As mentioned, lipid bilayer phospholipid membrane vesicles, such as extracellular vesicles, are loaded with an exogenous PTEN inhibitor.

[76] As used herein, the term “exogenous” refers to a molecule or substance (e.g., compound, nucleic acid, or protein) that originates outside of a given membrane vesicle, e.g., extracellular vesicles, and which is not naturally present in the vesicle. In relation to extracellular vesicles, this term refers to molecules or substances that are not naturally present in vesicles, but not in the cells from which extracellular vesicles originate. In some embodiments, the term "exogenous" refers to synthetic non-natural molecules. In some embodiments, the substance is artificially loaded into extracellular vesicles or into cells from which extracellular vesicles originate. In relation to peptides, proteins and nucleic acids, this term means that the compound is artificially loaded into extracellular vesicles or into the cells from which the vesicles are derived, or artificially expressed in the cells from which the vesicles are derived, however, the specified compound is not naturally expressed in the original cells. .

[77] The term "loaded" means that the particles, ie. membrane vesicles are artificially supplemented or filled with an inhibitor. In relation to the location of the inhibitor relative to the membrane vesicle, this term means "included within the vesicle, brought to the surface of the vesicle (inner and/or outer surface) or present on it, embedded in the membrane of the vesicle (outer, inner or intermediate)". In one embodiment, the inhibitor is enclosed within the outer membrane of the vesicle. According to another implementation variant, the inhibitor is enclosed within the inner membrane of the vesicle. According to another implementation variant, the inhibitor is enclosed in the liquid phase of the vesicle.

[78] In one embodiment, the PTEN inhibitor inhibits or suppresses the activity of PTEN. According to another implementation variant, the PTEN inhibitor inhibits or suppresses the expression of PTEN.

[79] In one embodiment, the PTEN inhibitor is a protein or peptide. According to another implementation variant, the PTEN inhibitor is a small molecule compound. Examples of protein-based PTEN inhibitors include those described in US Patent Applications No. 20160074472 and 20160311857, the contents of which are incorporated herein by reference, but are not limited to them. In some embodiments, protein-based PTEN inhibitors include peptides whose sequence is selected from sequence 1, 2, 5, 6 described in WO 2011/044701 and peptides with sequences 1-5 described in WO 2015/105957.

[80] Protein-based inhibitors of the present invention can be synthesized biochemically, for example, using standard solid phase techniques. These methods include exclusively solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical synthesis from solutions. Procedures for solid phase synthesis of polypeptides are well known in the art. Synthetic peptides can be purified by preparative HPLC and their composition can be confirmed by amino acid sequencing.

[81] In addition, recombinant techniques can be used to create protein-based inhibitors of the present invention. To obtain a polypeptide inhibitor of the present invention using recombinant technology, a polynucleotide encoding a peptide of the present invention is ligated into an expression nucleotide vector containing a polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., a promoter sequence) suitable for controlling constitutive, tissue-specific, or inducible transcription of the polypeptides of the present invention in host cells. Expression vectors are further described herein below.

[82] In addition to being synthesized in host cells, the peptides of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and components of this system are commercially available.

[83] In a specific embodiment, protein-based inhibitors are expressed in cells from which EVs originate. Thus, for example, the present invention contemplates the expression of a protein inhibitor in a population of MSCs, followed by the generation of EVs from genetically modified MSCs.

[84] In some embodiments, the exogenous PTEN inhibitor is an inhibitor of PTEN expression. In one embodiment, the PTEN expression inhibitor is a polynucleotide or oligonucleotide. In another embodiment, the inhibitor is an RNA interference (RNAi) oligonucleotide. In one embodiment, the RNA interference oligonucleotide is an siRNA. In another embodiment, the RNA interference oligonucleotide is shRNA.

[85] The term "polynucleotide" as used herein refers to a long nucleic acid containing more than 150 nucleotides. The term "oligonucleotide" as used herein refers to a short single-stranded or double-stranded nucleotide sequence, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or mimetics thereof, said nucleic acid typically containing no more than 150 nucleotides. In some embodiments, the oligonucleotide contains 2 to 150, 10 to 100, or 15 to 50 nucleotides. In other embodiments, the oligonucleotide contains 15 to 40, 17 to 35, or 18 to 30 nucleic acids.

[86] In one embodiment, the polynucleotide or oligonucleotide agent is an RNA silencing agent. As used herein, the term "RNA silencing" refers to a group of regulatory mechanisms (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), downregulation, co-suppression and repression of translation) mediated by RNA molecules. , which leads to inhibition or "silencing" of the expression of the corresponding gene encoding the protein. RNA silencing has been observed in many organisms, including plants, animals, and fungi.

[87] As used herein, the terms “RNA silencing agent”, “RNA silencing molecule”, and “RNA silencing oligonucleotide” are used interchangeably and refer to RNA that is capable of inhibiting or causing “silencing” of gene expression. -targets. In some embodiments, an RNA silencing agent is capable of preventing complete processing (eg, complete translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, such as double-stranded RNAs containing paired strands, as well as precursor RNAs from which such small non-coding RNAs can be derived. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs, and shRNAs. In one embodiment, the RNA silencing agent is capable of causing RNA interference. In yet another embodiment, the RNA silencing agent is capable of mediating translational repression.

[88] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNAs (miRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing, and in fungi as "suppression". The process of post-transcriptional gene silencing is believed to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly present in various plants and taxa of organisms. Such protection against foreign gene expression may have evolved in response to the production of double-stranded RNA (dsRNA) resulting from a viral infection, or as a result of the random integration of transposon elements into the host genome, through a cellular response that specifically degrades the homologous single-stranded RNA. or viral genomic RNA.

[89] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme called dicer. Dicer is involved in the processing of dsRNA to form short dsRNA fragments known as small interfering RNAs (siRNAs). The small interfering RNAs resulting from Dicer activity are typically about 21 to about 23 nucleotides in length and contain double-stranded structures of about 19 base pairs. The RNAi reaction also involves an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of a single-stranded RNA with a sequence complementary to the antisense strand of a double-stranded siRNA. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the double-stranded siRNA.

[90] Accordingly, the present invention contemplates the use of dsRNA to suppress protein expression from mRNA. In one embodiment, the dsRNA is larger than 30 bp. The use of long dsRNAs (i.e., dsRNAs greater than 30 bp) is very limited due to the notion that these longer double-stranded RNA regions result in the induction of interferon and a PKR response. However, the use of long dsRNAs can provide numerous advantages because the cell can choose the optimal silencing sequence, eliminating the need to test multiple siRNAs; long dsRNAs can reduce the complexity of silencing libraries compared to the complexity required for siRNAs; and, perhaps most importantly, long dsRNA can prevent virus escape mutations when used as therapeutic agents.

[91] Various studies demonstrate that long dsRNAs can be used to silence gene expression without causing a stress response or significant non-specific effects.

[92] In particular, the present invention also considers the introduction of long dsRNA (transcripts longer than 30 bases) for gene silencing in cells in which the interferon pathway is not activated (eg, embryonic cells and oocytes).

[93] The present invention also contemplates the introduction of a long dsRNA, specifically designed to avoid induction of the interferon and PKR pathways, to suppress gene expression. For example, Shinagwa and Ishii [ Genes & Dev. 17 (11): 1340-1345, 2003] developed a vector called pDECAP for expressing a long double-stranded RNA from an RNA polymerase II (Pol II) promoter. Since the pDECAP transcripts lack both the 5'-cap and the 3'-poly(A) tail, which facilitate the export of dsRNA to the cytoplasm, long dsRNA from pDECAP does not induce an interferon response.

[94] Another way to bypass the pathways of interferon and PKR in mammalian systems is the introduction of small inhibitory RNA (siRNA) through transfection or by endogenous expression.

[95] The term "siRNA" refers to double-stranded small inhibitory RNAs (typically 18-30 base pairs long) that induce an RNA interference (RNAi) pathway. Typically, miRNAs are chemically synthesized as 21-mer molecules with a 19 bp central double-stranded region. and symmetrical 2-base 3' sticky ends, although it has recently been described that chemically synthesized double-stranded RNAs of 25-30 bases in length can provide up to a 100-fold increase in efficiency compared to a 21-mer molecule in the same position. The observed increased RNAi triggering efficiency obtained with longer RNAs is theoretically the result of providing the dicer with a substrate (27mer) instead of a product (21mer), which increases the rate or efficiency of entry of the double stranded siRNA into RISC.

[96] The position of the 3' sticky end has been found to affect siRNA efficiency, and asymmetric double-stranded structures with a 3' sticky end on the antisense strand are generally more efficient than double stranded structures with a 3' sticky end on the sense strand. This may be due to asymmetric loading of the strand in RISC, since the opposite pattern of efficiency is observed when targeting the antisense transcript.

[97] Exemplary siRNA sequences considered for PTEN inhibition include 5'-GUUAGCAGAAACAAAAGGAGAUAUCAA-3' (SEQ ID NO: 2; sense strand); 5'-UUGAUAUCUCCUUUUGUUUCUGCUAAC-3' (SEQ ID NO: 3; antisense strand); or 5'-CAGCCGUUCGGAGGAUUAUUCGUCUTT-3' (SEQ ID NO: 4; sense strand), 5'-AGACGAAUAAUCC UCCGACGGCUGTT-3' (SEQ ID NO: 5 antisense strand). In one embodiment, the siRNA that inhibits PTEN expression contains the nucleotide sequence 5'-GAGUUCUUCCACAAAACAGAA-3' (SEQ ID NO: 10; sense). In one embodiment, the siRNA that inhibits PTEN expression comprises the nucleotide sequence 5'-UUCUGUUUGUGGAAGAACUC-3' (SEQ ID NO: 11; antisense strand). In one embodiment, the siRNA is a double-stranded siRNA comprising SEQ ID NOS: 10 and 11.

[98] One exemplary sequence that can be targeted by PTEN siRNA is shown in SEQ ID NO: 6 (5'-GAGTTCTTCCACAAAACAGAA-3'). Another exemplary sequence that can be targeted by PTEN siRNA is shown in SEQ ID NO: 7 (5'-GTATAGAGCGTGCAGATAA-3').

[99] Thus, in one embodiment, the PTEN inhibitor is an siRNA. In some embodiments, the siRNA may have several modifications that improve the penetration of vesicles or cells into the membrane.

[100] In one embodiment, the siRNA comprises a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4, and 5. In one embodiment, the PTEN inhibitor is an siRNA with the sequence of SEQ ID NO: 2 and/ or 3. In another embodiment, the PTEN inhibitor is an siRNA of SEQ ID NO: 4 and/or 5. In some embodiments, the siRNA contains a nucleotide sequence that targets SEQ ID NO: 6. In other embodiments, the siRNA contains a nucleotide sequence whose target is SEQ ID NO: 7. In one embodiment, the PTEN inhibitor is an siRNA with the sequence of SEQ ID NO: 10. In another embodiment, the PTEN inhibitor is an siRNA with the sequence of SEQ ID NO: 11. According to an additional implementation variant, the PTEN inhibitor is a double-stranded siRNA containing the sequence of SEQ ID NOS: 10 and 11.

[101] According to another implementation variant, the PTEN inhibitor is an siRNA that is a variant of the nucleotide sequence selected from SEQ ID NOS: 10, 11, 2, 3, 4 and 5. According to one implementation variant, this variant is characterized by at least 70 % sequence identity with the original sequence. In yet another embodiment, the variant has at least 80%, 85%, or 90% sequence identity with the original sequence. In one embodiment, the variant has the activity of the parent sequence.

[102] The terms "homolog", "variant", "DNA variant", "sequence variant", and "polynucleotide variant" are used interchangeably herein and refer to a DNA polynucleotide or oligonucleotide characterized by at least 70% sequence identity with the original sequence. A variant may include mutations, such as a deletion, addition or substitution, wherein said mutations do not change the open reading frame and said polynucleotide encodes a peptide or protein that is substantially similar in structure and function to the peptide or protein encoded by the parent polynucleotide. In some embodiments, the options are conservative options. As used herein, the term "conservative variants" refers to variants in which a change in one or more nucleotides at a given codon position does not result in a change in the amino acid encoded at that position. Thus, a peptide or protein encoded by conservative variants has 100% sequence identity with the peptide or protein encoded by the parent polynucleotide. In some embodiments, the variant is a non-conservative variant encoding a peptide or protein that is a conservative peptide analog of the protein encoded by the parent polynucleotide. In some embodiments, the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. with the original sequence.

[103] In another embodiment, the siRNA is an siRNA that is complementary to the PTEN encoding sequence and inhibits its expression and/or translation. In one embodiment, the siRNA is complementary to a sequence selected from SEQ ID NOs: 6 and 7. In one embodiment, the siRNA contains a sequence complementary to a nucleic acid fragment encoding a PTEN protein and inhibits its expression. In one embodiment, the siRNA contains a sequence complementary to a nucleic acid fragment encoding SEQ ID NO: 1. In one embodiment, the siRNA is characterized by 85%, 88%, 90%, 92%, 95%, 98%, 99%, or 100% complementarity of the specified sequence.

[104] It should be appreciated that more than one miRNA agent can be used to suppress a target gene. Thus, for example, the present invention contemplates the use of at least two siRNAs that target PTEN.

[105] The terms "have", "has", "having", and "comprising" may also include the meanings of "consisting of" and "consisting essentially of" and may be replaced by these terms.

[106] Chains of double-stranded interfering RNA (eg, siRNA) can be connected to form a hairpin or rod-loop structure (eg, shRNA). Thus, as mentioned, the RNA silencing agent of the present invention can also be short hairpin RNA (shRNA).

[107] The term "shRNA" as used herein refers to an RNA agent containing a rod-loop structure comprising first and second regions of a complementary sequence, wherein the degree of complementarity and orientation of the regions is sufficient for base pairing between said regions, wherein the first and second the regions are connected by a loop region, said loop being due to a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop ranges from 3 to 23 inclusive, or from 5 to 15, or from 7 to 13, or from 4 to 9, or from 9 to 11. Some nucleotides in the loop region may participate in base pair interactions with other nucleotides in loop area. Examples of oligonucleotide sequences that can be used to form a loop include 5'-UUCAAGAGA-3' (SEQ ID NO: 8;) and 5'-UUUGUGUAG-3' (SEQ ID NO: 9). One skilled in the art will understand that the resulting single stranded oligonucleotide forms a rod-loop or hairpin structure containing a double stranded region capable of interacting with the RNAi machinery. In some embodiments, the shRNA contains the nucleic acid of SEQ ID NO: 2. In other embodiments, the shRNA contains the nucleic acid of SEQ ID NO: 4. In one embodiment, the shRNA contains the nucleic acid of SEQ ID NO: 2 and 3. In another embodiment, the shRNA contains the nucleic acid of SEQ ID NO: 2 and 3. in an embodiment, the shRNA contains the nucleic acid of SEQ ID NOs: 4 and 5. In some embodiments, the shRNA contains the nucleic acid of SEQ ID NOs: 10 or 11. In other embodiments, the shRNA contains the nucleic acid of SEQ ID NOs: 10 and 11.

[108] In another embodiment, the RNA silencing agent may be a microRNA. MicroRNAs are small RNAs composed of genes encoding primary transcripts of various sizes. They are found in both animals and plants. The primary transcript (referred to as "pri-miRNA") is processed through various nucleolytic steps to a shorter miRNA precursor, or "pre-miRNA". The pre-miRNA is present in the form of a tertiary structure, so that the finished (mature) miRNA is present in a double-stranded structure, with the two strands being called miRNA (the strand that eventually pairs with the target). The pre-miRNA is a substrate for a form of dicer that removes the double-stranded miRNA from the precursor, after which, as in the case of miRNAs, the double-stranded structure can be incorporated into the RISC complex. It has been demonstrated that microRNAs can be expressed transgenically and be effective due to the expression of the precursor form, rather than the entire primary form.

[109] Unlike miRNAs, miRNAs bind to transcript sequences with only partial complementarity and repress translation without affecting equilibrium levels of RNA. Both miRNAs and miRNAs are processed by a dicer and bind to the components of the RNA-induced silencing complex. It has been hypothesized that the regulation of genes by the miRNA pathway versus the miRNA pathway is determined solely by the degree of complementarity of the target transcript. It is assumed that miRNAs, which are characterized by only partial identity with the target mRNA, function by causing translational repression similar to miRNAs, and do not trigger the destruction of RNA.

[110] It should be appreciated that the RNA silencing agent of the present invention is not necessarily limited to molecules containing only RNA, but also includes chemically modified nucleotides and non-nucleotide compounds.

[111] Additional agents capable of inhibiting PTEN include ribozymes, DNA enzymes, and agents of the CRISPR system (eg, CRISPR/Cas).

[112] Ribozymes are increasingly being used to inhibit the expression of sequence-specific genes by cleaving the mRNAs encoding the proteins in question. The ability to design ribozymes to cleave any particular target RNA has made them valuable tools in both basic research and therapeutic applications. In the field of therapy, ribozymes are used to target viral RNAs in infectious diseases, dominant oncogenes in cancer, and specific somatic mutations in genetic disorders. In particular, several ribozyme gene therapy protocols for HIV patients are already in phase 1 trials. More recently, ribozymes have been used for transgenic animal studies, target gene validation, and metabolic pathway studies. Some ribozymes are at various stages of clinical research. ANGIOZYME® is the first chemically synthesized ribozyme studied in human clinical trials. ANGIOZYME® specifically inhibits the formation of VEGF-r (vascular endothelial growth factor receptor), a key component of the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other companies, have demonstrated the importance of anti-angiogenic therapeutics in animal models. HEPTAZYME®, a ribozyme designed to selectively destroy hepatitis C virus (HCV) RNA, has been shown to be effective in reducing levels of hepatitis C virus RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - home page).

[113] Another agent capable of inhibiting PTEN is RNA-dependent endonuclease technology, such as the CRISPR system.

[114] An additional way to regulate the expression of the PTEN gene in cells are oligonucleotides that form three-stranded structures (TFO). Research has shown that it is possible to construct TFOs that can recognize and bind to polypurine/polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. Modification of oligonucleotides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentrations) have helped to overcome natural obstacles to TFO activity, such as charge repulsion and instability, and it has recently been shown that certain sequences can be targets of synthetic oligonucleotides.

[115] Thus, for any given sequence in the PTEN gene, a sequence can be designed to form a three-stranded structure. The length of the oligonucleotides forming the three-strand structure is preferably at least 15, more preferably 25, even more preferably 30 or more nucleotides, up to 50 or 100 bp.

[116] As already mentioned, the extracellular vesicles of the present invention can be loaded with a polynucleotide or oligonucleotide agent that directly inhibits PTEN expression.

[117] To facilitate loading of membrane vesicles (eg, exosomes), the polynucleotide or oligonucleotide cargo may contain one or more of the hydrophobic modifications. Hydrophobic modifications increase the hydrophobicity of the polynucleotide or oligonucleotide cargo compared to native (unmodified) RNA or DNA. In some embodiments, hydrophobic modifications increase the hydrophobicity of a polynucleotide or oligonucleotide by at least two orders of magnitude (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more orders of magnitude) compared to native (unmodified) RNA or DNA. In other embodiments, hydrophobic modifications increase the hydrophobicity of a polynucleotide or oligonucleotide by at least 10 orders of magnitude compared to native (unmodified) RNA or DNA. In other embodiments, hydrophobic modifications increase the hydrophobicity of the polynucleotide or oligonucleotide by at least two orders of magnitude (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more orders of magnitude) compared to an unmodified polynucleotide or oligonucleotide . In other embodiments, hydrophobic modifications increase the hydrophobicity of a polynucleotide or oligonucleotide by at least ten orders of magnitude compared to an unmodified polynucleotide or oligonucleotide. The increase in hydrophobicity can be assessed using any suitable method. For example, hydrophobicity can be determined by measuring the percentage solubility in an organic solvent, such as octanol, compared to the solubility in an aqueous solvent, such as water.

[118] In some embodiments, the hydrophobic nature of a polynucleotide or oligonucleotide cargo can be increased by increasing the proportion of hydrophobically modified nucleotides in the polynucleotide or oligonucleotide molecule. For example, in one embodiment, 20% or more of the nucleotides in an oligonucleotide or polynucleotide molecule are hydrophobically modified, e.g., 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more , 80% or more, 90% or more, 95% or more, 99% or more, etc. nucleotides in the oligonucleotide or polynucleotide molecule are hydrophobically modified. In one embodiment, 100% of the nucleotides in an oligonucleotide or polynucleotide molecule are hydrophobically modified. In a typical embodiment, 30% or more of the nucleotides in an oligonucleotide or polynucleotide molecule contain hydrophobic modifications. Increasing the proportion of hydrophobically modified nucleotides in an oligonucleotide or polynucleotide molecule can be beneficial when, for example, the hydrophobic modification is weakly hydrophobic (eg, 2'O-methyl modification). In embodiments using a highly hydrophobic modification, eg via a sterol, lipid, etc., the hydrophobic modification alone may be sufficient to facilitate loading into exosomes.

[119] In some embodiments, the hydrophobic modification is a covalent modification. Hydrophobic modifications of nucleic acid molecules may include, for example, backbone modifications, carbohydrate modifications, base modifications, and/or conjugate modifications, and combinations thereof.

[120] Framework modifications include changes in the ester phosphate bonds in the nucleic acid molecule. Examples of suitable backbone modifications include phosphorothioate modifications, phosphorodithioate modifications, p-ethoxy modifications, methylphosphonate modifications, methylphosphorothioate modifications, alkyl and aryl phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), alkyl phosphotriesters (in which the charged oxygen moiety is alkylated), peptide nucleic acid (PNA) backbone modifications, locked nucleic acid (LNA) backbone modifications, and the like, but are not limited to. These modifications can be used in combination with each other and/or in combination with phosphodiester backbone bonds.

[121] In one embodiment, the hydrophobic modification is a phosphorothioate (PS) modification in which one of the non-bridging oxygen atoms of the phosphate group is replaced by sulfur to form a PS group. This modification provides significant resistance to cleavage by nucleases and has favorable pharmacokinetic properties. PS bonds can be incorporated into oligonucleotide molecules using standard techniques such as solid phase oligonucleotide synthesis.

[122] In yet another embodiment, the hydrophobic modification is a phosphonate modification in which one non-bridging oxygen atom is replaced by an alkyl group. In other embodiments, the hydrophobic modification is a peptide nucleic acid (PNA) modification. PNAs are mimics of oligonucleotides with a neutrally charged peptide backbone compared to the highly charged carbohydrate-phosphate backbone of native RNA and DNA (see, for example, In other embodiments, the hydrophobically modified nucleic acid molecule is a phosphorodiamidate morpholino oligonucleotide (PMO).

[123] In other embodiments, the oligonucleotide cargo molecules can be hydrophobically modified at a carbohydrate moiety (eg, ribose, deoxyribose, etc.). Carbohydrate modifications often occur at the 2' position of the sugar ring, where, for example, the 2' moiety may be modified or replaced with a hydrophobic moiety, such as a halogen atom, alkoxy, aminoalkoxy, alkyl, azide, or amino group. In non-limiting examples, carbohydrate modifications may include O-methyl-, F-, methoxyethyl-, and 2'-fluoro-β-D-arabinonucleotide (FANA). Other 2' modifications include, for example, 2'O-allyl, 2'O-ethylamine and 2'O-cyanoethyl modifications. In addition, modifications can be made at other sites, including the 4' position on the carbohydrate moiety.

[124] In other embodiments, the oligonucleotide cargo molecules may contain hydrophobic base modifications. In exemplary embodiments, these modifications include phenyl, naphthyl, and isobutyl modifications. Other embodiments include C-5 propynyl bases, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, and hypoxanthine.

[125] In addition to increasing the hydrophobic character of the oligonucleotide payload, the aforementioned backbone, carbohydrate, and base modifications increase the stability of oligonucleotides in the presence of particles (eg, exosomes) and minimize degradation that can occur during loading.

[126] In some embodiments, an RNA interference oligonucleotide, such as siRNA or shRNA, contains a hydrophobic moiety. Hydrophobic moieties can also be chemically conjugated to an oligonucleotide to enhance its hydrophobicity. Thus, in one embodiment, the RNA interference oligonucleotide is conjugated to a hydrophobic moiety. In one embodiment, said hydrophobic moiety is selected from the group consisting of a sterol, ganglioside, lipid, vitamin, fatty acid, peptide, and combinations thereof. In one embodiment, the RNA interference oligonucleotide is conjugated to a sterol. In exemplary embodiments, the fragment is a cholesterol molecule, therefore, according to such embodiments, the RNA interference oligonucleotide is conjugated to cholesterol. In some embodiments, one of the strands of the double-stranded RNAi molecule is conjugated to a hydrophobic molecule, such as cholesterol. In other embodiments, the two strands of the double-stranded RNAi molecule are conjugated to a hydrophobic molecule, such as cholesterol. In other embodiments, the RNA interference oligonucleotide is conjugated to a molecule selected from monosialotetrahexosyl ganglioside (GM1), a lipid, a vitamin, a small molecule, a peptide, or a combination thereof. In some embodiments, said fragment is a lipid. For example, in some embodiments, said moiety is a palmitoyl group. In some embodiments, said moiety is a sterol, such as cholesterol. Additional hydrophobic moieties include, for example, phospholipids, vitamin D, vitamin E, squalene, and fatty acids. In another exemplary embodiment, the oligonucleotide cargo is conjugated to myristic acid or a derivative thereof (eg, a myristoylated oligonucleotide cargo). In some embodiments, the hydrophobic fragment is conjugated to the ends of the oligonucleotide cargo (i.e., "terminal modification"). In other embodiments, the hydrophobic fragment is conjugated to other regions of the oligonucleotide molecule.

[127] In some specific embodiments, a PTEN inhibitor is an siRNA molecule containing a nucleotide sequence selected from SEQ ID NOs: 10, 11, 2, 3, 4, and 5 and conjugated to a hydrophobic molecule, such as cholesterol. In some embodiments, the siRNA contains a nucleotide sequence whose target is a nucleotide sequence selected from SEQ ID NOs: 6 and 7 conjugated to a hydrophobic molecule, such as cholesterol. In some embodiments, the siRNA contains a nucleotide sequence whose target is a nucleotide sequence selected from SEQ ID NOs: 6 and 7 conjugated to a hydrophobic molecule, such as cholesterol. In one embodiment, the PTEN inhibitor is an siRNA of SEQ ID NO: 10 conjugated to cholesterol. In another embodiment, the PTEN inhibitor is a double-stranded siRNA containing the sequence of SEQ ID NO: 10 conjugated to cholesterol along with SEQ ID NO: 11. In specific embodiments, the siRNA contains a sequence complementary to a nucleic acid fragment encoding a PTEN protein, and inhibits its expression, wherein at least one of the miRNA sequences is conjugated to a hydrophobic molecule, such as cholesterol. In one embodiment, the PTEN protein comprises SEQ ID NO: 1.

[128] In specific embodiments, the oligonucleotide is stabilized by including one or more of the backbone modifications, carbohydrate modifications, and/or base modifications described herein, and is further conjugated to a hydrophobic moiety. For example, an oligonucleotide in certain embodiments may contain one or more of backbone modifications, carbohydrate modifications, and/or base modifications in up to at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or more nucleotides, and further conjugated to a hydrophobic moiety as described herein, e.g. conjugated to a sterol, GM1, lipid , vitamin, low molecular weight compound or peptide, or a combination thereof. In a typical embodiment, the oligonucleotide cargo is conjugated to a sterol, such as cholesterol. In yet another exemplary embodiment, the oligonucleotide cargo is conjugated to GM1. In yet another exemplary embodiment, the oligonucleotide cargo is conjugated to myristic acid or a derivative thereof.

[129] In some embodiments, the hydrophobically modified oligonucleotide may include a detectable label. Typical labels include fluorescent labels and/or radioactive labels. In embodiments where the hydrophobically modified oligonucleotides contain a fluorescent label, the detectable label may be, for example, Cy3. The addition of a detectable label to hydrophobically modified oligonucleotides can be used as a method for labeling exosomes and tracking their biodistribution. In other embodiments, a detectable label can be attached directly to exosomes, for example, by labeling an exosomal lipid and/or an exosomal peptide.

[130] Nucleic acids can be synthesized using any number of procedures known in the art. A number of automated nucleic acid synthesizers are commercially available for this purpose. In some embodiments, the nucleic acid cargo is a synthetic oligonucleotide. In other embodiments, nucleic acids can be generated using, for example, restriction enzymes, exonucleases, or endonucleases.

[131] According to any of the above embodiments, the pharmaceutical composition of the present invention further comprises lyase-chondroitinase ABC.

[132] Chondroitin-ABC-lyase (EC 4.2.2.4, chondroitinase, chondroitin-ABC-eliminase, chondroitinase ABC) is an enzyme systematically named chondroitin-ABC-lyase. This enzyme catalyzes the following chemical reaction:

Eliminating destruction of polysaccharides containing 1,4-beta-D-hexosaminyl and 1,3-beta-D-glucuronosyl or 1,3-alpha-L-iduronosyl bonds to disaccharides containing 4-deoxy-beta-D-glucose 4-enuronosyl groups. In one embodiment, the lyase-chondroitinase ABC is from Proteus vulgaris.

[133] Chondroitinase ABC can be presented as a protein itself or as part of a carrier, for example, in the form of particles (as described above in this document). In one embodiment, the pharmaceutical composition comprises extracellular vesicles loaded with an exogenous PTEN inhibitor and chondroitinase ABC as a protein. In yet another embodiment, the pharmaceutical composition comprises extracellular vesicles loaded with an exogenous PTEN inhibitor and chondroitinase ABC in the same type of extracellular vesicles (eg, exosomes derived from mesenchymal stem cells). In a further embodiment, the pharmaceutical composition comprises extracellular vesicles loaded with an exogenous PTEN inhibitor and other different extracellular vesicles loaded with chondroitinase ABC. In some embodiments, these different extracellular vesicles may be of the same type (e.g., exosomes derived from mesenchymal stem cells) or different types (exosomes derived from different cells, e.g., a PTEN inhibitor is contained in exosomes derived from mesenchymal stem cells). stem cells, and chondroitinase ABC is found in exosomes derived from dental pulp, or vice versa).

[134] In accordance with some implementation options, the pharmaceutical composition further contains additional therapeutic agents that can be used in the treatment of neurological disorders. Non-limiting examples of such active agents are gangliosides; antibiotics, neurotransmitters, neurohormones, toxins, molecules that promote axon development; and antimetabolites, small molecule agents, and precursors of neurotransmitter molecules, such as L-DOPA. In addition or alternatively, cells capable of alleviating at least one symptom of a neurological disease are additional therapeutic agents.

[135] As used herein, the term "pharmaceutical composition" refers to a composition containing membrane vesicles loaded with an exogenous PTEN inhibitor, in particular, extracellular vesicles, eg, exosomes, formulated with one or more pharmaceutically acceptable carriers.

[136] The composition of the pharmaceutical composition can be adjusted in accordance with the fields of application. In particular, the pharmaceutical composition can be formulated using a method known in the art to provide fast, continuous or delayed release of the active ingredient after administration to a mammal. For example, the composition may be any composition selected from patches, granules, lotions, ointments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, liquid extracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, alcohol solutions, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[137] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to various solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, lubricants, pH adjusting agents, buffering agents, action enhancers, humectants, solubilizing agents, surfactants, antioxidants, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing additional, additional or enhanced therapeutic functions, solid carriers or excipients such as lactose, starch or talc, or liquid carriers such as water, fatty oils or liquid paraffins. Other examples of carriers include culture media such as DMEM or RPMI; a hypothermic storage medium containing components that scavenge free radicals, provide pH buffering, oncotic/osmotic support, energy substrates, and ion concentrations that balance the intracellular state at low temperatures; as well as mixtures of organic solvents with water.

[138] Examples of excipients, without limitation, include albumin, plasma, serum and cerebrospinal fluid (CSF), antioxidants, such as N-acetylcysteine (NAC) or resveratrol. Typically, the pharmaceutical carrier retains the particle number and activity of the PTEN inhibitor (eg, the amount is not reduced by more than 90%) in the composition for at least 24 hours, at least 48 hours, or even at least 96 hours.

[139] According to any of the above embodiments, the pharmaceutical composition is formulated for administration by a route selected from intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral, and intracerebral routes of administration. In one embodiment, the pharmaceutical composition is formulated for intranasal administration. In some embodiments, such a pharmaceutical composition is in the form of a liquid solution, nasal drops, spray, metered spray. According to another implementation variant, the pharmaceutical composition is formulated for injection, for example, intralesional, intrathecal or intravenous injection. In such embodiments, the pharmaceutical composition is in the form of a sterile injectable solution.

[140] In some embodiments, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN), said extracellular vesicles being of cellular origin. In one embodiment, EVs are selected from exosomes, microvesicles, and combinations thereof. In another embodiment, the extracellular vesicles are exosomes. In some embodiments, the extracellular vesicles are derived from MSCs. In one embodiment, the PTEN inhibitor is an inhibitor of PTEN expression. In yet another embodiment, the PTEN expression inhibitor is an siRNA. In yet another embodiment, the PTEN expression inhibitor is shRNA. In some embodiments, the siRNA comprises a nucleotide sequence selected from SEQ ID NOs: 10, 11, 2, 3, 4, and 5. In one embodiment, the PTEN inhibitor is an siRNA containing the nucleic acid of SEQ ID NO: 10. According to still in one embodiment, the PTEN inhibitor is a double-stranded siRNA containing the sequence of SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the siRNA contains a nucleotide sequence that targets SEQ ID NO: 6. In other embodiments, the siRNA contains a nucleotide sequence whose target is SEQ ID NO: 7. In one embodiment, the siRNA contains a sequence complementary to a nucleic acid fragment encoding a PTEN protein and inhibits its expression. In one embodiment, the siRNA contains a sequence complementary to the nucleic acid fragment encoding SEQ ID NO: 1. In some embodiments, the siRNA is cholesterol-conjugated. In some embodiments, the present invention provides a pharmaceutical composition comprising extracellular vesicles selected from exosomes, microvesicles, or combinations thereof, loaded with a cholesterol-conjugated siRNA molecule capable of inhibiting PTEN expression. In such embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NOs: 2 and 3. In other such embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NOs: 4 and 5. In another such embodiment, the PTEN inhibitor is a double-stranded siRNA containing SEQ ID NO: 10 and SEQ ID NO: 11. In some embodiments, the siRNA contains a nucleotide sequence that targets SEQ ID NO: 6. In other embodiments, the siRNA contains a nucleotide sequence that targets SEQ ID NO: 7 In one embodiment, the siRNA contains a sequence complementary to the nucleic acid fragment encoding SEQ ID NO: 1. In some embodiments, the pharmaceutical composition is formulated for intranasal administration. According to another implementation variant, the pharmaceutical composition is formulated for intralesional administration. According to another implementation variant, the pharmaceutical agent is formulated for oral administration. In some embodiments, the pharmaceutical composition is co-administered with chondroitinase ABC.

[141] In one embodiment, the present invention provides a pharmaceutical composition comprising extracellular vesicles selected from exosomes, microvesicles, and combinations thereof, wherein said extracellular vesicles are loaded with siRNA or shRNA capable of inhibiting PTEN expression, and the pharmaceutical composition is formulated for intranasal or intrathecal administration. introductions. In one embodiment, the siRNA or shRNA is conjugated to cholesterol. In one embodiment, the PTEN inhibitor is a double-stranded siRNA comprising SEQ ID NO: 10, optionally conjugated to cholesterol, and SEQ ID NO: 11, or variant(s) thereof. In another embodiment, the siRNA or shRNA contains the nucleotide sequence of SEQ ID NO: 2 and/or 3, or a variant thereof. In a further embodiment, the siRNA or shRNA contains the nucleotide sequence of SEQ ID NO: 4 and/or 3 or a variant thereof.

[142] According to any of the above implementation options, the pharmaceutical composition according to the present invention can be used to treat a neurological disease, disorder, injury or condition. In one embodiment, the neurological condition is a spinal cord injury.

[143] In one embodiment, the membrane vesicles are liposomes. Thus, in one embodiment, the present invention provides a pharmaceutical composition comprising liposomes loaded with an exogenous phosphatase and tensin homologue inhibitor (PTEN). The term "liposome" refers to a microscopic closed vesicle containing an internal phase enclosed in a lipid bilayer. The liposome can be a small single membrane liposome, such as a small unilamellar vesicle (SUV), a large unilamellar liposome, such as a large unilamellar vesicle (LUV), an even larger unilamellar liposome, such as a giant monolamellar vesicle (GUV), a multilamellar liposome containing several concentric membranes, such as a multilamellar vesicle (MLV), or a liposome containing several irregular and non-concentric membranes, such as a multivesicular vesicle (MVV). In one embodiment, the liposomes are SUVs. According to another implementation variant, the liposomes are MLV.

[144] In one embodiment, the liposomes are SUVs. According to another implementation variant, the liposomes are MLV. In one embodiment, the liposomes contain a liposome-forming lipid selected from the group consisting of phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingophospholipids, distearoyl compounds, and any combination thereof. In yet another embodiment, the liposome-forming lipid is phosphatidylcholine. In one embodiment, the phosphatidylcholine is hydrogenated soy phosphatidylcholine (HSPC). In other embodiments, the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and said distearoyl compound is distearoyl glycol (DSG) or oxycarbonyl-3-amino-1,2-propanediol distearoyl ether ( D.S.). According to another embodiment, the liposomes contain a stabilizing polymer molecule, such as polyalkyl ether, polysialic acid, polylactic acid, and polyglycolic acid. In one embodiment, the liposomes contain PEG. In some embodiments, the liposomes further contain cholesterol.

[145] In one embodiment, the liposomes contain a PTEN inhibitor. All terms and embodiments relating to a PTEN inhibitor are used throughout this document. In one embodiment, the PTEN inhibitor is an inhibitor of PTEN expression. In some embodiments, the PTEN expression inhibitor is an RNA interference (RNAi) oligonucleotide. In one embodiment, the RNAi oligonucleotide is selected from siRNA and shRNA. In another embodiment, the siRNA contains a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4, and 5. In some embodiments, the siRNA contains a nucleotide sequence whose target is SEQ ID NO: 6. In other embodiments, implementation, the siRNA contains a nucleotide sequence whose target is SEQ ID NO: 7. According to an additional implementation variant, the specified oligonucleotide for RNAi, for example, siRNA or shRNA, is conjugated to a hydrophobic fragment. In yet another embodiment, the hydrophobic moiety is selected from the group consisting of a sterol, a ganglioside, a lipid, a vitamin, a fatty acid, a hydrophobic peptide, and combinations thereof. In one embodiment, the siRNA or siRNA is conjugated to cholesterol.

[146] In accordance with the idea of the present invention, the pharmaceutical composition according to the present invention is for use in the treatment of a neurological disease, disorder or condition in a subject. Thus, in some embodiments, a pharmaceutical composition comprising particles loaded with an exogenous phosphatase and tensin homolog inhibitor (PTEN) is for use in the treatment of a neurological disease, disorder, or condition in a subject in need thereof. In some embodiments, the particles are membrane vesicles. In some preferred embodiments, the membrane vesicles are extracellular vesicles.

[147] Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous phosphatase and tensin homologue inhibitor (PTEN) for use in the treatment of a neurological disease, disorder, or condition in a subject in need thereof.

[148] The term "neurological disease, disorder, or condition" refers to a disease, disorder, or condition of the brain, spinal cord, and/or the nerves that connect them.

[149] According to a specific implementation variant, the specified condition is caused by damage. In one embodiment, the injury is in the spinal cord, i. is a spinal cord injury (SCI). In another embodiment, the neurological disease, disorder, or condition is damage to neurons.

[150] The terms "spinal cord injury" and "SCI" are used interchangeably herein and refer to spinal cord injury. According to one implementation variant, the specified damage is the result of trauma. According to another implementation variant, the lesion or damage is the result of degeneration or disease. Depending on the area of damage to the spinal cord and nerve roots, symptoms can vary widely, for example, from pain to paralysis and urinary incontinence. Spinal cord injuries are described as varying levels of "incomplete damage" that can range from no impact on the patient to "complete" damage, meaning complete loss of function. Spinal cord injuries have a variety of causes, but are commonly associated with major trauma from traffic accidents, falls, sports injuries, and violence. Thus, according to one implementation variant, the PSM is selected from the complete and incomplete PSM. In some embodiments, spinal cord injury is selected from acute or chronic SCI. Spinal cord injury can be susceptible to secondary tissue damage, including but not limited to glial scarring, inhibition of myelin formation, demyelination, cell death, lack of neurotrophic support, ischemia, free radical generation, and excitotoxicity.

[151] Diseases of the spinal cord include, but are not limited to, autoimmune diseases (eg, multiple sclerosis), inflammatory diseases (eg, arachnoiditis), neurodegenerative diseases, polio, spina bifida, and tumors of the spinal cord.

[152] Subjects that can be treated in accordance with the principles of the present invention include mammalian subjects, such as humans, mice, rats, monkeys, dogs and cats. In one embodiment, the subject is a human.

[153] The term "treatment" of a condition or patient refers to taking steps to obtain beneficial or desirable outcomes, including clinical outcomes. Beneficial or desirable clinical results include improvement, cessation, significant inhibition, retardation, or reversal of the progression of a disease, condition, or disorder, significant improvement or alleviation of the clinical or aesthetic symptoms of a condition, substantial prevention of the onset of clinical or aesthetic symptoms of a disease, condition, or disorder, and protection from dangerous or annoying symptoms, but are not limited to them. Treatment also refers to doing one or more of the following: (a) lessening the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting the worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients previously suffering from said(m) disorder(s); and/or (e) limiting recurrence of symptoms in patients previously symptom-free of the disorder(s). In some embodiments, the term "treatment" includes nerve regeneration, axonal expansion, reduction of astrogliosis and microgliosis at the site of injury. In other embodiments, the term encompasses improvement in symptoms associated with a disease or condition. According to one implementation variant, the term "treatment" includes the improvement of motor parameters. In one embodiment, the improvement in motor parameters includes a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% improvement in motor parameters compared to an untreated subject. In some embodiments, treatment includes reducing astrogliosis and/or microgliosis at the site of injury. In one embodiment, the reduction in astrogliosis and/or microgliosis comprises a 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% reduction in astrogliosis and/or microgliosis compared to an untreated subject.

[154] The pharmaceutical composition according to the present invention can be administered by any known method. "Administration" of a substance, compound, or agent to a subject can be accomplished using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intranasally (eg, by inhalation), intrathecally (into the spinal canal or into the subarachnoid space), arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by mouth), intracerebral, and transdermally. (by absorption, for example, through the skin ducts). The compound or agent may also be appropriately administered with rechargeable or biodegradable polymeric articles or other articles such as patches and pumps or formulations that provide sustained, slow or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times and/or over one or more extended periods. In some embodiments, the composition is administered 1, 2, 3, 4, 5, or 6 times per day. In other embodiments, the composition is administered 1, 2, 3, 4, 5, or 6 times per month. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including prescribing activities. For example, herein, a doctor who instructs a patient to self-administer a drug or administer a drug to another person, and/or who gives a patient a prescription for a drug, administers the drug to the patient. According to one implementation variant, the pharmaceutical composition according to the present invention is administered intranasally. According to another implementation variant, the pharmaceutical composition according to the present invention is administered intralesionally. In one embodiment, the pharmaceutical composition is administered orally.

[155] In a specific embodiment, the neurological disease is a degenerative disease of the nervous system. In one embodiment, the degenerative disease of the nervous system is selected from Parkinson's disease, essential tremor, Huntington's disease, Alzheimer's disease, multiple sclerosis, ALS, and organic psychosis. In one embodiment, the disease is a memory disorder. In one embodiment, the disease is a developmental disorder, such as autism or schizophrenia. In another embodiment, the disease is a behavioral disorder, such as schizophrenia, attention deficit hyperactivity disorder, autism, Tourette's syndrome, obsessive compulsive disorder, and symptoms associated with neurobehavioral aspects. In a specific embodiment, the neurological disorder is an autism spectrum disorder (ASD).

[156] In any of the above embodiments, the present invention provides a pharmaceutical composition comprising membrane vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN) for use in the treatment of neuronal injury or injury in a subject. In some embodiments, the neuronal injury is spinal cord injury. According to another implementation variant, the damage is damage to the nerve cells of the brain. In one embodiment, the injury is brain injury. According to another implementation variant, the damage is caused by a neurodegenerative disease. In yet another embodiment, the neurodegenerative disease is selected from Parkinson's disease, essential tremor, Huntington's disease, Alzheimer's disease, multiple sclerosis, ALS, and organic psychosis.

[157] Thus, in accordance with some embodiments, the present invention provides a pharmaceutical composition comprising extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN) for use in the treatment of neuronal injury or injury in a subject, wherein the extracellular vesicles have extracellular origin.

[158] In one embodiment, the extracellular vesicles are exosomes. In another embodiment, the EVs are microvesicles. In one embodiment, EVs are selected from exosomes, microvesicles, and combinations thereof. In some embodiments, the extracellular vesicles are derived from MSCs. In one embodiment, the exogenous PTEN inhibitor is an inhibitor of PTEN expression. In yet another embodiment, the PTEN expression inhibitor is an siRNA or shRNA. In some embodiments, the siRNA contains a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4, and 5. In one embodiment, the siRNA is complementary or specific to a sequence selected from SEQ ID NO: 6 and 7. In one embodiment, the siRNA contains a sequence complementary to a nucleic acid fragment encoding a PTEN protein and inhibits its expression. In one embodiment, the PTEN contains SEQ ID NO: 1. In one embodiment, the PTEN inhibitor is an siRNA containing the nucleotide sequence 10. In another embodiment, the PTEN inhibitor is a double-stranded siRNA containing the sequences of SEQ ID NO: 10 and SEQ ID NO: 11. According to an additional implementation variant, the PTEN inhibitor is a shRNA containing a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4 and 5. According to one implementation variant, the shRNA is specific or complementary with respect to a sequence selected from SEQ ID NOs: 6 and 7. In some specific embodiments, the shRNA is cholesterol-conjugated.

[159] According to some embodiments, the present invention provides a pharmaceutical composition comprising EVs selected from exosomes, microvesicles, or combinations thereof, loaded with a cholesterol-conjugated miRNA oligonucleotide capable of inhibiting PTEN expression, for use in the treatment of spinal cord injury. . In such embodiments, the siRNA contains a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4, and 5. In one embodiment, the siRNA contains the sequences of SEQ ID NO: 10 and SEQ ID NO: 11. According to some embodiments, the pharmaceutical composition is administered intranasally. According to another implementation variant, the pharmaceutical composition is administered intralesionally. According to another implementation variant, the pharmaceutical composition is administered orally.

[160] According to any of the above embodiments, the treatment comprises the co-administration of the pharmaceutical composition of the present invention with lyase-chondroitinase ABC.

[161] In one embodiment, a polynucleotide encoding lyase-chondroitinase ABC is administered to a patient (via gene therapy). The lyase-chondroitinase ABC expression constructs are further described herein below. The polynucleotide encoding lyase-chondroitinase ABC can be loaded into particles. In yet another embodiment, the patient is administered the enzyme itself.

[162] In a particular embodiment, both the PTEN inhibitor and chondroitinase ABC are co-included in the same particles. In another embodiment, the PTEN inhibitor and chondroitinase ABC are loaded into the same type of particles (eg, exosomes derived from mesenchymal stem cells), but not into the same particle. In yet another embodiment, the PTEN inhibitor and chondroitinase ABC are loaded into different types of particles (eg, the PTEN inhibitor is contained in exosomes derived from mesenchymal stem cells and chondroitinase ABC is contained in exosomes derived from dental pulp, or vice versa).

[163] In some embodiments, the use further comprises co-administration with therapeutic agents that can be used to treat neurological disorders, eg, gangliosides; antibiotics, neurotransmitters, neurohormones, toxins, molecules that promote the development of axons; and small molecule antimetabolite agents and neurotransmitter molecule precursors such as L-DOPA, or with cells capable of alleviating at least one symptom of a neurological disease.

[164] In some embodiments, the particles loaded with an exogenous phosphatase and tensin homologue inhibitor (PTEN) are liposomes, as defined above. Thus, in some embodiments, the present invention provides a pharmaceutical composition comprising inhibitor-loaded exosomes (PTEN) for use in the treatment of a neurological disease, disorder, or condition in a subject in need thereof. In some embodiments, the condition is a spinal cord injury.

[165] In accordance with yet another aspect, the present invention provides a method of treating a lesion, injury, disease, or disorder of neurons in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of membrane vesicles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN) that results in the treatment of a lesion, injury, disease, or disorder. All provisions of the above embodiments and aspects also apply to this document. In some embodiments, the membrane vesicles are the extracellular vesicles described above.

[166] Membrane vesicles according to the present invention can be administered to an individual receiving treatment using various routes of administration as defined above, including transplant-based approaches, the nature of which depends on the site of implantation.

[167] The terms or phrases "transplantation", "cell replacement", or "transplantation" are used interchangeably herein and refer to the introduction of particles according to the present invention into a target tissue, for example, brain, gray matter, etc. The mesenchymal stem cells from which the particles are derived can be obtained from the body of the recipient (allogeneic cells) or from a non-allogeneic or xenogeneic donor.

[168] Membrane vesicles can be transplanted directly into the spinal cord (intrathecally), intravenously, directly into the brain, or combinations of the above can be used to allow the vesicles to reach the brain. In one embodiment, the implementation of the particles are delivered non-invasively, for example, intranasally. In addition, other routes of administration are contemplated, such as systemic administration.

[169] A typical dose of membrane vesicles (eg, exosomes) that can be administered (eg, intranasally) per treatment session can be from 1 x 10 ⁶ - 1 x 10 ²⁰ to or between 1 x 10 ⁹ - 1 x 10 ¹⁵ for person weighing 70 kg.

[170] The term "therapeutically effective amount" of membrane vesicles refers to an amount that, when administered to a subject, will provide the intended therapeutic effect, for example, in the treatment of neuronal injury, such as SCI. A full therapeutic effect is not necessarily achieved with the introduction of a single dose, but may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject depends, for example, on the size, health and age of the subject, the nature and extent of the cognitive impairment, as well as on the therapeutic agents or combination of therapeutic agents chosen to be administered, and on the route of administration. One skilled in the art can determine the effective amount for a given situation by routine experimentation.

[171] In some embodiments, membrane vesicles, such as EV, are administered intranasally, intralesionally, parenterally, topically, systemically, or orally.

[172] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro , cell culture or experimental animals.

[173] Data from these in vitro and cell culture assays, as well as animal studies, can be used to determine the dosage range for human use. Additional information can be obtained from clinical studies - see, for example, Salem HK et al., Stem Cells 2010; 28:585-96; and Uccelli et al. Lancet Neurol. 2011; 10:649-56).

[174] The dosage may vary depending on the dosage form used and the route of administration used. The exact composition, route of administration and dosage may be selected by the physician on an individual basis, taking into account the condition of the patient.

[175] For injection, formulations of the active ingredients of the pharmaceutical composition in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or buffered saline, as well as additional agents described herein above, can be used.

[176] The amount and interval of administration can be adjusted on an individual basis to achieve levels of the active ingredient sufficient to effectively treat a neurological disorder. The dosages necessary to achieve the desired effect depend on individual characteristics and the route of administration.

[177] Depending on the severity and sensitivity of the condition to be treated, the administration of the particles can be single or multiple, and the course of treatment can last from several days to several weeks or months, depending on the time to achieve improvements in the disease state.

[178] The amount of particles to be administered will, of course, depend on the individual being treated, the severity of the disease, the route of administration, the opinion of the attending physician, and so on. The dosage and timing of administration depend on careful and continuous monitoring of changes in the individual's condition.

[179] After the introduction of the particles can be tracked to ensure that they have reached the target area. This can be done, for example, using gold nanoparticles - see WO 2013/186735 A3.

[180] In some embodiments, the present invention provides extracellular vesicles loaded with an exogenous phosphatase and tensin homolog inhibitor (PTEN) for use in the manufacture of a medicament for the treatment of a neurological disease, disorder, or condition in a subject in need thereof. In some embodiments, the extracellular vesicles are isolated extracellular vesicles. In one embodiment, the neurological condition is a spinal cord injury.

[181] According to another aspect, the present invention provides bilayer phospholipid membrane lipid vesicles loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN). In some embodiments, the membrane vesicles are isolated extracellular vesicles.

[182] Thus, according to some embodiments, the present invention provides extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN), wherein the extracellular vesicles are of cellular origin. In one embodiment, the extracellular vesicles are selected from extracellular vesicles, liposomes, ectosomes, and transferosomes. In one embodiment, the extracellular vesicles are selected from exosomes, microvesicles, and combinations thereof. In one embodiment, the EVs are exosomes. In one embodiment, EVs are microvesicles. According to one implementation variant, the diameter of the microvesicles is from 100 to 1000 nm, from 120 to 800 nm, from 150 to 600 nm, from 200 to 400 nm. According to another implementation variant, the size of the microvesicles is from 100 to 300 nm or from 150 to 250 nm. In some embodiments, the EV diameter is 30 to 250 nm, or 50 to 200 nm. In some embodiments, the EV diameter is 70 to 170 nm, or 80 to 150 nm.

[183] In any of the above embodiments, EVs are of cellular origin. In one embodiment, EVs, such as exosomes, are derived from cells expressing mesenchymal markers. In one embodiment, the cells are adherent cells. In some embodiments, the cells are selected from mesenchymal stem cells, oral mucosal stem cells, and olfactory parietal cells. In some embodiments, the cells are mesenchymal stem cells (MSCs). In one embodiment, the mesenchymal stem cells are derived from a region selected from bone marrow, adipose tissue, umbilical cord, dental pulp, oral mucosa, peripheral blood, and amniotic fluid. In some embodiments, EV is derived from bone marrow-derived MSCs. According to another implementation variant, EV is obtained from MSCs derived from adipose tissue. In some of these embodiments, EVs are selected from exosomes, microvesicles, and combinations thereof. In some embodiments, cells express markers CD105, CD166, CD29, CD90, and CD73. In an additional embodiment, the cells express CD105, CD166, CD29, CD90 and CD73 and do not express CD34, CD45 and CD133. In some embodiments, the cells are selected from dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP), and dental follicle progenitor cells (DFPC).

[184] EVs may have at least one property of mesenchymal stem cells. The particle may have a biological property, such as biological activity. The particle may have any of the biological activities of the MSC. The particle may, for example, have the therapeutic or restorative activity of the MSC.

[185] In some embodiments, EVs are derived from cells expressing neural crest cell (NCC) markers. In one embodiment, the cells are neural crest cells. In some embodiments, the neural crest cells are cranial neural crest cells. In one embodiment, cranial neural crest cells are selected from dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP), and dental follicle progenitor cells ( DFPC). In another embodiment, the cells express the mesenchymal markers defined above.

[186] In any of the above embodiments, EVs are separated from cells. Therefore, in some embodiments, EVs are isolated EVs, eg, isolated exosomes and/or isolated microvesicles. In one embodiment, the ratio of EV to residual original cells is at least 2, 3, 4, 5, 6, 8, or 10 times higher, or, in some preferred embodiments, at least 50, 100, or 1000 times higher than in the original material. In some embodiments, EVs are cell-free EVs.

[187] EV can be obtained or allocated in several ways. Such a method may include isolation of EV from mesenchymal stem cells (MSC) or from neural crest cells (NCC). In some embodiments, the isolated extracellular vesicles do not contain cells.

[188] In any of the above embodiments, the extracellular vesicles contain a PTEN inhibitor as defined in any of the above aspects and embodiments. In one embodiment, the extracellular vesicles are loaded with a PTEN inhibitor. In one embodiment, isolated extracellular vesicles are loaded with said PTEN inhibitor. In one embodiment, the PTEN inhibitor inhibits or suppresses PTEN activity. According to another implementation variant, the PTEN inhibitor inhibits or suppresses the expression of PTEN.

[189] In one embodiment, the exogenous PTEN inhibitor is a protein or peptide. In another embodiment, the exogenous PTEN inhibitor is a small molecule compound. In some embodiments, the exogenous PTEN inhibitor is an inhibitor of PTEN expression. In one embodiment, the exogenous inhibitor of PTEN expression is a polynucleotide agent or an oligonucleotide agent. In another embodiment, the exogenous PTEN inhibitor is an oligonucleotide for RNAi. In one embodiment, the RNA interference oligonucleotide is an siRNA. In another embodiment, the RNA interference oligonucleotide is shRNA. In one embodiment, the PTEN inhibitor is an siRNA. In one embodiment, the siRNA comprises a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4, and 5. In one embodiment, the PTEN inhibitor is an siRNA with the sequence of SEQ ID NO: 10. In another embodiment, implementation, the PTEN inhibitor is a double-stranded siRNA containing the sequence of SEQ ID NO: 10 and SEQ ID NO: 11. According to one implementation variant, the siRNA contains a sequence complementary to a nucleic acid fragment encoding a PTEN protein and inhibits its expression. In one embodiment, the siRNA contains a sequence complementary to the nucleic acid fragment encoding SEQ ID NO: 1. Thus, in one embodiment, extracellular vesicles are loaded with siRNA containing the nucleotide sequence of SEQ ID NO: 2 and 3 or SEQ ID NO: 4 and 5. In some embodiments, the isolated extracellular vesicles are loaded with siRNA containing the nucleotide sequences of SEQ ID NOs: 10 and 11. In one embodiment, the siRNA contains a sequence that is a variant of the nucleotide sequence selected from SEQ ID NOs: 10, 11, 2 , 3, 4 and 5, and characterized by at least 80% sequence identity with the original sequence. In another embodiment, isolated extracellular vesicles are loaded with shRNA containing the nucleotide sequence of SEQ ID NOs: 10 and 11. In one embodiment, the shRNA contains the nucleotide sequences of SEQ ID NOs: 2 and 3. SEQ ID NOs: 4 and 5. In an additional embodiment, the siRNA contains a nucleic acid complementary to the sequences of SEQ ID NOs: 6 or 7. In another embodiment, the shRNA contains a sequence selected from SEQ ID NOs: 8 and 9. According to In some embodiments, the inhibitor oligonucleotide, such as siRNA or shRNA, contains a hydrophobic moiety. In some embodiments, the hydrophobic moiety is a sterol, ganglioside, lipid, vitamin, fatty acid, peptide, or a combination thereof. In one embodiment, the sterol is cholesterol.

[190] In one specific embodiment, the present invention provides isolated exosomes derived from mesenchymal stem cells and loaded with siRNA molecules that inhibit PTEN expression. In one embodiment, the siRNA contains, eg, is conjugated to, a cholesterol moiety. In one embodiment, the siRNA contains a nucleotide sequence selected from SEQ ID NOs: 10, 11, 2, 3, 4, and 5 and is conjugated to cholesterol. In another embodiment, the siRNA contains the nucleotide sequences of SEQ ID NO: 10 and 11. In some embodiments, the siRNA contains a nucleotide sequence that targets SEQ ID NO: 6 conjugated to cholesterol. In other embodiments, the siRNA contains a nucleotide sequence whose target is SEQ ID NO: 7 conjugated to cholesterol.

[191] In some embodiments, isolated extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN) can be used to treat a neurological disease, disorder, injury, or condition. In one embodiment, the neurological condition is a spinal cord injury. Thus, in one embodiment, the present invention provides extracellular vesicles for use in the treatment of spinal cord injury, said membrane vesicles being loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN). In a specific embodiment, the neurological disease is a degenerative disease of the nervous system. In one embodiment, the degenerative disease of the nervous system is selected from Parkinson's disease, essential tremor, Huntington's disease, Alzheimer's disease, multiple sclerosis, ALS, and organic psychosis. In one embodiment, the disease is a memory disorder. In one embodiment, the disease is a developmental disorder, such as autism or schizophrenia. In another embodiment, the disease is a behavioral disorder, such as schizophrenia, attention deficit hyperactivity disorder, autism, Tourette's syndrome, obsessive compulsive disorder, and symptoms associated with neurobehavioral aspects.

[192] In some embodiments, the extracellular vesicles further comprise chondroitinase ABC or a polynucleotide encoding it. In yet another embodiment, the PTEN inhibitor loaded EVs further comprise chondroitinase ABC or a polynucleotide encoding it.

[193] In some embodiments, the present invention provides a pharmaceutical composition comprising isolated extracellular vesicles according to any of the above embodiments. In some embodiments, the pharmaceutical composition is for use in the treatment of a neurological disease, disorder, injury, or condition. In one embodiment, the pharmaceutical composition is for use in the treatment of spinal cord injury.

[194] In some embodiments, the membrane vesicles are liposomes. In one embodiment, the liposomes are SUVs. According to another implementation variant, the liposomes are MLV. In one embodiment, the liposomes contain a liposome-forming lipid selected from the group consisting of phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingophospholipids, distearoyl compounds, and any combination thereof. In yet another embodiment, the liposome-forming lipid is phosphatidylcholine. In one embodiment, the phosphatidylcholine is hydrogenated soy phosphatidylcholine (HSPC). In other embodiments, the phosphatidylethanolamine is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and said distearoyl compound is distearoyl glycol (DSG) or oxycarbonyl-3-amino-1,2-propanediol distearoyl ether ( D.S.). According to another embodiment, the liposomes contain a stabilizing polymer molecule, such as polyalkyl ether, polysialic acid, polylactic acid, and polyglycolic acid. In one embodiment, the liposomes contain PEG. In some embodiments, the liposomes further contain cholesterol.

[195] According to another aspect, the present invention provides a method for obtaining extracellular vesicles loaded with an exogenous PTEN inhibitor, according to the present invention. In some embodiments, the extracellular vesicles are exosomes. In another embodiment, EVs are microvesicles. In a further embodiment, EVs are a combination of microvesicles and exosomes. Exosomes and microvesicles are as defined in any of the above embodiments and aspects. In some embodiments, EV is derived from cells expressing mesenchymal markers. In one embodiment, cells expressing mesenchymal markers are selected from mesenchymal stem cells, oral mucosal stem cells, and olfactory parietal cells. In yet another embodiment, EV is derived from neural crest cells, for example from cranial neural crest cells. In one embodiment, EVs, such as exosomes, are derived from mesenchymal stem cells. In some embodiments, MSCs are isolated from tissue selected from bone marrow, adipose tissue, dental pulp, oral mucosa, peripheral blood, and amniotic fluid. In some embodiments, mesenchymal stem cells typically express the following markers: CD105, CD166, CD29, CD90, and CD73, and do not express CD34, CD45, and CD133.

[196] The method may include isolation of EV from mesenchymal stem cell conditioned medium (MSC-CM) or from neural crest cell conditioned medium (NCC-CM). An EV can be isolated, for example, by separating from unrelated components based on any property of the EV. For example, EVs can be isolated based on their molecular weight, size, shape, composition, or biological activity.

[197] The conditioned medium can be filtered and/or concentrated during, before or after separation. For example, it can be filtered through a membrane, for example, limited by size or molecular weight. It can be filtered using shear stress or ultrafiltration.

[198] For example, filtration using a membrane with a suitable molecular weight or size restriction can be used, as described in the Molecular Weight Analysis section elsewhere herein.

[199] The conditioned medium, optionally filtered and/or concentrated, can be subjected to additional separation means, such as column chromatography. For example, high performance liquid chromatography (HPLC) can be used with different columns. The columns may be size-exclusion columns or binding columns.

[200] One or more of the properties or biological activities of the particle can be used to monitor the activity of the particle during fractionation of the medium conditioned by the cells. For example, light scattering, refractive index, dynamic light scattering, or UV and visible radiation detectors can be used to track particles. For example, therapeutic activity, such as cardioprotective activity, can be used to track activity during fractionation.

[201] The following paragraphs provide a specific, non-limiting example of a method for producing EVs, such as exosomes, from mesenchymal stem cells.

[202] EV can be obtained from mesenchymal stem cells by culturing mesenchymal stem cells in a conditioning medium. The medium may contain DMEM. DMEM may not contain phenol red. Insulin, transferrin or selenoprotein (ITS) or any combination thereof can be added to the medium. It may contain FGF2. It may contain PDGF AB. The concentration of FGF2 may be approximately 5 ng/ml. The concentration of PDGF AB may be approximately 5 ng/ml. The medium may contain glutamine-penicillin-streptomycin or α-mercaptoethanol, or any combination thereof.

[203] Cells can be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, for example, for 3 days. The conditioned medium can be obtained by separating the cells from the medium. The conditioned medium can be centrifuged, for example, at 500 g; it can be concentrated by membrane filtration. The membrane may include a membrane with a mass limit of >1000 kDa. The conditioned medium can be concentrated by about 50 times or more.

[204] The conditioned medium can be subjected to liquid chromatography, such as HPLC. The conditioned medium can be separated with a size limitation. Any size exclusion matrix can be used, such as Sepharose. As an example, a TSK Guard SWXL column, 6 x 40 mm, or a TSK G4000 SWXL, 7.8 x 300 mm gel can be used. The elution buffer may contain any physiological medium, such as saline. It may contain 20 mM phosphate buffer with 150 mM NaCl at pH 7.2. The chromatography system can be equilibrated at a flow rate of 0.5 ml/min. The elution mode may be isocratic. UV absorption at 220 nm can be used to monitor the progress of the elution. Fractions can be examined for dynamic light scattering (DLS) using a quasi-elastic second scattering (QELS) detector.

[205] Fractions in which dynamic light scattering is detected can be saved. For example, a fraction obtained by the general method described above and eluting with a retention time of 11-13 minutes, eg 12 minutes, has been found to exhibit dynamic light scattering. The R _h of the particles in this peak is approximately 45-55 nm. Such fractions include EVs from mesenchymal stem cells, such as exosomes.

[206] In one embodiment, the exogenous PTEN inhibitor is an inhibitor of PTEN expression. In one embodiment, the exogenous PTEN is an oligonucleotide for RNAi. In one embodiment, the RNA interference oligonucleotide is an siRNA. In another embodiment, the RNA interference oligonucleotide is shRNA. In some embodiments, the RNAi oligonucleotide is conjugated to a hydrophobic moiety. In one embodiment, said hydrophobic moiety is selected from a sterol, a ganglioside, a lipid, a vitamin, a fatty acid, a peptide, and combinations thereof. In some embodiments, the RNA interference oligonucleotide is conjugated to cholesterol. Thus, in one embodiment, an RNAi agent, such as siRNA, is conjugated to cholesterol.

[207] It should be appreciated that polynucleotides or oligonucleotides, such as siRNA or shRNA, can also be loaded directly into an EV. In one embodiment, direct loading of an RNAi oligonucleotide into an EV is performed by electroporation and/or using transfection agents. In alternative embodiments, loading is performed in the absence of electroporation and/or in the absence of transfection agents.

[208] In one embodiment, the EV is incubated with the RNAi oligonucleotide inhibitor for a period of time sufficient to allow loading of the particles with the nucleic acid inhibitor. The length of time sufficient to load the EV with the nucleic acid inhibitor cargo can be optimized for the specific type of cargo, and if the modification includes a hydrophobic modification, the type of modification. Typically, an incubation time of approximately 1 hour or less is sufficient to ensure efficient loading of nucleic acid particles. In many cases, the hydrophobically modified cargo is efficiently loaded into exosomes in a very short period of time, for example within 5 minutes. Accordingly, in some embodiments, effective loading occurs during an incubation time of 5 minutes or less, such as 1 to 5 minutes. In typical embodiments, effective loading occurs over an incubation period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, and so on. In other embodiments, effective loading may occur within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 24 hours, etc.

[209] EV loading with oligonucleotides is not strongly dependent on temperature. In typical embodiments, loading of exosomes occurs at a temperature equal to or approximately equal to 37°C. In other embodiments, EVs (eg, exosomes) can be loaded at or around room temperature. In other embodiments, the exosomes can be loaded at a temperature equal to or approximately equal to 4°C.

[210] In some embodiments, EVs can be loaded without the use of ultracentrifugation. According to another implementation variant, the download further includes ultracentrifugation. In some embodiments, the method of obtaining further includes the step of purifying or isolating loaded EVs. According to one implementation variant, the selection is carried out by centrifugation, for example, ultracentrifugation. According to another implementation variant, the selection is carried out by filtration. In one embodiment, the ratio of EV to residual parental cells after purification is at least 2, 3, 4, 5, 6, 8, or 10 times higher, or, in some preferred embodiments, at least 50, 100, or 1000 times higher than in the original material. In some embodiments, EVs are cell-free EVs.

[211] In some embodiments, the present invention provides a method for producing EVs, e.g., exosomes, the method comprising incubating the EV with cholesterol-conjugated RNAi oligonucleotides, e.g., siRNA or shRNA, for 0.5-5 hours at temperature from 25 to 42°C.

[212] According to one implementation variant, the method further includes the step of isolating loaded EVs using centrifugation, for example, ultracentrifugation. In some embodiments, another hydrophobic moiety can be used instead of cholesterol. In one embodiment, the RNAi oligonucleotide is an siRNA. In one embodiment, the siRNA contains the nucleotide sequence of SEQ ID NO: 2. In another embodiment, the siRNA contains the nucleotide sequence of SEQ ID NO: 3. In a certain embodiment, the siRNA contains the nucleotide sequence of SEQ ID NO: 4. In another embodiment, the siRNA contains the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the siRNA contains the nucleotide sequence of SEQ ID NO: 10. In other embodiments, the siRNA contains the nucleotide sequence of SEQ ID NO: 11. In an additional embodiment, the siRNA is complementary to the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the target of the siRNA is the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the RNAi oligonucleotide is shRNA. In one embodiment, the siRNA or shRNA contains the nucleotide sequences of SEQ ID NO: 2 and 3. In another embodiment, the siRNA or shRNA contains the nucleotide sequences of SEQ ID NO: 4 and 5. In a further embodiment, the siRNA or shRNA contains the nucleotide sequences SEQ ID NOs: 10 and 11. In an additional embodiment, the shRNA contains nucleotide sequences complementary to SEQ ID NOs: 6 or 7. In another embodiment, the shRNA contains a sequence selected from SEQ ID NOs: 8 and 9. In some embodiments, implementation, shRNA or siRNA contain a sequence complementary to the nucleic acid fragment encoding the PTEN protein and inhibit its expression. According to one implementation variant, PTEN contains the amino acid sequence of SEQ ID NO: 1.

[213] In some embodiments, more than 50% of the hydrophobically modified oligonucleotide cargo is loaded into exosomes using the methods described herein. Accordingly, in some embodiments, the hydrophobically modified oligonucleotide cargo is loaded into exosomes with an efficiency of 5 to 40%. Accordingly, in one embodiment, the hydrophobically modified oligonucleotide cargo is loaded into exosomes with an efficiency of 10 to 35%, 15 to 30%, or 20 to 25%. For example, a hydrophobically modified oligonucleotide cargo is loaded into exosomes with an efficiency of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more , or 50% or more. The methods described herein result in the incorporation of the hydrophobically modified oligonucleotide into all or nearly all of the exosomes being treated. For example, at least 80% of the exosomes incubated with the hydrophobically modified oligonucleotide are loaded with the oligonucleotide. In some embodiments, the hydrophobically modified oligonucleotide cargo is included in at least 90% of the exosomes incubated with the oligonucleotide. Thus, exosome populations can be obtained in which at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%, at least 97%, at least 98% or more of the exosomes are loaded with oligonucleotide cargo. In one embodiment, at least 99% of the exosomes are loaded with a hydrophobically modified oligonucleotide.

[214] EVs (e.g., exosomes) can be loaded with more than 500 oligonucleotide molecules per particle (e.g., exosome), e.g., at least 500, at least 600, at least 700, at least 800, at least 900 , at least 1000, at least 1200, at least 1500, at least 2000, at least 2500, at least 3000 or more oligonucleotides per exosome. In one embodiment, exosomes contain an average of about 500-3000 oligonucleotides per exosome. In yet another embodiment, exosomes contain an average of approximately 500-1000 oligonucleotides per exosome. In yet another embodiment, exosomes contain an average of approximately 1000-1500 oligonucleotides per exosome. In another embodiment, exosomes contain an average of approximately 1000-2000 oligonucleotides per exosome. In yet another embodiment, exosomes contain an average of approximately 1000-3000 oligonucleotides per exosome. In yet another embodiment, exosomes contain an average of approximately 3000 oligonucleotides per exosome. The amount of hydrophobically modified oligonucleotide cargo that can be loaded into exosomes allows the production of exosomes in which the hydrophobically modified oligonucleotide cargo occupies a significant portion of the exosome membrane. For example, EVs (e.g., exosomes) can be made in which the oligonucleotide cargo occupies approximately 1-10% of the surface area of the particle, e.g., approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8 %, 9% or 10% of the particle surface area. In some embodiments, the cargo oligonucleotide is an RNAi oligonucleotide, such as siRNA or shRNA. In other embodiments, the hydrophobically modified oligonucleotide is an RNAi oligonucleotide conjugated to a hydrophobic moiety, such as a cholesterol moiety.

[215] In some embodiments, the lipid bilayer phospholipid membrane vesicles are liposomes. In such embodiments, liposomes are loaded in the same manner as described for EV.

[216] In another embodiment, RVs loaded with an RNAi inhibitor of PTEN can be obtained from cells artificially loaded with an RNAi oligonucleotide or polynucleotide encoding and capable of expressing or generating said RNAi inhibitor in the cell. In this case, the polynucleotide/oligonucleotide agent is ligated into a nucleotide construct under the control of a cis-acting regulatory element (eg, a promoter) capable of directing the expression of the agent in a constitutive or inducible manner.

[217] Delivery of the nucleotide agent can be performed using a suitable carrier/gene delivery method (transfection, transduction, etc.). Optionally, an appropriate expression system is used. Examples of suitable constructs include pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto, all of which are available from Invitrogen Co but not limited to them.

[218] The expression construct can also be a virus. Examples of viral constructs include, but are not limited to, adenovirus vectors, retroviral vectors, vaccinia virus vectors, adeno-associated virus vectors, polyoma virus vectors, alphavirus vectors, rhabdovirus vectors, lentiviral vectors, and herpesvirus vectors.

[219] A viral construct, e.g., a retroviral construct, contains at least one transcriptional promoter/enhancer or locus-determining element(s), or other elements to control gene expression in other ways, for example, via alternative splicing, nuclear RNA export, or post-transcriptional messenger RNA modifications. Such vector constructs also contain a packaging signal, long terminal repeats (LTRs) or fragments thereof, and positive and negative strand primer binding sites suitable for the virus being used, if they are not already present in the viral construct. In addition, such a construct typically contains a signal sequence for the secretion of the peptide from the host cell into which it is placed. The signal sequence for this purpose is preferably a mammalian signal sequence or a signal sequence of peptide variants according to the present invention. The construct may also optionally include a polyadenylation control signal, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs typically contain a 5'-LTR, a tRNA binding site, a packaging signal, a start site for second strand DNA synthesis, and a 3'-LTR or fragments thereof.

[220] The dose of virus for infection is preferably at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or more PFU or viral particles.

[221] Double-stranded RNA can be synthesized by adding two opposite promoters to the ends of gene segments, with one promoter placed directly 5' to the gene and the opposite promoter placed directly 3' to the gene segment. The dsRNA can then be transcribed with the appropriate polymerase.

[222] In another embodiment, polynucleotide or oligonucleotide agents can be incubated with cells in culture, resulting in efficient uptake of the nucleic acid by the cells. For such an embodiment, it is preferred that the nucleic acid agents be hydrophobically modified, as further described herein below.

[223] Regardless of the method used to load the particles with the nucleotide agents described herein, the cells are then incubated for a time sufficient to produce EVs, such as exosomes. Exosomes isolated from the culture medium contain exosomes loaded with a nucleic acid molecule taken up, produced or expressed by the cells. Accordingly, in one embodiment, a method is provided for loading an EV with an oligonucleotide cargo, comprising incubating cells capable of producing EVs (e.g., exosomes) with the oligonucleotide for a time sufficient for the cells to internalize the oligonucleotide, culturing the cell for a time sufficient for secretion of the exosomes, and isolation of exosomes loaded with oligonucleotide from the culture medium.

[224] In some embodiments, the present invention provides isolated extracellular vesicles prepared according to any of the above embodiments. According to another implementation variant, the present invention provides a pharmaceutical composition containing extracellular vesicles obtained in accordance with any of the above implementation options. In some embodiments, such isolated extracellular vesicles and pharmaceutical compositions can be used in the treatment of neurological diseases or conditions, such as spinal cord injury.

[225] The particles or pharmaceutical composition according to the present invention, in at least some embodiments, can be pre-packaged into dosage forms in the form of a syringe, ready for use. As part of the labeling of the syringe, you can specify the name of the particles and their source. The marking may also contain information relating to the function of the particles. The syringe can be packaged in a package that also contains particle information as part of the labeling.

[226] The terms "comprising", "include(s)", "having", "has", and "comprises(at)" are used interchangeably herein and have the meaning of "at least partially consisting of". When interpreting each statement in this document that includes the term "comprising", there may also be other features than those preceded by this term. Related terms such as "comprise" and "comprises" should be interpreted in the same way. The terms "have", "has", "having" and "comprising" may also include the meanings of "consisting of" and "consisting essentially of" and may be replaced by these terms. The term "consisting of" excludes any component, step, or procedure not specifically identified or listed. The term "consisting essentially of" means that the composition or component may contain additional ingredients, but only if these additional ingredients do not lead to a significant change in the basic and new characteristics of the claimed compositions or methods.

[227] As used herein, the term "approximately" when used in relation to a measurable quantity, such as quantity, time, etc., means that it includes variability in the range of +/- 10% or +/- 5%, + // 1% or even +/- 0.1% of the specified value.

[228] As used herein, singular forms include plural forms, unless otherwise clearly indicated by the context. For example, the term "compound" or "at least one compound" can include a variety of compounds, including mixtures thereof.

[229] In the present application, various embodiments of the present invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the spirit of the invention. Accordingly, the description of a range should be considered to include all possible specific subranges as well as individual numerical values within that range. For example, a description of a range, such as 1 to 6, should be considered as a description of specific subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers in this range, such as 1, 2, 3, 4, 5, and 6. This does not depend on the latitude of the range.

[230] Any reference to a numerical range herein is intended to include any referenced number (fractional or integer) within the specified range. The phrases "varies between" the first specified number and the second specified number and "ranges from" the first specified number "to" the second specified number are used interchangeably herein and are intended to include the first and second specified numbers, as well as all fractional and integer numbers. between them.

[231] As used herein, the term “method” refers to methods, means, techniques, and procedures for accomplishing a given task, including methods, means, techniques, and procedures known to, or derivable from, known methods, means, techniques, and procedures by practicing chemists, but not limited to pharmacologists, biologists, biochemists and medical specialists.

[232] It should be understood that certain features of the invention, which are described in the context of separate embodiments for clarity, may also be presented in combination in one embodiment. Conversely, various features of the invention, which for brevity are described in the context of one embodiment, may also be presented separately or in any suitable subcombination or in any other suitable described embodiment of the invention. Certain features described in the context of various implementations should not be considered important features of those implementations unless the implementation operates without those elements.

[233] The various embodiments and aspects of the present invention described above and in the following section of the claims find experimental support in the following examples.

[234] According to another aspect, the present invention provides neural crest cell-derived exosomes for use in the treatment of spinal cord injury.

EXAMPLES

[235] This section provides references to the following examples, which, together with the above description, illustrate some embodiments of the invention in a non-limiting manner.

[236] As a rule, the nomenclature and laboratory procedures used herein include molecular, biochemical, microbiological and recombinant DNA technology techniques. Such techniques are described in detail in the literature.

[237] Materials and methods

[238] Obtaining mesenchymal stem cells

[239] Human bone marrow MSCs were purchased from Lonza (Basel, Switzerland). Cells were cultured and propagated as described previously. Before exosome harvesting, cells were cultured in exosome-free platelets and medium was harvested 3 days later. MSC-exo were labeled with PKH26 (Sigma) for 5 minutes and washed by ultracentrifugation (100,000 g, 2 hours, 4°C). The pellet was resuspended in 200 µl PBS.

[240] Exosome Purification Protocol

[241] Human MSC was purchased from Lonza (Basel, Switzerland). Cells were cultured and expanded. The cells were cultured with exosome-free platelet lysate (Rabin Medical Center, Israel) and the medium was collected 3 days later. Exosomes were purified using a standard differential centrifugation protocol, which included isolation of culture fluid and centrifugation for 10 min at 300 g. The supernatant was collected and centrifuged for 10 minutes at 2000 g and then re-centrifuged for 30 minutes at 10000 g. Then the supernatant was passed through a filter with a pore size of 0.22 μm and centrifuged for 70 min at 100,000 g. The precipitate containing exosomes and proteins was washed with PBS and then centrifuged for 70 min at 100,000 g. The pellet was resuspended in 200 µl of sterile PBS. All centrifugation steps were performed at 4°C. Exosomes were characterized by NanoSight technology, electron microscopy and Western blotting using calnexin as a negative marker and CD9 and CD81 as a positive marker.

[242] Fluorescent labeling of exosomes from mesenchymal stem cells (MSC-Exo)

[243] MSC-Exo were labeled with PKH26/PKH67 (Sigma) for 5 minutes and then washed with PBS using ultracentrifugation (100,000 g, 2 hours, 4°C). The pellet was resuspended in 200 µl PBS. For PTX experiments, MSC-Exo was suspended with 5 µl of PTX for 10 minutes at 37°C, then labeled with PKH26 and centrifuged at 100,000 g for 2 h. and the same rat (n = 3).

[244] Synthesis and conjugation of gold nanoparticles (GNP)

[245] To 30 ml of DDW was added a mixture of 3.75 ml of linoleic acid (18%), oleic acid (65%), palmitic acid (16%) and ethanol (1%), 200 mg of NaOH and 15 ml of ethanol. The solution was stirred for 5 minutes. Then fifty milligrams of HAuCl ₄ was added with stirring, followed by 5 ml of ascorbic acid (0.05 M) and stirred for another minute. Then a PEG7 solution (2.26 x 10 ^-3 g) was added to the GNP solution and the mixture was stirred for 1 hour. The pH was then adjusted to 9 using NaOH and the solution was stirred for another hour. After adding 80 ml of n-hexane, the solution was stirred for 1 hour. Then the pH of the resulting mixture was adjusted to 7 using a solution of HCl (37%). The mixture was placed in a funnel to separate the phases (organic and aqueous). The aqueous phase was evaporated in vacuo. An excess of carbodiimide (1.87 x 10 ^-3 g) and N-hydroxysuccinimide (Thermo Fisher Scientific, Inc., Rockford, IL, USA) (2.12 x 10 ^-3 g) were then added to the solution, followed by the addition of 2GF ( Sigma-Aldrich, Israel Ltd.) (1.75 x 10 ^-3 g). A total of 2.8 x 10 ¹⁰ exosomes (200 μl exosomes in 1 ml saline) were incubated at 37° C. for 3 h with glucose-coated GNPs (35 mg/ml, 100 μl). Then the exosomes were centrifuged for 2 h (100,000 g, 4°C).

[246] GNP Characterization Study

[247] Transmission electron microscopy (JEM-1400, JEOL) was used to measure the size and shape of GNPs, and further characterization was performed using UV-visible spectroscopy (UV-1650 PC; Shimadzu Corporation, Kyoto, Japan), ζ -potential (ZetaSizer 3000HS; Malvern Instruments, Malvern, UK) and dynamic light scattering.

[248] GNP Quantifications

[249] Flame gas was used to determine the amount of gold in several major organs (heart, lungs, kidneys, and spleen). atomic absorption spectroscopy (SpectrAA 140; Agilent Technologies, Santa Clara, CA, USA). The excised tissues were dissolved in 1 ml of aqua regia (a 1:3 mixture of nitric and hydrochloric acids), allowed to evaporate, and then diluted to a total volume of 4 ml. After filtering the samples through syringe filters with a pore size of 0.45 nm, the gold concentration was determined from calibration curves constructed using a solution with known concentrations of gold. Inductively coupled plasma (ICP) spectrometry was used to determine the amount of gold in the brain and spinal cord lesions. Fresh tissues were extracted and burned at 550°C for 5 h. Then 5 ml of 1% HNO was added to the molten tissues.₃ and filtered through filters with a pore size of 0.45 μm. The concentrations of gold in the samples were determined by correlation with the standard curve for gold.

[250] Exosome Loading

[251] Exosomes (40 μl, approximately 10 ¹⁰ exosomes) were loaded with 0.1 nmol cy3-MAPK-siRNA (Advirna Company) in an EPPENDORF® tube and incubated for 1-3 hours at 37°C. The suspension was then subjected to Nanosight analysis. The concentration ratio of fluorescent and non-fluorescent exosomes was determined as the loading efficiency.

[252] For in vivo experiments, 40 µl of exosomes were loaded with 0.1 nmol PTEN-miRNA (Advirna Company) for 2-3 hours at 37° C. prior to intranasal administration to SCI rats. Exosomes loaded with PTEN miRNAs were called ExoPTEN.

[253] Axonal outgrowth in dorsal root ganglion (DRG) neurons

[254] dorsal root ganglia at the level of the thoracic and lumbar spine of adult Sprague-Dawley rats (200-250 g) were dissected and immersed in Hank's ice-cold balanced salt solution (HBSS). DRGs were washed with HBSS, transferred to a small dish containing 4 ml 2.5 mg/ml collagenase type II (Worthington), cut into small pieces with a scalpel, and incubated at 37°C for 40 min. The solution was centrifuged at 200 g for 3 min to remove collagenase, resuspended in 2 ml culture medium containing F12 culture medium (Gibco, 11765), 10% fetal bovine serum (Gibco), 100 U/ml penicillin (Biological Industries, Beit ha-Emek, Israel), 100 µg/ml streptomycin, minced ~20 times by passing through 1 ml pipette tips. Cells were seeded on a 15 mm coverslip pre-coated with laminin (Sigma, L2020) inside a 12-well plate at a density of 5 x 10 ⁴ per well for 5 hours. The DRG medium was then changed to one containing normal DRG medium, 40 μl exosomes, 0.1 nmol non-specific control (NTC) siRNA, 0.1 nmol PTEN siRNA, or 0.1 nmol PTEN siRNA loaded into 40 μl exosomes.

[255] The siRNA target sequence of PTEN is shown in SEQ ID NO: 6 (5'-GAGTTCTTCCACAAAACAGAA-3'). To inhibit PTEN expression, a double-stranded siRNA containing a single strand was used, containing the cholesterol-conjugated nucleotide sequence 5'-GAGUUCUUCCACAAAACAGAA-3' (SEQ ID NO: 10) and the nucleotide sequence 5'-UUCUGUUUGUGGAAGAACUC-3' (SEQ ID NO: 11 ).

[256] 24 hours after seeding, cells were fixed for 10 minutes with 4% paraformaldehyde (PFA), washed three times for 5 minutes with PBS, permeabilized for 10 minutes with 0.3% Triton X-100, washed three times for 5 minutes with PBS, blocked for 2 hours in 5% bovine serum and incubated overnight with mouse anti-tubulin antibody (Promega, 1:500) in 5% bovine serum at 4°C. Samples were then incubated with donkey anti-mouse labeled with Alexa488 (Invitrogen, 1:800) and DAPI (1:1000) for 3 hours. After three washes with PBS, samples were mounted and examined by confocal microscopy (LSM700). The images were processed using the IMARIS software (version 8.20) to quantify the level of axon branches, the maximum level of branches, the number of axons, and the total length of axons. The parameters were set as follows and applied to each image: maximum diameter 60 µm, minimum diameter 0.6 µm, starting point threshold from 10 µm to 60 µm, starting point threshold 10 µm, removal of origin points around the origin points with a spherical region diameter of 24 µm , the maximum allowable gap length is 60 µm.

[257] Ex vivo CT of the brain and spinal cord:

[258] Samples of the brain and spinal cord were collected and placed in 4% paraformaldehyde for 3 days for fixation, then impregnated with a non-ionic iodinated contrast agent (iopamidol, Bayer Schering Pharma, Japan) at a concentration of 150 mg/ml, diluted with 7.5% paraformaldehyde, at 4°C for 7 days to distinguish between gray and white matter. Prior to CT imaging, the samples were removed from the solution, blotted dry, and placed in a sample holder for imaging. The sample holder was sealed with a plastic film to prevent dehydration. Micro-CT scans of ex vivo samples were performed using a micro-CT scanner (Skyscan High Resolution Model 1176) with a nominal resolution of 35 µm, a 0.2 mm aluminum filter, and a tube voltage of 45 kV. Reconstruction was performed using a modified Feldkamp algorithm using SkyScanNRecon software and GPU acceleration. Ring artifact removal, Gaussian smoothing (3%), and beam gain correction (25%) were used. Three-dimensional (3D) volumetric images were generated using the RGBA transfer function in SkyScan CT-Volume (“CTVol”) and SkyScan CT-Voxel (“CTVox”) software.

[259] Immunofluorescence analysis

[260] The spinal cord was dissected, fixed in 4% PFA overnight, treated with cryoprotectant (30% sucrose solution) overnight, placed in an optimum cutting temperature (OCT) composite, and cut longitudinally (20 µm) using a cryostat ( Leica CM1850, Germany). Sections were permeated in 0.5% Tween, blocked with 5% bovine serum, and then incubated with rabbit anti-tubulin (Abcam, 1:500), rabbit anti-GFAP (1:1000, Millipore), mouse anti-CD11b (1 :500, BioRad) or mouse anti-CD31 antibody (1:50, BD) overnight at 4°C. Sections were then incubated with goat anti-rabbit labeled with Alexa647 (Invitrogen, 1:800) and DAPI (1:1000) for 3 hours, covered with coverslips, and visualized with a confocal microscope (LSM700).

[261] Spinal Cord Injury Model

[262] All animal experiments were performed in strict accordance with protocols approved by the Technion-Israel Institute of Technology. The animals were randomized by the experimenter unaware of the treatment. Adult female Sprague-Dawley rats were anesthetized with a mixture of xylazine (10-15 mg/kg) and ketamine (60-90 mg/kg) and a maintenance dose of 1-2% isoflurane (Harvard Apparatus, USA) during surgery. After laminectomy at the level of 9-11 thoracic vertebrae, the spinal cord was completely dissected at the T10 level using microscissors (Kent Scientific, USA). The rostral and caudal stumps were raised to ensure complete dissection, and a hook (Kent Scientific, USA) was inserted into the resulting gap in a circular motion to confirm the absence of remaining fibers in the lower part of the spinal canal. The groups evaluated in this study were designated as “control dissection” (n = 15), “intranasal administration of control exosomes” (n = 10), “intranasal administration of ExoPTEN” (n = 7), “intralesional administration of PTEN-siRNA” ( n = 3) and "intralesional injection of ExoPTEN" (n = 4). The muscle layers and skin were then sutured and the rats were placed in temperature controlled incubation chambers. For intranasal treatment, rats were intranasally injected with 40 μl of saline, 40 μl of exosomes, or 40 μl of ExoPTEN 2-3 hours after surgery and every 24 hours for 5 days. For intralesional treatment, rats were injected with 40 μl of PTEN-miRNA (0.1 nmol) or 40 μl of ExoPTEN once upon inflicting spinal cord injury. Bladder massage was performed twice a day until the bladder function was restored. Antibiotics (cefazolin, 25 mg/kg, twice daily) were administered daily for 1 week. Buprenorphine (Bayer) was administered at a dose of 0.01-0.05 mg/kg before and 3 days after surgery. Cyclosporine (10 mg/kg/day) (Novartis) was administered to all rats for 1 week.

[263] Behavior Analysis

[264] Motor recovery was assessed by an experimenter unaware of the treatment using a method based on the Basso, Beatty and Bresnahan (BBB) motor function scale. Measurements were taken 2-3 days after implantation and then once every 7 days. All measurements were taken at the same time of day to avoid circadian variability. Baseline BBB was set as the value recorded in the first analysis after surgery. The weekly score was the minimum score received during each calendar week. Sensory recovery was assessed using the von Frey filament test. Threads (Bioseb) with a gradient of bending forces were applied to the paws to stimulate nociceptive reactions (rapid withdrawal of the paw from the stimulus). The withdrawal threshold was set as the minimum effort that produces a positive response in at least 2 of 3 tests with an interval of at least 30 seconds between tests.

[265] MRI

[266] MRI was performed with a 9.4 T scanner (Bruker Biospec, Ettlingen, Germany) using a cylindrical volume coil (86 mm inner diameter) for signal excitation and a single-channel surface coil (20 mm diameter) for signal reception. The animals were anesthetized (0.5-1.5% isoflurane) and received oxygen (0.5 l/min). Breathing was monitored during imaging (Small Animal Instruments, Stony Brook, New York, NY, USA) and body temperature was maintained with thermostatically controlled circulating hot water. Prior to DTI imaging, sagittal and axial T2-weighted anatomical scans were obtained using the RARE sequence to determine the slice geometry of the DTI scan. The following RARE registration parameters were used: thickness 0.8 mm, field of view = 3x3 cm, matrix size = 192x192, (spatial resolution = 156x156 μm ² ), repetition/echo time (TR/TE) = 1200/16.22 ms, 4 average values. DTI imaging was performed using an EPI-DTI sequence with the following parameters: 30 non-collinear gradient directions, 3 images A ₀ , b = 1000 s/mm ² , diff. duration (δ) = 2.6, diff. separation (Δ) = 11, 2 averages, thickness 0.8 mm, field of view = 3x3 cm, matrix size = 192x192, (spatial resolution = 156x156 µm ² ). DTI calculations and fiber tracking were performed using ExploreDTI software (Leemans et al., 2009). The resulting tensors were spectrally decomposed into their own components. Eigenvalues were used to calculate fractional anisotropy (FA) maps. The areas of interest (AR) of the spinal cord (white and gray matter) were manually segmented in each section. Tractography was applied using deterministic (smooth) tracking of a fiber terminating in voxels with FA below 0.2 or after reorienting the tract by more than 30° (Basser et al. mm. Fibers that passed through manually selected areas of interest were plotted on a graph in the form of directions.

[267] Electrophysiology

[268] At week 8, an electrophysiological study was performed to record the propagation of the neural signal through the lesion. After anesthesia with ketamine/xylazine, the rats were placed in a stereotaxic apparatus and a midline incision was made on the scalp. The skull was exposed and two helical electrical stimulation electrodes were implanted 2 mm to the right of the midline, at -1.0 mm and +4.0 mm anterior and posterior to the bregma, respectively. Screw electrodes were connected to the output terminals of the stimulator. The sciatic nerve at the back of the leg was exposed and two silver wire hook electrodes were inserted. Another wire was inserted into the ball of the foot and used as a ground electrode. The amplified signals were bandpass filtered from 0.1 to 3 kHz (7P511 AC wideband preamplifier with 7DA power preamplifier, Grass Technologies, Warwick, RI, USA), digitized (NI USB-6341 analog-to-digital converter, National Instruments), recorded at 10 kHz and stored on a personal computer with the WinWCP software package (courtesy of Dr. John Dempster, University of Strathclyde, UK). The intensity of stimulation was chosen according to the contraction of the hind limbs and the appearance of a reliable total action potential of the sciatic nerve (CAP) in the first animal and was maintained throughout the experiment.

[269] Western blotting

[270] Affected spinal cord segments and liver were placed in RIPA solution ((1% sodium deoxycholate (DOC) (Sigma), 1% 100x-Triton (Biolab), 50 mM Tris-HCl (Sigma), 150 mM NaCl (Sigma) , 5 mM EDTA (Sigma)), containing a mixture of protease inhibitors (100 mM PMSF, 2 mg/ml leupeptin, 50 mM Na ortavanadate, 50 mM aprotinin, Sigma). 12% SDS-PAGE, and then transferred to a nitrocellulose membrane for 90 minutes After blocking with 2% BSA in saline with Tris buffer and 0.1% Tween20 (TBST) for 3 hours, the membranes were incubated overnight with antibody against PTEN , 1:1000 (mouse monoclonal antibody; catalog # 9556, Cell Signaling) or anti-β-actin antibody, 1:1000 (rabbit monoclonal antibody; catalog # 8457, Cell Signaling) as loading control in 2% BSA in TBST The following day, after three washes with TBST, the membranes were incubated with anti-rabbit IgG labeled with HRP (1:5000, NA934V, GE Healthcare) or anti-mouse IgG antibody labeled with HRP (1:5000, NA931V, GE Healthcare), for 2 hours at room temperature. Signals were developed by exposing the blot to enhanced chemiluminescent reagents (catalog # 1705061, BioRad) and then exposure to a LAS-3000 imaging system (FujiFilm).

[271] Real-time qPCR

[272] Total RNA was isolated from damaged spinal cord and liver tissues using the Qiagen RNeasy mini kit, first strand DNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Real-time quantitative PCR was performed on a QuantStudio 12K Flex Real-Time PCR System. Primers included PTEN (Thermo Fisher, assay ID: Rn00477208) or GAPDH (Thermo Fisher, assay ID: Rn01775763-g1). The following amplification reaction conditions were used: 95°C for 20 s, then 40 cycles at 95°C for 3 s, 60°C for 30 s in 10 μl of the reaction mixture in triplicate. Relative expression levels were determined using the ΔΔCt method, in which the gene of interest was standardized for GAPDH expression.

[273] Neuroanatomical Tracking

[274] To track corticospinal axons, rats were subjected to anterograde tracking using biotinylated dextranamine (BDA) at week 6. Under ketamine and xylazine anesthesia as described above, rats were placed in a stereotaxic frame. The hair on the scalp was shaved, the shaved area was treated with ethanol, and small holes were drilled in the skull above the sensorimotor cortex. A Hamilton microsyringe with an extended glass micropipette was used to inject 1 µl of 10% BDA (MW 10,000, D1956, Molecular Probes) into 8 areas per hemisphere for 2 minutes per area with 2-minute intervals between injections using the following coordinates: AP + 1.0, ML ± 2.0; AP 0, ML ± 1.5; AP -1.0, ML ± 1.5; AP -2.0, ML ± 1.5; DV 1.5 mm. After all injections were completed, the scalp was sutured and the rats were left in the recovery chamber. At week 8, rats were deeply anesthetized with ketamine and xylazine and transcardially perfused with 4% PFA. Injured segments of the spinal cord were collected, exposed to cryoprotectant (30% sucrose), included in OCT, and longitudinal sections of 20 μm were obtained. Sections were washed three times with PBS and 0.1% Triton X-100, incubated overnight at 4°C with streptavidin conjugated to Alexa Fluor594 (1:500, Invitrogen, S32356), washed three times with PBS, mounted and covered with a coverslip.

[275] Statistical Analysis

[276] Statistical analysis was performed using MATLAB/Prism 5 software (USA). Two-tailed unpaired Student's t-test was used to compare between the two groups. Unless otherwise indicated, the remaining data were analyzed using one-way or two-way ANOVA with Tukey's post hoc multiple comparison. All pooled data were presented as "mean ± standard error of the mean". Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[277] Example 1 - MSC-Exo, when administered intranasally, crosses the BBB and migrates to the spinal cord

[278] The average size of exosomes isolated from exosomes of mesenchymal stem cells (MSC-Exo) was 111 ± 64 nm, and the concentration was 40.43 × 10 ⁸ particles/ml (Fig. S1D-E). MSC-Exo, visualized using cryo-TEM, showed a typical spherical shape. To test the ability of MSC-Exo to pass through the BBB, MSC-Exo was labeled with gold nanoparticles (GNP) as described in (Betzer et al., 2017, ACS Nano 11, 10883-10893). Cryo-TEM imaging of MSC-Exo loaded with GNP showed uptake of GNP by exosomes ( Fig. 1 ). To test the ability of MSC-Exo to cross the blood-brain barrier after intranasal (IN) administration, GNP-loaded MSC-Exo was intranasally administered three hours after complete spinal cord dissection. Micro-CT scan 24 hours after administration showed significant accumulation of GNP in the area of spinal cord injury, but not in the brain ( Fig. 2 , top panel). In contrast, in healthy control animals, GNPs were located mainly in the brain and olfactory bulbs ( Fig. 2 , bottom panel). Evaluations by inductively coupled plasma (ICP) spectrometry confirmed that the amount of GNP in the region of the T10 segment of the spine was significantly higher in injured rats compared to healthy controls ( Fig. 3A , one-way analysis of variance, F(3, 8) = 27, 5, p < 0.001, Tukey's test). Similarly, traumatized rats accumulated significantly more PKH26 (red lipophilic dye) labeled exosomes in the region of the T10 segment of the spine after intranasal injection compared to healthy rats ( Fig. 4 , t(14) = 3.21, p < 0 .01). Qualitative micro-CT and quantitative ICP analysis in the CNS, along with immunofluorescent staining of spinal cord lesions, showed that MSC-Exo crosses the blood-brain barrier and is homing to spinal cord injury. Surprisingly, MSC-Exo was significantly more common in the area of injury in rats with spinal cord injury, while in healthy rats they predominated in the brain.

[279] In order to study the biodistribution of MSC-Exo after intranasal administration, flame atomic absorption spectroscopy (FAAS) was used to quantify GNP in major organs (liver, lung, heart, kidney, and spleen) 24 hours after administration. Significant differences between the levels of MSC-Exo in healthy and injured rats in the lungs, heart and spleen were not found. However, higher levels were found in the liver and kidneys of healthy rats compared to injured rats, indicating faster clearance of MSC-Exo in intact rats ( Fig. 4 , right panel, one-way analysis of variance, F(9, 10) = 12.92, p < 0.001, Sidak's multiple comparison test).

[280] In addition, the authors demonstrated that blocking the chemokine receptors of exosomes labeled with PKH26 (red dye) with pertussis toxin dramatically reduces the ability of MSC-Exo to migrate to the site of injury. To test for the existence of preferential uptake of MSC-Exo by specific cell types in the area of spinal cord injury, neurons, astrocytes, and microglia in damaged segments were subjected to immunochemical staining 24 hours after IN administration of PKH26-Exo to rats 2-3 hours after injury. MSC-Exo was found to co-localize mainly in neurons and to a much lesser extent in astrocytes and activated microglia ( Fig. 5 , one-way analysis of variance, F(2, 13) = 11.59, p > 0.01, test Tukey).

[281] Example 2 PTEN siRNA loaded into MSC-Exo promotes sustained growth of DRG neurons in vitro

[282] MSC-Exo was loaded with cholesterol-conjugated non-coding cy3-MAPK siRNA as described in the Materials and Methods section. Nanosight analysis demonstrated efficient loading of cy3-MAPK-siRNA (33.64%) ( Fig. 6 ).

[283] To test the ability to regulate axonal growth by inhibiting PTEN by siRNA-containing MSC-Exo, self-delivering PTEN siRNA was loaded into MSC-Exo, hereinafter referred to as ExoPTEN, for delivery to dorsal root ganglion (DRG) neurons in culture. One day after ExoPTEN treatment, DRG neurons were characterized by more complex, branched, and elongated morphology compared to neurons treated with medium alone, non-specific control siRNA (NTC-siRNA), MSC-Exo, or PTEN-siRNA alone ( Fig. 7 ). At medium branch levels, ExoPTEN-treated neurons exhibited significantly more branch points ( Figure 8 , one-way analysis of variance, p < 0.001, Tukey's test). Total axon length, number of axons, branch points, and maximum level of branches were significantly higher in neurons treated with ExoPTEN; more specifically, they were 6.6, 6.6, 6.8, and 2.3 times greater than those in untreated neurons, respectively (Fig. 9, one-way analysis of variance, all p < 0.001, Tukey's test). Both MSC-Exo and PTEN-siRNA alone induced DRG neuronal outgrowth to the same but less pronounced extent than with ExoPTEN treatment.

[284] Example 3 Intranasal administration of ExoPTEN causes silencing of PTEN expression in lesions of the spinal cord

[285] To test the silencing effect of ExoPTEN in vivo, ExoPTEN was administered intranasally for 5 consecutive days, starting from the day of complete dissection of the spinal cord. At week 8, Western blotting of proteins isolated from the SCI region demonstrated a 59% reduction in PTEN protein levels in ExoPTEN-treated rats compared to untreated SCI rats ( Figure 10A , Kruskal-Wallis test, p = 0.0627). No significant changes in liver tissue protein levels were found between groups ( Figure 10B , p = 0.8643). In parallel, RT-qPCR analysis demonstrated a 32% reduction in PTEN gene expression after ExoPTEN treatment compared to untreated SCI rats (Fig. 10C, p = 0.1520), while the level of liver gene expression was comparable ( Fig. 10D , p > 0.9999).

[286] Example 4 Intranasal ExoPTEN Provides Significant Functional Restoration in Complete SCI Rats

[287] To evaluate the neuroprotective potential of ExoPTEN treatment, complete SCI rats were divided into five groups: (1) no treatment, (2) intralesional (IL) administration of PTEN-siRNA, (3) intralesional single administration of ExoPTEN, (4) IN administration of exosomes for 5 consecutive days and (5) IN administration of ExoPTEN for 5 consecutive days (starting from the day of injury). The rats were subjected to BBB motor function assessment every week for 8 weeks. IN administration of ExoPTEN for 5 days resulted in a significant recovery of motor functions ( resulting in F(4, 300) = 35.72, p < 0.0001, F(8, 300) = 2.931, p < 0.001, respectively) compared with all other experimental groups. More specifically, at week 8, the mean BBB motor function score in the ExoPTEN (IN) group ( Fig. 11A ) reached 7.75 ± 2.14, which differs sharply from the mean score of 0.39 ± 0.14 in untreated rats with dissection (p < 0.001). Statistically significant differences were observed between groups receiving intranasal ExoPTEN and groups receiving intranasal empty exosomes (FIG. 11A). Insignificant, statistically insignificant (all p > 0.05) improvements were noted at week 8 in the PTEN miRNA (IL), ExoPTEN (IL) and exosome (IN) groups, with a BBB motor function score of 2.17 ± 1, 48, 2.50 ± 1.21, 2.00 ± 1.23, respectively ( Fig. 11A ). At 8 weeks, 28.6% of ExoPTEN (IN)-treated rats exhibited a BBB motor function score ≥ 14, reflecting persistent plantar treading, toe separation, and robust coordination between fore and hind limbs, while none from other treated groups failed to achieve this level of motor function ( Fig. 11B ). The ExoPTEN (IN) treated rats also gained weight faster than the other groups and reached constant weights at week 5 ( FIG. 11C ). In addition, at week 8, there was a positive correlation between rat weight and BBB motor function score ( Fig. 11D , Pearson's correlation coefficient = 0.6819, p < 0.0001). Von Frey filament tests with a bending force gradient (6, 8, 10, 15, 26, 60, 100, 180, 300 g) were applied to the hind limbs to determine paw withdrawal threshold as an indicator of sensory recovery. The threshold for stimulating paw withdrawal in healthy rats was set at 60 g, below which the withdrawal response was considered allodynic. None of the rats responded to the thread with a force of 300 g after SCI, which indicates the complete disappearance of sensory functions. Normal sensory response was achieved in 25% and 33.3% of the animals in the ExoPTEN (IL) and ExoPTEN (IN) groups, respectively, at week 4 and remained the same at week 8. In addition, 25% of the rats in the exosome (IN) group achieved sensory recovery at week 8. In contrast, the untreated and PTEN-siRNA (IL) groups did not show recovery of sensory functions within 8 weeks ( FIG. 11E ). Time to urinary reflex responses reflecting bladder function recovered fastest in ExoPTEN (IN)-treated animals (7.0 ± 0.7 days) and then in untreated rats (8.9 ± 0. 7 days) ( Fig. 11F , one-way analysis of variance, p = 0.4079, Tukey's test). In FIG. 11G shows differences in recovery in rats treated with control agent, exosomes, and ExoPTEN within 8 weeks of injury.

[288] Example 5 Intranasal administration of ExoPTEN stimulates structural organization and electrophysiological conduction

[289] To assess the structural integrity of the spinal cord in untreated SCI animals, animals with SCI treated with exosomes (IN) or ExoPTEN (IN), and intact rats, conventional MRI at 9.4 T induction and diffuse tensor imaging (DTI) were performed. . In the dissection control group, severe spinal cord atrophy was observed 4 mm caudal to the injury epicenter, while in the exosome and ExoPTEN groups, such degeneration was less obvious. Cross-sectional areas measured 4 mm rostral to the lesion epicenter did not differ significantly between SCI groups (Fig. 12A, one-way analysis of variance, F(3, 11) = 0.6120, p = 0.6212), but decreased sharply by 4 mm caudal to epicenter in the control dissection group compared to the ExoPTEN group (Fig. 12B, 2.5 ± 0.5 mm ² versus 4.2 ± 0.3 mm ² , p < 0.05). Diffusion tensor imaging (DTI) tractography showed complete separation of the rostral and caudal stumps in the dissection group, partial bridging in the exosome group, and most pronounced bridging in the ExoPTEN group. Fractional anisotropy (FA) values rostral and caudal with respect to the injury site were higher in ExoPTEN-treated rats compared to the exosome-treated or untreated SCM group, but still lower than in intact rats (average FA values within the lesion in the control dissection, exosomes, ExoPTEN, and intact rats groups were 0.2856, 0.2938, 0.3293, 0.5055, respectively, Fig. 12C ). Mean diffusivity (MD) scores showed no difference between the untreated SCI groups and those treated with exosomes and ExoPTEN. In addition, ExoPTEN-treated rats exhibited high-amplitude motor-stimulus potentials (MEPs) propagating from the motor cortex through the lesion to the sciatic nerve, while low-amplitude signals were recorded in the exosome group ( Fig. 12D , 1.283 ± 0.085 mV vs. 0.662 ± 0.162 mV, respectively, p = 0.0273 MEP latency was shorter in the ExoPTEN treated group than in the exosome treated group ( Fig. 12E , 31.03 ± 3.05 ms vs. s 38.49 ± 6.78 ms, p = 0.3726) All signals disappeared after recrossing rostral to the spinal cord injury.

[290] Example 6 Intranasal administration of ExoPTEN improves the microenvironment at the site of damage and restores corticospinal axons

[291] To identify the cellular mechanisms underlying motor function recovery, spinal cord lesions in untreated animals and animals treated with exosomes (IN) and ExoPTEN (IN) were stained for beta-III-tubulin, CD11b, CD31, and GFAP to detect axonal regeneration, microgliosis, astrogliosis and angiogenesis, respectively. The expression level of β-III-tubulin was significantly higher in ExoPTEN-treated rats than in exosome-treated rats, while it was barely detectable in untreated SCI rats (Fig. 13A, p < 0.0001). CD11b-positive microglial cells were the most common cell type in the untreated dissection group and were present in significantly lower numbers in the exosomes and ExoPTEN groups (F(2, 24) = 25.18, p < 0.0001), with significant there was no difference between them ( Fig. 13B , panel 2, p = 0.9818). A similar expression profile was observed for GFAP ⁺ astrocytes (Fig. 13C ). Found that the minimum level of angiogenesis, according to the measurement of the number of CD31 ⁺ cells, was observed in untreated rats with dissection (Fig. 13D ).

[292] The corticospinal tract (CST) is responsible for voluntary motor control of the body and hind limbs. To investigate whether this particular pathway could be regenerated by intranasal administration of ExoPTEN, the inventors tracked its progression using biotinylated dextranamine (BDA) administered to the somatomotor cortex of untreated, exosome-treated (IN) or ExoPTEN (IN) rats with SCI. It was found that in the untreated SCM group, BDA-positive fibers failed to enter the lesion ( Fig. 14 , upper panel), while in the exosome-treated group, BDA-positive fibers were noted inside the lesion but did not cross over. into the tail stump ( Fig. 14 , middle panel). In contrast, in the ExoPTEN group, BDA-positive fibers were clearly observed infiltrating and extending beyond the lesion ( Fig. 14 , bottom panel).

[293] While the present invention has been described in connection with specific embodiments, various alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to cover all such alternatives, modifications and variations that are included within the spirit of the appended claims. Although the present invention has been described above in this document through its preferred embodiments, it can be modified without departing from the spirit of the present invention as defined in the appended claims.

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SEQUENCE LIST

<110> Technion Research & Development Foundation Limited

RAMOT AT TEL-AVIV UNIVERSITY LTD.

<120> VESICLES CONTAINING PTEN INHIBITOR AND THEIR USE

<130> TECH-075 PCT

<150> 62/649648

<151> 2018-03-29

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 403

<212> PROTEIN

<213> Homo sapiens

<400> 1

Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr

1 5 10 15

Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile

20 25 30

Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn

35 40 45

Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His

50 55 60

Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys

65 70 75 80

Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro

85 90 95

Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu

100 105 110

Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys

115 120 125

Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys

130 135 140

Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr

145 150 155 160

Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr

165 170 175

Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala

180 185 190

Leu Leu Phe His Lys Met Met Glu Glu Thr Ile Pro Met Phe Ser Gly

195 200 205

Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile

210 215 220

Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr

225 230 235 240

Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu

245 250 255

Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His

260 265 270

Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu

275 280 285

Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys

290 295 300

Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu

305 310 315 320

Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr

325 330 335

Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu

340 345 350

Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp

355 360 365

Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp

370 375 380

Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile

385 390 395 400

Thr Lys Val

<210> 2

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Representative siRNA sequences considered

for PTEN inhibition

<400> 2

guuagcagaa acaaaaggag auaucaa 27

<210> 3

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Representative siRNA sequences considered

for PTEN inhibition

<400> 3

uugauaucuc cuuuuguuuc ugcuaac 27

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Representative siRNA sequences considered

for PTEN inhibition

<400> 4

cagccguucg gaggauuauu cgucutt 27

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Representative siRNA sequences considered

for PTEN inhibition

<400> 5

agacgaauaa uccuccgaac ggcugtt 27

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Typical sequence that can be targeted

miRNA PTEN

<400> 6

gagttcttcc acaaacagaa 20

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Typical sequence that can be targeted

miRNA PTEN

<400> 7

gtatagagcg tgcagataa 19

<210> 8

<211> 10

<212> RNA

<213> Artificial sequence

<220>

<223> Examples of oligonucleotide sequences that can be

use to form a loop

<220>

<221>n

<222> (10)..(10)

<223> n=a, u, t, c, g or missing

<220>

<221> other

<222> (10)..(10)

<223> n is a, c, g, or u

<400> 8

uucaagagan 10

<210> 9

<211> 10

<212> RNA

<213> Artificial sequence

<220>

<223> Examples of oligonucleotide sequences that can be

use to form a loop

<220>

<221>n

<222> (10)..(10)

<223> n=a, u, t, c, g or missing

<220>

<221> other

<222> (10)..(10)

<223> n is a, c, g, or u

<400> 9

uuuguguagn 10

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> artificial sequence

<400> 10

gaguucuucc acaaacagaa 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> artificial sequence

<400> 11

uucuguuugu ggaagaacuc 20

<---

Claims (29)

1. Pharmaceutical composition for the treatment of damage or damage to neurons, containing extracellular vesicles loaded with an exogenous inhibitor of phosphatase and tensin homologue (PTEN), and a pharmaceutically acceptable carrier, where said extracellular vesicles are obtained from mesenchymal stem cells (MSC) and said PTEN inhibitor is miRNA . 2. Pharmaceutical composition according to claim 1, characterized in that the extracellular vesicles are selected from exosomes, microvesicles, ectosomes, exovesicles, and combinations thereof. 3. Pharmaceutical composition according to claim 1 or 2, characterized in that the extracellular vesicles are exosomes. 4. Pharmaceutical composition according to any one of paragraphs. 1-3, characterized in that the adhesive cells are selected from the group consisting of dental pulp stem cells (DPSC), deciduous tooth stem cells (SHED), periodontal ligament stem cells (PDLSC), apical papilla stem cells (SCAP) and cells progenitors of dental follicles (DFPC). 5. Pharmaceutical composition according to any one of paragraphs. 1-4, characterized in that the adhesive cells express neural crest cell markers. 6. Pharmaceutical composition according to claim 5, characterized in that the neural crest cells are cranial neural crest cells. 7. Pharmaceutical composition according to claim 1, characterized in that the PTEN inhibitor is selected from (i) miRNA containing a nucleotide sequence selected from SEQ ID NO: 10, 11, 2, 3, 4 and 5; (ii) miRNA, the target of which is a nucleotide sequence selected from SEQ ID NO: 6 and 7; and (iii) an siRNA containing nucleic acid variants from (i) and (ii) having at least 80% sequence identity with the original sequence. 8. Pharmaceutical composition according to claim 7, characterized in that said RNAi oligonucleotide additionally contains a hydrophobic fragment. 9. Pharmaceutical composition according to claim 8, characterized in that said hydrophobic moiety is selected from the group consisting of a sterol, ganglioside, lipid, vitamin, fatty acid, hydrophobic peptide, and combinations thereof. 10. Pharmaceutical composition according to claim 9, characterized in that said sterol is cholesterol. 11. Pharmaceutical composition according to any one of paragraphs. 1-10, characterized in that the extracellular vesicles are isolated extracellular vesicles. 12. Pharmaceutical composition according to any one of paragraphs. 1-11, additionally containing chondroitinase ABC or extracellular vesicles containing chondroitinase ABC. 13. Pharmaceutical composition according to any one of paragraphs. 1-12 presented in a form for administration by a route selected from intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral, and intracerebral routes of administration. 14. A method of treating a lesion or injury to neurons in a subject, comprising administering to said subject a pharmaceutical composition according to any one of paragraphs. 1-13. 15. The method according to claim 14, characterized in that the lesion or damage to the neurons is a spinal cord injury (SCI). 16. The method according to claim 15, characterized in that the siRNA contains a nucleotide sequence selected from SEQ ID NOs: 10, 11, 2, 3, 4 and 5. 17. The method according to claim 16, characterized in that miRNA is conjugated to a cholesterol fragment. 18. The method according to any one of paragraphs. 14-17, characterized in that the composition is administered intranasally. 19. The method according to any one of paragraphs. 14-18, characterized in that the method further comprises administering chondroitinase ABC. 20. The method of claim 19, wherein the extracellular vesicles loaded with an exogenous PTEN inhibitor and chondroitinase ABC are administered as a single pharmaceutical composition or as different pharmaceutical compositions. 21. An isolated extracellular vesicle loaded with an exogenous inhibitor of phosphatase homologue and tensin (PTEN) to deliver a PTEN inhibitor, said extracellular vesicle is derived from mesenchymal stem cells (MSC) and said PTEN inhibitor is an siRNA. 22. An isolated extracellular vesicle according to claim 21, characterized in that the extracellular vesicle is selected from exosomes, microvesicles, and combinations thereof. 23. An isolated extracellular vesicle according to claim 21 or 22, characterized in that the RNAi oligonucleotide is selected from (i) miRNA containing a nucleotide sequence selected from SEQ ID NOs: 10, 11, 2, 3, 4 and 5; (ii) miRNA, the target of which is a nucleotide sequence selected from SEQ ID NO 6 and 7; and (iii) an siRNA containing nucleic acid variants from (i) and (ii) having at least 80% sequence identity with the original sequence. 24. Selected extracellular vesicle according to any one of paragraphs. 21-23, characterized in that said RNAi oligonucleotide contains a hydrophobic fragment. 25. An isolated extracellular vesicle according to claim 24, characterized in that said hydrophobic fragment is selected from a sterol, ganglioside, lipid, vitamin, fatty acid, peptide, and combinations thereof. 26. An isolated extracellular vesicle according to claim 25, characterized in that said sterol is cholesterol. 27. An isolated extracellular vesicle according to claim 26, characterized in that the vesicle is an exosome loaded with cholesterol-conjugated siRNA molecules that inhibit PTEN expression, and said exosomes originate from mesenchymal stem cells. 28. An isolated vesicle according to claim 27, characterized in that the siRNA contains a nucleic acid sequence selected from SEQ ID NOs: 10, 11, 2 and 4. 29. Selected vesicle according to any one of paragraphs. 21-28, additionally containing chondroitinase ABC or a polynucleotide encoding it.