RU2727540C1 - Application of membrane vesicles of multipotent stromal cells induced by b cytochalasin, for restoration and increase of mitochondrial function - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention refers to biotechnology, namely to application of membrane vesicles of multipotent stromal cells for restoration and enhancement of mitochondrial function by delivering functionally active mitochondria into cells of mammals for therapy of mitochondrial dysfunction.EFFECT: membranous vesicles of multipotent stromal cells are induced by cytochalasin B, surrounded by bilipid cytoplasmic membrane, carry superficial cytoplasmic receptors (CD90, CD29, CD44, CD73), have diameter ranging from 100 to 2,000 nanometers and contain functionally active mitochondria.1 cl, 8 dwg

Description

The invention generally relates to medicine and veterinary medicine, in more detail to the development of drugs and medicaments in their production on an industrial scale. The claimed technical solution makes it possible to deliver functionally active mitochondria as part of membrane vesicles of multipotent human stromal cells induced by cytochalasin B (MB-CV MSCs) into mammalian cells in vitro, in order to increase / restore mitochondrial function in cells, which makes it possible to increase / restore mitochondrial function in cells with reduced activity or / and dysfunction of mitochondria.

The pharmaceutical composition developed on the basis of the claimed technical solution can be used for the treatment of diseases caused by mutations in mitochondrial DNA, for example, degeneration, muscle atrophy and myopathy, as well as for the treatment of diseases associated with impaired mitochondrial function (type 2 diabetes, ischemic heart disease, stroke, cancer), neurodegenerative diseases, in which there is a problem of using mitochondrial replacement therapy for adults, can be used to slow down the aging of the body.

Further in the text, the applicant provides an explanation of a number of terms , which is necessary to facilitate an unambiguous understanding of the essence of the declared materials and to exclude contradictions and / or controversial interpretations when conducting an examination on the merits.

Dysfunction of mitochondria - in the context of the present description, the applicant means by the specified term a violation of the functional activity of mitochondria, as a result of mutations, any diseases or aging of cells, characterized by a decrease in mitochondrial potential.

Defective cells - in the context of the present description, the applicant means by the specified term cells with low mitochondrial potential: with dysfunction (not functioning) of mitochondria or decreased functioning of mitochondria.

Fibroblasts are a type of cell found in the connective tissues of the body. Fibroblasts perform a repair function in case of injury and any other tissue damage. Elongated or irregularly sized cells are components of the fibrous protein collagen and other substances that make up the connective tissue [https://dic.academic.ru/contents.nsf/ntes].

A549 are human adenocarcinomic alveolar basal epithelial cells and constitute a cell line that was first developed in 1972 by removing and culturing cancerous lung tissue [https://en.wikipedia.org/wiki/].

Debris - in the context of the present description, the applicant means by the specified term debris formed after exposure to the substance cytochalasin B.

Lytic enzymes are enzymes that cause lysis, that is, the dissolution of cells and their systems, including microorganisms, under the influence of various agents [https://ru.wikipedia.org/wiki/].

Expression (expression of genes) - in the context of the present description, the applicant means by these terms the process during which the hereditary information from a gene (DNA nucleotide sequence) is converted into a functional product - RNA or protein. Some stages of gene expression can be regulated: these are transcription, translation, RNA splicing, and the stage of post-translational protein modifications. The process of activating gene expression by short double-stranded RNAs is called RNA activation [https://ru.wikipedia.org/wiki/].

Non - immunogenicity - in the context of the present description, the applicant means by this term the absence of the ability of an antigen to induce an immune response, regardless of its immune specificity.

Membrane vesicles of multipotent stromal cells induced by cytochalasin B - in the context of the present description, the applicant uses as the specified term the abbreviation MV-CV MCT.

As of the filing date of the claimed technical solution, the applicant conducted studies of the prior art and trends in the development of technology in this area on scientific and patent information, identified existing problems associated with the lack of a method for delivering functionally active mitochondria into human cells in order to replenish the insufficient function of mitochondria Thus, there is a still unresolved problem of creating new drugs characterized by the ability to restore mitochondrial function in human cells, to increase the functional activity of mitochondria in places with reduced activity and / or dysfunction of mitochondria.

From the investigated prior art, an invention was revealed according to patent No. WO2018140726 [1] (analogue No. 1) "Method for identifying mitochondrial dna in extracellular vesicles and treatment of mtDNA-related disorders and cancer" "Method for identifying mitochondrial DNA in extracellular vesicles and treatment of diseases associated with mitochondrial DNA and cancer. "

The essence of the known technical solution is a method for detecting a state associated with mitochondrial DNA (hereinafter referred to as mtDNA) in a subject, which includes the stages: (a) isolation of extracellular vesicles (hereinafter referred to as BB) from plasma or other biological fluids obtained from the subject ;

(b) detecting levels of one or more mtDNA genes and / or 70%, 80%, 90% or 95% of the entire mtDNA genome in the isolated BB;

determining the likelihood of having a condition associated with mtDNA based on the result of step (b), where the condition associated with mtDNA is selected from the group consisting of cancer, metabolic disorders, heart failure, brain damage, and neurological syndromes.

The method of claim 1, further comprising the step of (c) identifying specific genetic variants of mtDNA; and determining the likelihood of having the mtDNA related condition based on the result of step (b) and step (c), where the mtDNA related condition is selected from the group consisting of cancer, metabolic disorders, heart failure, brain damage, and neurological syndromes ...

Thus, in brief, the essence of the known technical solution is a method of detecting (diagnosing), as well as monitoring a disease associated with mitochondrial DNA (hereinafter referred to as mtDNA): cancer, metabolic disorders, heart failure, brain damage, lung / kidney damage, neurological syndromes , by isolating extracellular vesicles (IVs) from plasma or other biological fluids obtained from the subject, followed by analysis of mtDNA in the IV. The essence of the known method is also the detection of specific genetic variants of the patient's mtDNA to assess the likelihood of developing resistance to hormone therapy in a patient with breast cancer. The essence of the known method is also the generation of explosives loaded with wild-type mtDNA, and the introduction of an effective amount of functional explosives containing mtDNA to the subject. In more detail, the known method relates to the possibility of treating an mtDNA-associated disorder by administering explosives loaded with functional wild-type mtDNA molecules. Indications for the use of the known method are disorders associated with mtDNA, namely, mtDNA deficiency (reduced number of mtDNA copies) or a genetic mutation of mtDNA in humans. The method consists in isolating explosives loaded with wild-type mtDNA and introducing these functional mtDNAs into the bloodstream (plasma) and / or topically (inside the tissue) of the subject.

The disadvantage of the known invention according to patent No. WO2018140726 is the low effectiveness of the intended action, due to the absence of functionally active mitochondria in the explosive composition. mtDNA performs the function of storing genetic information without having the function of a cellular organelle, that is, a mitochondrial function, namely, mtDNA is not capable of supporting oxidative phosphorylation reactions, while the claimed technical solution ensures the implementation of the delivery of functionally active mitochondria into cells.

At the same time, the claimed technical solution solves the problem of low effectiveness of the intended action due to the fact that their use for a new purpose (to restore and increase mitochondrial function) provides the possibility of delivering whole and functionally active organelles to mammalian cells - mitochondria, as part of multipotent stromal membrane vesicles cells induced by cytochalasin B, which, in fact, provide the implementation of the delivery of functionally active mitochondria into cells.

From the investigated prior art, an invention was revealed according to patent No. US2018036344 [2] (analogue No. 2) "Targeted transplantation of mitochondria to hepatocytes"

The essence of the known technical solution is a pharmaceutical composition containing: asialoglycoprotein (AsG) covalently linked to a polycation; and functional mammalian mitochondria that are at least partially purified; where AsG, covalently bound to the polycation, electrostatically forms a complex with mitochondria. The composition of claim 1, wherein the mitochondria are obtained from a cell from a healthy donor or isolated from a mammalian cell or tissue. The composition of claim 11, wherein the donor mammalian cell is a hepatocyte, leukocyte, stem cell or tissue. The composition of claim 1, wherein the mitochondria are human or rat mitochondria. A method of making a composition, including: at least partial purification of functional mitochondria from a cell; enabling AsG to covalently attach to a highly positively charged polycation; and by allowing AsG, covalently attached to a highly positively charged polycation, to electrostatically bind to mitochondria. A method of transplanting mitochondria into a hepatocyte, comprising providing functional mammalian mitochondria, complexed with AsG-PL, an electrostatic and endosomolytic agent that is covalently attached to AsG through a cleavable bond; and also delivery of the specified composition to the hepatocyte.

Thus, briefly, the essence of the known technical solution is a pharmaceutical composition containing: asialoglycoprotein (AsG) covalently linked to a polycation (polylysine, polyarginine and polyornithine); which electrostatically form a complex with functional mammalian mitochondria. Mitochondria are obtained from cells of a healthy donor or isolated from mammalian cells / tissues. The method of manufacturing the composition includes: partial purification of functional mitochondria from the cell; enabling AsG to covalently attach to a highly positively charged polycation; and providing electrostatic binding to mitochondria. The essence of the known method is a method of treating a patient with liver disease, which can benefit from enhancing cellular mitochondrial function. A method of treating a patient with liver disease comprises administering a therapeutically effective amount of a pharmaceutical composition to a patient in need thereof. Liver cells (hepatocytes) have unique cell surface receptors that recognize and bind proteins known as asialoglycoproteins (AsG), which contain galactose residues. Binding of AsG to its receptor triggers invagination of the cell surface membrane and, ultimately, isolates the receptor-glycoprotein complex in the membrane-limited intracellular membrane, the endosome. Endosomes fuse with lysosomes containing degrading enzymes, which leads to the cleavage of the glycoprotein. The asialoglycoprotein receptor system is a natural mechanism by which substances outside cells can access the inside of cells.

The disadvantage of the known invention according to patent No. US2018036344 is the absence of a membrane, which jeopardizes the maintenance of the functional activity of mitochondria. Another disadvantage is that purified mitochondria (not surrounded by the cytoplasmic membrane of the cell) induce an immune response in the human body, namely, stimulation of macrophages and neutrophils has been shown (Zhu et al., 2018) [3] “Mitochondria Released by Apoptotic Cell Death Initiate Innate Immune Responses ".

Thus, the known invention is recognized by the immune system and removed from the human body, due to which the known technical solution cannot be used to achieve the stated goal (s), namely, is not effective when used for its intended purpose.

While the claimed technical solution solves the problem of the absence of a membrane, since it provides for the transfer of functionally active mitochondria inside the membrane vesicles of multipotent stromal cells induced by cytochalasin B (MV-CV MSCs), which are protected by the cell membrane, which allows to maintain mitochondrial function, that is, provides the possibility of their use for a new purpose, namely, to restore and increase the mitochondrial function of a living cell.

In addition, according to the claimed technical solution, MV-CV MSCs express little or do not express molecules of the main histocompatibility complex at all, therefore, they are non-immunogenic, that is, they are not rejected by the body, in contrast to the known technical solution.

From the investigated prior art, an invention was revealed according to patent No. IL247447 [4] (analogue No. 3) "An oocyte comprising exogenous, autologous functional mitochondria obtained from an oogonial stem cell (osc) or the progeny of an osc" "Oocyte containing exogenous, autologous functional mitochondria derived from the oogonial stem cell (CSC) or offspring from the CSC ”.

The essence of the known technical solution is a method of preparing an egg for in vitro fertilization (IVF) or artificial insemination, and this method includes transferring a composition containing i) mitochondria of oogonial stem cells (CSC) or ii) mitochondria obtained from the offspring of CSC into an autologous oocyte, thus by preparing the specified oocyte for IVF or artificial insemination. The method of claim 1, wherein the composition comprising i) CSC mitochondria or ii) mitochondria derived from CSC offspring is a purified mitochondrial preparation. Composition containing isolated mitochondria or mitochondria of CSC obtained from the offspring of CSC.

Thus, briefly, the essence of the known technical solution are compositions obtained from oogonial stem cells (CSCs), such as nuclear-free cytoplasm or isolated mitochondria, and the use of compositions obtained from CSCs in procedures aimed at increasing fertility is described. In one aspect, the invention relates to a method for preparing an oocyte for in vitro fertilization (IVF) or artificial insemination. The method involves transferring a composition containing CSC mitochondria or mitochondria obtained from the CSC offspring into an autologous oocyte, thereby preparing the oocyte for in vitro fertilization. CSC is an isolated non-embryonic stem cell that is a competent cell and expresses embryonic stage specific antigens: Vasa, Oct-4, Dazl, Stella, SSEA (eg SSEA-1, -2, -3 and -4). CSC can be obtained from ovarian or non-ovarian tissue, as well as bone marrow, peripheral and umbilical cord blood.

The disadvantage of the known invention according to patent No. IL247447 is the complexity of obtaining a biomaterial. The material can only be taken from women of childbearing age.

In addition, the use of pure mitochondria carries the risk of their death due to the absence of a protective membrane and, as a consequence, the known technical solution has a low efficiency of action as intended.

At the same time, the claimed technical solution solves the problem of age and gender restrictions due to the fact that the biomaterial according to the claimed technical solution can be obtained from the MSC of any healthy person. The claimed technical solution solves the problem of low efficiency of action for prescribing analogs by delivering whole and functionally active mitochondria to animal cells, enclosed in membrane vesicles of multipotent stromal cells induced by cytochalasin B.

The claimed technical solution eliminates the problem of the loss of the functional activity of mitochondria by enclosing mitochondria in the membrane vesicles of multipotent stromal cells induced by cytochalasin B, which are surrounded by a cytoplasmic membrane, which makes it possible to use them for a new purpose, namely, to restore and increase mitochondrial function.

From the investigated prior art, an invention was revealed under the patent No. KR20190026045 [5] (analogue No. 4) "Compositions and methods for improving mitochondrial function and treating neurodegenerative diseases and cognitive disorders" "Compositions and methods for improving mitochondrial function and treatment of neurodegenerative diseases and cognitive disorders" ...

The essence of the known technical solution is:

For the treatment and prevention of conditions selected from the group consisting of obesity, decreased metabolic rate, metabolic syndrome, diabetes, cardiovascular diseases, hyperlipidemia, neurodegenerative disorders, cognitive disorders, mood disorders, stress and anxiety disorders; For weight control; Or to increase muscle performance or mental performance: a food or nutritional supplement containing an effective amount of pomegranate extract.

Contacting cells with an effective amount of uroritin or its precursor to increase the function of mitochondria, while the method includes the steps: increasing or maintaining mitochondrial function.

Administration of a therapeutically effective amount of evoritin or its precursor to an individual in need thereof for the treatment of a disease or condition associated with altered mitochondrial function or decreased mitochondrial density, including the stages: associated with altered mitochondrial function or decreased mitochondrial density. A method of treating, preventing, or treating a mitochondrial disease or condition.

Thus, in brief, the essence of the known technical solution are compositions that can be used for various therapeutic purposes, including the treatment and / or prevention of diseases or disorders associated with reduced or inadequate mitochondrial activity, including aging or stress, diabetes, obesity and neurodegenerative diseases. The known invention provides an extract of the fruit, its active fraction or one or more active ingredients that can be separated from them, for use as a factor in mitochondrial function. As used herein, the term "fraction" refers to purified or partially purified extracts. The known invention also relates to natural compounds with biological activity found in pomegranate and other fruits, as well as natural extracts with biological activity containing these compounds, also relates to polyphenolic compounds and their derivatives associated with elagitanin. These compounds include elagitanin, punatsirazine, and elaginic acid, which are found in pomegranate, but can also be isolated from other fruits and berries, as well as metabolites of these compounds. As described in the prior art, these compounds have been found to have beneficial effects on mitochondrial function, cellular metabolism and neuronal formation. The studies were carried out on a model of culture of neuronal cells in vitro. In one aspect, a known invention relates to a composition comprising a compound of a known invention, such as a pharmaceutical, medical food, functional food, herbal supplement, or food supplement, and mixtures thereof. The compositions may optionally contain additional therapeutic agents and may be administered in combination with another therapeutic compound. Also provided are packaged products containing the aforementioned compositions and labels and / or instructions for use to improve memory and cognitive functions or to treat diseases or conditions associated with typical brain damage in the case of conditions encountered in the elderly.

The disadvantage of the known invention according to patent No. KR20190026045 is the lack of a targeted effect on the mitochondrial activity of human cells, since plant mitochondria are not able to replace and take over the function of human mitochondria. The similarity of plant and animal mitochondria is speculation, since direct chemical analyzes to determine the homology of the composition and function of plant and animal mitochondria have not been identified by the applicant. Thus, the known invention is a biologically active food supplement, the use of which does not lead to the replacement of defective mitochondria with donor ones. The disadvantage of the known invention according to patent No. KR20190026045 is the low effectiveness of the intended action, since the drug is administered to the patient orally. Oral administration of analogue No. KR20190026045 leads to the destruction of plant mitochondria, without reaching the human bloodstream. In more detail: plant mitochondria that are part of the invention according to patent No. KR20190026045 do not enter the bloodstream, since they are destroyed when passing through the gastrointestinal tract, thus, according to the applicant, plant mitochondria cannot reach the focus of the disease and, as a consequence , cannot be used for its intended purpose, namely, to restore and increase the mitochondrial function of human cells.

In this case, the claimed technical solution solves the problem of increasing the efficiency of action by intravenous administration of membrane vesicles of multipotent human stromal cells induced by cytochalasin B, which contain functionally active mitochondria capable of increasing the functional activity of mitochondria.

The intravenous method is most effective when used as directed due to its fast action and the possibility of accurate dosing of MV-CV MSCs.

In addition, the claimed technical solution contains human mitochondria, protected by the cytoplasmic membrane from destructive environmental factors, which makes it possible to use them for a new purpose, namely, to restore and enhance mitochondrial function.

From the investigated prior art, an invention was revealed according to patent No. SG11201901689Y [6] (analogue No. 5) "Compounds for treating diseases associated with a mitochondrial dysfonction" "Compounds for the treatment of diseases associated with mitochondrial dysfunction"

The essence of the known technical solution is:

A compound or a pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 9 in combination with at least one additional compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction. A pharmaceutical composition containing, as an active substance, a compound of formula (A) or a pharmaceutically acceptable salt or hydrate thereof as defined in any one of claims 1 to 5, optionally in combination with at least one pharmaceutically acceptable excipient or carrier, for prophylactic use or treating a disease associated with mitochondrial dysfunction according to claim 1, 6 or 7 in the individual. Products containing: a compound of formula (A) according to any one of claims 1 to 5, at least one additional compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction, as a combined preparation for simultaneous, separate or sequential use in prevention or treatment of a disease associated with mitochondrial dysfunction, according to claim 1, 6 or 7 in the individual.

WO 2018/041919 33 PCT / EP2017 / 071812.

A nutritional supplement containing a compound of formula (A) according to any one of claims 1 to 5 for use in reducing the risk of a disease associated with mitochondrial dysfunction according to claim 1, 6 or 7 in an individual.

Thus, briefly, the essence of the known technical solution is a pharmaceutical composition, which is an organic compound of the formula (<br/> WO 2018/041919 4 PCT / EP2017 / 071812 NH 0 NH NN NH 2 R 1) (I) or a pharmaceutically acceptable salt thereof / a hydrate that has a neuroprotective effect in an in vitro model of Parkinson's disease involving mitochondrial dysfunction. In an embodiment, the aforementioned pharmaceutical composition further comprises at least one additional compound useful for preventing or treating a disease associated with mitochondrial dysfunction as a combination preparation for single, separate or sequential use in the prevention or treatment of the disease. The essence of the known method is to obtain a pharmaceutical composition by chemical synthesis, or by extraction, followed by purification from natural sources such as microorganisms, in particular bacteria, or from plants, in particular from plants that do not contain alpha-proteobacteria, such as bacteria Rhizobium, Mesorhizobium and Sinorhizobium genii.

A disadvantage of the known invention according to the patent is its limited application due to the use of a chemical substance that can adversely affect the patient's condition. There is also a danger of immune rejection of the synthesized substance by the body. Extraction of the pharmaceutical composition from natural sources carries the risk of contamination with toxins (phytotoxins, endotoxins). The toxins have a specific effect on the body, namely, they are neutralized only by the corresponding antitoxic serum, which implies the introduction of additional difficulties in the implementation of the invention under patent No. SG11201901689Y.

At the same time, the claimed technical solution is safe, technologically advanced for use, since membrane vesicles of multipotent stromal cells induced by cytochalasin B express little or no expression of the molecules of the main histocompatibility complex, therefore, they are non-immunogenic, and also do not contain any synthetic materials. Thus, the use of MSC membrane vesicles for the delivery of functionally active mitochondria to mammalian cells is biocompatible, non-immunogenic, and non-toxic, and also serves to restore and enhance mitochondrial function.

From the studied prior art, a method has been identified for transferring mitochondria from stromal cells obtained from bone marrow to the alveoli of the lungs, which protects against acute lung injury (Islam et al., 2012) [7] (analogue No. 6) “Mitochondrial transfer from bone-marrow- derived stromal cells to pulmonary alveoli protects against acute lung injury ".

The essence is that the culture of stromal cells (bone marrow-derived stromal cells (hereinafter referred to as BMSCs) is isolated from the bone marrow and cultured. Further, on the model of lipopolysaccharide-induced acute lung injury, delivery of human BMSCs into the alveoli of mice was carried out. extracellular environment natural microvesicles carrying mitochondria Real-time microscopic studies showed that murine BMSCs formed connective channels with the alveolar epithelium, releasing vesicles with mitochondria that invaded alveolar epithelial cells The presence of mitochondria transferred from the epithelial cells of BMSCs was observed in visually, due to the fluorescent label, as well as the presence of human mitochondrial DNA in the lungs of the mouse. As a result of mitochondrial transfer, the amount of ATP in the alveoli of the lungs increased. The known method partially coincides with the claimed technical solution in the region Part of the choice of delivery method for mitochondria is via vesicles.

The disadvantage of the known technical solution is the low efficiency of the intended use due to the limited yield of the product. Injection of cells (BMSCs) carries risks of unlimited division and tumor transformation. The natural release of vesicles is carried out in vitro and in vivo constantly, but in very small quantities, which requires a large amount of time and cellular material. In this regard, the achievement of a therapeutic effect will take a long time, which will adversely affect the patient's condition. As a result of the foregoing, the specified known method is ineffective and uncomfortable in the intended use for the patient, since it requires regular and frequent administration of this pharmaceutical composition to the patient. The disadvantage is also limited applicability, since the known technical solution is aimed at treating acute lung injury, while the claimed technical solution provides for a therapeutic effect on the entire body, since membrane vesicles of multipotent stromal cells obtained with cytochalasin B spread throughout the bloodstream and reach all organs and tissues. As a result, the known technical solution is less effective for its intended purpose and limited applicability for its intended purpose as compared to the declared technical solution.

At the same time, the claimed technical solution solves the problem of limited product yield, since it provides for the production of membrane vesicles from multipotent stromal cells using cytochalasin B, which increases the output of membrane vesicles, does not require a significant time interval and does not require the use of special expensive equipment. Also, the claimed technical solution is aimed at transferring mitochondria to all organs and tissues, which makes it possible to use it for a new purpose, namely, to restore and increase mitochondrial function.

From the investigated prior art, an invention was revealed according to patent No. RU2605853C1 [8] (analogue No. 7) "A method for producing a medicinal product based on human cell vesicles."

The essence of the known technical solution is a method of producing a drug based on human vesicles to stimulate angiogenesis in patients with ischemic tissue damage, characterized in that human cells of the SH-SY5Y line are grown in a culture flask, washed with isotonic buffer solution, and a sterile solution of cytochalasin B is added to the cells , the cells are washed with isotonic buffer solution, put into suspension, subjected to active stirring and a series of centrifugations is carried out to obtain the claimed drug. The method according to claim 1, characterized in that the obtained drug is filtered.

The disadvantage of the known technical solution is the applicant's lack of information about the property of membrane vesicles to contain functionally active mitochondria and the lack of knowledge of the properties of membrane vesicles associated with the transfer of functionally active mitochondria to other cells. The disadvantage is also limited applicability, since the known technical solution is aimed at stimulating angiogenesis in patients with ischemic tissue damage. As a result, the known technical solution is less effective for its intended purpose and limited applicability for its intended purpose as compared to the claimed technical solution.

At the same time, the claimed technical solution provides for the intended use for the entire body, since the membrane vesicles of multipotent stromal cells obtained with the help of cytochalasin B reach all organs and tissues and contain functionally active mitochondria, which makes it possible to use them for a new purpose, namely to restore and enhance mitochondrial function.

Thus, from the studied prior art for all analogues of the claimed technical solution in general, it is possible to draw general conclusions that:

- mtDNA (see analogue of the presented technical solution - 1), which is a part of extracellular vesicles, does not have mitochondrial function, namely, mtDNA is not capable of supporting oxidative phosphorylation reactions;

- transplantation of mitochondria (see analogs of the presented technical solution - 2,3), not protected by a membrane, endangers the preservation of their functional activity due to the impact of destructive factors of the extracellular environment;

- the oral route of administration of the pharmaceutical composition (see analogue of the presented technical solution - 4) is ineffective when used as directed, since part of the pharmaceutical composition is inevitably lost, dissolving in the contents of the gastrointestinal tract or absorbed, enters the liver with the bloodstream, where it is partially destroyed without reaching the focus of the disease;

- chemical compounds or chemically synthesized pharmaceutical compositions (see analogue of the presented technical solution - 5) are unsafe for use as a medicine for humans, since they can have a harmful effect on healthy organs;

- the introduction of cells (BMSCs) carries the risks of unlimited division, which can lead in the future to tumor transformation (see analogue of the presented technical solution - 5);

- natural vesicles of human cells (see analogue of the presented technical solution - 5) are not applicable on a large industrial scale, since the number of vesicles naturally secreted by cells is very small, and the method of isolating natural vesicles is complicated and time-consuming, therefore there is no possibility of their wide clinical application;

- the applicant's lack of information on the property of membrane vesicles to contain functionally active mitochondria and the lack of knowledge of the properties of membrane vesicles associated with the transfer of functionally active mitochondria to other cells (see analogue of the presented technical solution - 6).

The purpose and technical result of the claimed technical solution is to eliminate the disadvantages of analogues, namely:

1 - the use of safe, effective in use, non-immunogenic membrane vesicles of multipotent stromal cells induced by cytochalasin B, which contain functionally active mitochondria and express little or no expression of the molecules of the main histocompatibility complex;

2 - delivery of functionally active mitochondria located inside the MV-CV MCT, and protected by the cytoplasmic membrane from external factors of the extracellular environment, which increases the life of mitochondria and, as a consequence, increases the effectiveness of the claimed technical solution for its intended purpose;

3 - increasing the effectiveness of the intended action, increasing the speed of action and dosing accuracy of MV-CV MCT due to intravenous administration;

4 - obtaining membrane vesicles from multipotent stromal cells of mammals that do not contain any toxins and components not characteristic of an animal cell;

5 - replacement of cell therapy with vesicular therapy, which will avoid the threat of unlimited division and tumor transformation;

6 - the use of a highly effective, competitive, import-substituting new generation means, which does not require a significant time interval, does not require the use of special expensive equipment;

7 - obtaining membrane vesicles of multipotent stromal cells containing functionally active mitochondria and confirming their presence in the composition of membrane vesicles.

The claimed technical solution, according to the applicant, provides an overall increase in the quality of human life by virtue of providing the possibility (full or partial) of replacement of the means and methods of therapy known at the date of submission of the application materials for such intractable diseases as degeneration of the body as a whole (deterioration of the biological characteristics of the body) ), muscle atrophy, myopathy, type 2 diabetes, ischemic heart disease, stroke, cancer, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease) on the possibility of a significant improvement in the health of patients at the first stages of using the claimed technical solution, followed by reaching the stage of complete recovery above presented diseases in the process of improving methods of treatment of these diseases, including through the use of the claimed technical solution.

The essence of the claimed technical solution lies in the use of membrane vesicles of multipotent stromal cells induced by cytochalasin B containing functionally active mitochondria to restore and increase mitochondrial function and deliver functionally active mitochondria to mammalian cells.

Thus, according to the applicant, for the first time in the world, the property of MSC membrane vesicles obtained using cytochalasin B, previously unknown from the prior art studied by the applicant, has been revealed to contain functionally active mitochondria, which provide the possibility of using the claimed technical solution for the treatment of previously incurable diseases. such as degeneration, muscle atrophy and myopathy caused by mutations in mitochondrial DNA, as well as for the treatment of diseases associated with dysfunction of mitochondria (type 2 diabetes, coronary heart disease, stroke, cancer), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, etc. etc.), in which there is a problem of using mitochondrial replacement therapy for adults, and can also be used to slow down the aging of the body.

Thus, in more detail the purpose of the claimed technical solution is to use membrane vesicles of multipotent stromal cells induced by cytochalasin B, obtained from human cells with an increased mitochondrial potential - multipotent stromal cells (MSC).

The claimed technical solution is illustrated by Fig. 1 - Fig. 8.

Figure 1 shows the data of confocal microscopy of human MSCs, where:

1 A - functionally active mitochondria in the cell are colored green (indicated by arrows);

1 B - cell nuclei are painted in blue;

1B - Overlay MitoTracker Green + Hoechst 33342. Scale 20 micrometers.

Figure 2 shows a scanning electron microscopy of human cell vesicles isolated from MSC cells. The micrograph shows individual vesicles of human cells - rounded microstructures surrounded by a membrane (indicated by arrows in the figure), the size of which is in the range of 100-820 nm. Magnification 20 times, scale section 20 micrometers.

Figure 3 shows the analysis of the population of MV-CV MSC for the presence of mitochondria, where:

3 A - the cytoplasmic membrane of membrane vesicles is colored red (DiD dye, indicated by arrows);

3 B - functionally active mitochondria in MV-CV M SC are colored green (dye MitoTracker Green, indicated by insoles);

3 B - MitoTracker Green + DiD photo overlay.

Scale bar 2 micrometers.

Figure 4 shows an analysis of a population of MV-CV MSCs using flow cytometry with an enhanced detector (BD FACS Aria III), where:

4 А - position of MV-CV MSCs containing mitochondria on the dot-plot plot (X-axis - (SSC - side scatter - lateral light scatter) - proportional to the granularity of membrane vesicles, Y-axis (FSC - forward scatter - forward light scatter) - proportional to the size of microvesicles ) (arrows indicate MSC membrane vesicles).

4B - dot-plot plot of microvesicles containing mitochondria (DiD + TMRE +).

Figure 5 shows the analysis of recipient cells (MSCs) incubated with MB-CV MSCs (DiD + TMRE +), where:

5 A - the surface of the recipient cell with inclusions of the MB-CV MSC membrane. Fluorescence in the dark red region of the spectrum - DiD (indicated by arrows),

5 B - mitochondria of the recipient cell. Fluorescence in the green region of the spectrum - MitoTracker Green (indicated by arrows),

5B - introduced mitochondria from MV-CV MSCs. Fluorescence in the red region of the spectrum - TMRE (indicated by arrows),

5 G - the nucleus of the recipient cell. Fluorescence in the blue region of the spectrum - Hoechst 33342),

5 D - overlay photos. Scale section 10 micrometers.

FIG. 6 - presents a comparison of mitochondrial potentials of donor and recipient cells, where:

6 А - mitochondrial potential of MSCs;

6 B - mitochondrial potential of Fb;

6 B - mitochondrial potential A549;

o.e. - relative units;

Δψ is the potential of the mitochondrial membrane.

FIG. 7 shows the assessment of the mitochondrial potential of recipient cells before and after treatment with MV-CV MSCs, where

7 А - mitochondrial potential of MSC recipient cells before and after treatment with MV-CV MSCs,

7B - mitochondrial potential of Fb recipient cells before and after treatment with MV-CV MSCs,

7B - mitochondrial potential of A549 recipient cells before and after treatment with MV-CV MSCs.

* - statistically significant difference (p <0.05);

o.e. - relative units;

Δψ is the potential of the mitochondrial membrane;

24 hours, 2 days, 4 days - the growth period of the cell culture.

FIG. 8 - the table of results of the mitochondrial potential assessment is presented.

o.e. - relative units;

Δψ is the potential of the mitochondrial membrane;

K ^- - control cells, untreated with MV-CV MSC.

In the tests presented below, the presence of functionally active mitochondria in the membrane vesicles of multipotent stromal cells induced by cytochalasin B (MV-CV MSC) was shown.

This application presents experimental data confirming that the claimed technical solution performs the function of delivering mitochondria contained in it to recipient cells. That is, MV-CV MSCs have the effectiveness of the intended action in the field of increasing mitochondrial function.

It should be noted that, in contrast to analogs, the claimed technical solution meets the requirements for pharmaceuticals in terms of biological safety, effectiveness and industrial applicability.

Further, the applicant shows the implementation of the claimed technical solution .

The objectives and technical result of the claimed technical solution are achieved by the fact that MSCs isolated from human subcutaneous adipose tissue are used to obtain membrane vesicles (Fig. 1).

Next, human cells are grown on culture dishes, then detached from the culture vessel, a substance is added - cytochalasin B. Cytochalasin B disrupts the structure of the cytoskeleton, which allows further separation of microvesicles from whole viable cells using a series of centrifugations [9 - Pick, N. et al., Investigating Cellular Signaling Reactions in Single Attoliter Vesicles. JACS, 2005. V.127: p. 2908-2912]. This will make it possible to obtain microvesicles on an industrial scale. Then the cells are subjected to mechanical stress in order to separate the vesicles from human cells. The resulting suspension containing human cells and vesicles is subjected to a series of sequential centrifugations in order to separate the vesicles from whole human cells and cellular debris. The vesicles obtained after the isolation process are called cytochalasin B-induced membrane vesicles of multipotent stromal cells (MV-CV MSCs), while the resulting suspension does not contain viable cells (Figure 2). MV-CV MSCs protect mitochondria from destruction and maintain them in a functionally active state, as well as facilitate the efficient penetration of mitochondria into recipient cells. The proposed development will increase the yield of membrane vesicles.

It should be noted that there is a possibility of introducing a sorting step for the obtained membrane vesicles in order to obtain a uniformly sized fraction containing functionally active mitochondria in the composition, which, according to the applicant's assumption, will lead to the possibility of standardizing the form of the resulting pharmaceutical composition (since at the date a corresponding trend was revealed), however, supporting data can be submitted by the applicant only after completing additional studies, which at the date of submission of the application materials had not been brought to the final stage.

Next, the applicant gives a detailed description of the implementation of the claimed technical solution, which is carried out, for example, in four stages .

The most preferred steps are shown below.

First step. Isolation and cultivation of MSCs.

At the first stage, multipotent human stromal cells are isolated from subcutaneous adipose tissue. Adipose tissue is obtained from healthy donors with written consent after liposuction surgery. The collection of adipose tissue is carried out under sterile conditions. Isolation of stem cells is carried out by standard enzymatic treatment with 0.2% collagenase II solution (Dia-M, Russia). Tissue incubation is carried out at 37 ° C on a shaker at 120 rpm for an hour. The cells were washed three times with PBS (from the English phosphate buffered saline) (PanEko, Russia). The resulting cell pellet is resuspended in α-MEM medium supplemented with 10% fetal bovine serum (Sigma, USA), 2 mM L-glutamine (PanEco, Russia). All work with the cell culture is carried out in a sterile laminar flow hood in accordance with the generally accepted rules of work in a laboratory of the 2nd biosafety class.

MSCs are maintained in DMEM medium (PanEko, Russia), to which 10% fetal bovine serum (FBS - fetal bovine serum) (Sigma-Aldrich, USA), 2 mM L-glutamine (PanEko, Russia) and a mixture of penicillin antibiotics and streptomycin (10 U / ml penicillin and 100 U / ml streptomycin) (PanEco, Russia). Cell cultures are cultured in an inCusaFeMCO-15AC incubator (SANYO Electric Co., Ltd., USA) at 37 ° C in a humidified atmosphere containing 5% CO2.

When passaging cell cultures, the nutrient medium is removed from them, the cell monolayer is washed with a PBS solution (PanEko, Russia) in order to remove the remnants of the nutrient medium and Ca2 + and Mg2 + ions, which inhibit the action of the enzyme. A 0.25% trypsin-EDTA solution in a volume of 2 ml is added to the culture flask to cover the cell monolayer. The culture flask is incubated in an inCusaFeMCO-15AC incubator (SANYO Electric Co., Ltd., USA) at 37 ° C in a humidified atmosphere containing 5% CO2 for 5 minutes. Then trypsin is inactivated by adding complete nutrient medium in a 1: 2 ratio. To get rid of the remains of trypsin, the cells are centrifuged at 1400 rpm for 5 minutes. The cell pellet is resuspended in 1 ml of complete culture medium. Cell counting is carried out using a Goryaev chamber (Lenza, Russia) and the required number of cells is added to the culture flask.

Using the method of flow cytometry, immunophenotyping of MSCs is carried out in order to confirm the expression of surface markers characteristic of human MSCs. Multipotent stromal cells were suspended in PBS solution. Next, monoclonal antibodies are added to the cell suspension - specific surface markers of stem cells, in dilutions recommended by the manufacturer, are incubated for 20 minutes. Then centrifuged for 5 min at a speed of 500 g. Washed off antibodies using PBS. The analysis of the results obtained is carried out using a flow cytometer - sorter: BD FACS Aria III (BD Bioscience, USA). Obtaining multipotent stromal cells with increased mitochondrial potential, complete the first stage of the proposed method and proceed to the second stage.

Second phase. Allocation of MV-TsV MSC.

Then, membrane vesicles induced by cytochalasin B are obtained from cells with increased mitochondrial potential. Obtaining membrane vesicles of human cells that do not contain whole viable cells eliminates the risk of tumor formation as a result of uncontrolled cell division. Thus, using the potential of MSCs, a biologically safe preparation of human cell membrane vesicles is obtained.

Since it is known that the number of membrane vesicles naturally secreted by cells does not provide the possibility of their wide clinical use, as a result of which it can be used exclusively for research purposes, the claimed technical solution proposes to eliminate this drawback. The deficiency is eliminated by stimulating human cells to produce more vesicles with a substance that disrupts the structure of the cytoskeleton - cytochalasin B.

Obtaining membrane vesicles induced by cytochalasin B from a culture of MSC cells is carried out as follows.

When the cell culture reaches a monolayer density of 90%, the culture medium is taken, the culture is washed twice with PBS (PanEko, Russia) from the medium residues. The cells are detached with 0.25% trypsin-EDTA solution (PanEko, Russia), then the cells are washed with PBS (PanEko, Russia).

The cells are incubated in Dulbecco's modified Eagle DMEM medium without blood serum, containing 10 μg / ml cytochalasin B (Sigma-Aldrich, USA) for 30 min at 37 ° C in a humid atmosphere containing 5% CO ₂ , subjected to active stirring for 30 sec.

Then a series of sequential centrifugations is carried out: 500 rpm for 10 minutes, the supernatant is taken and centrifuged at 700 rpm for 10 minutes, the supernatant is taken and centrifuged at 4000 rpm for 25 minutes. The precipitate containing MV-CV is washed with a large amount of PBS (PanEko, Russia), centrifuged at 4000 rpm for 25 min. The resulting precipitate MV-TsV is used further in the work.

The resulting biologically safe product - human cell membrane vesicles is a pharmaceutical product created on the basis of the claimed technical solution. If necessary, it is possible to introduce a sorting step for the obtained product in order to obtain a more uniform in size fraction of membrane vesicles of human cells containing functionally active mitochondria. The results of the second stage - obtaining vesicles of human cells are presented in Fig. 2. Obtaining vesicles of human cells complete the second stage and proceed to the implementation of the third stage of the proposed use as directed.

Thus, at the second stage, a sufficient number of vesicles is obtained for wide clinical use. At the same time, the method of stimulating human cells with a high mitochondrial potential, as well as isolating vesicles of human cells, is not difficult from a technological point of view, does not require much time and special expensive equipment, which meets the criterion of industrial applicability. The number of obtained vesicles of human cells as a result of the second stage of manufacturing the claimed technical solution depends only on the amount of raw materials (human cells with functioning mitochondria).

Stage three. Assessment of the presence of functionally active mitochondria in MV-CV MSCs.

The composition of the membrane vesicles induced by cytochalasin B is analyzed for the presence of functionally active mitochondria. Membrane vesicles of multipotent stromal cells are stained with mitochondrial and membrane dyes to visualize the mitochondria they contain. The dyes DiD and MitoTracker Green are used to achieve the goal.

MitoTracker Green - designed for labeling mitochondria in living cells, has fluorescence in the green region of the spectrum (Ex / Em = 490/520 nm). The dye is retained in mitochondria for a long time. This key feature greatly enhances the efficiency of staining. To stain mitochondria with MitoTracker Green, the dye is added at a final concentration of 200 nM [10-Fu, D. et al., Monitoring the Effects of Pharmacological Reagents on Mitochondrial Morphology. Current Protocols in Cell Biology, 2018. V.79: p. 34-37].

Cells (106 cells / 1 ml) are incubated in DMEM (PanEco, Russia) in an inCusaFeMCO-15AC incubator (SANYO Electric Co., Ltd., USA) at 37 ° C in a humidified atmosphere containing 5% CO ₂ for 20 minutes ... Analyze the fluorescent signal using a flow cytometer - sorter: BD FACS Aria III (BD Bioscience, USA). For staining the cytoplasmic membrane (CPM) of human cells, a vital, DiD dye (cat. No. V22889, Life technologies, USA) is used. This is a lipophilic compound of a non-lipid nature, are persistent dyes and are found in the CPM cells even after three weeks [12 - Yumoto, K. et al., A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD. Cytometry A, 2014. V.85, no. 6: p. 548-555].

Human cells are washed from the culture medium twice with PBS, resuspended in a medium without serum at the rate of 106 cells / 1 ml. The dye is added to a final concentration of 5 mM and the cells are incubated in an inCusaFeMCO-15AC incubator (SANYO Electric Co., Ltd., USA) at 37 ° C in a humidified atmosphere containing 5% CO ₂ for 15 minutes. The cells are washed three times with complete culture medium. Analyzed using a Zeiss LSM780 laser confocal microscope (Carl Zeiss, Germany). DiD dye stains the cytoplasmic membrane of the vesicles (Fig. 3), while MitoTracker Green penetrates functionally active mitochondria, resulting in mitochondria fluorescence in the green region of the spectrum (Fig. 3).

An overlay of photos from MitoTracker Green and DiD shows that the resulting human vesicles contain functionally active mitochondria and are surrounded by a membrane.

In this way, the assessment of the presence of functionally active mitochondria in the membrane vesicles of multipotent stromal cells is completed and the fourth stage is started.

Fourth stage. Delivery of MV-CV MSCs into human cells and assessment of mitochondrial potential.

An analysis of the effectiveness of the action of the obtained membrane vesicles, induced by cytochalasin B, is carried out for the transfer of functionally active mitochondria into recipient cells in vitro by applying vesicles of human cells isolated from MSC to the following types of cells: MSC, fibroblasts and A549.

In order to assess the biological activity of human MSC membrane vesicles carrying functionally active mitochondria, MSC membrane vesicles are applied to recipient cells and the change in the mitochondrial potential of recipient cells is assessed.

At the first stage, the mitochondrial potential of donor cells - MSCs, as well as recipient cells - fibroblasts and A549 is assessed. According to the data obtained (Fig. 6), the mitochondrial potential of MSCs was 1.85 ± 0.2 p.u., of fibroblasts - 1.03 ± 0.1 p.u., A549 - 0.72 ± 0.2 p.u. ... Applicant has found that fibroblasts and A549 have decreased mitochondrial potential (similar to senescent and defective cells). MSCs have an increased mitochondrial potential, which exceeds the mitochondrial potential of fibroblasts by 1.8 times, and the mitochondrial potential of A549 by 2.5 times (Fig. 6).

Thus, the applicant has established that donor MSCs, from which CF-CVs are obtained, carrying mitochondria of donor cells, have an increased mitochondrial potential. At the second stage, recipient cells (MSCs, fibroblasts, A549) with equal (MSC) and reduced (fibroblasts, A549) mitochondrial potentials are treated in order to assess the activity of mitochondrial MSCs introduced with the help of MV-CV MSCs and, as a consequence, a change in mitochondrial potential in recipient cells.

Next, the mitochondrial potential of the recipient cells is assessed before and after treatment with MV-CV MSCs. Mitochondrial transfer using cytochalasin B-induced membrane vesicles from multipotent human stromal cells was performed on three cell lines: MSCs, human fibroblasts and A549 in vitro (Fig. 7). Measurements are carried out 24 hours, 2 days and 4 days after treatment of recipient cells with MV-CV MSCs. The cells are stained with the fluorescent dye jc-1 (Thermo Fisher Scientific, USA) (Fig. 7).

jc-1 is a cationic dye that exhibits voltage-dependent uptake in mitochondria, as indicated by a shift in fluorescence emission from green (~ 525 nm) to red (~ 590 nm). jc-1 is more mitochondrial specific and more accurate in its response to depolarization than other cationic dyes. This dye in monomeric form stains the cytoplasm of cells in green, and the dye aggregates stain mitochondria in red. The ratio of green to red fluorescence depends only on the membrane potential and not on other factors, such as the size, shape, and density of mitochondria, which can influence fluorescence.

The calculation of the mitochondrial potential (Δψ) in the cell population is carried out according to the formula:

Δψ = R / G

Δψ is the potential of the mitochondrial membrane;

R is the number of cells with fluorescence in the red region of the spectrum (staining with the jc-1 dye of the cell mitochondria);

G - the number of cells with fluorescence in the green region of the spectrum (staining of the monomeric form of the dye jc-1 of the cell cytoplasm).

According to the data obtained (Fig. 7 - Fig. 8), the mitochondrial potential of untreated MSC recipients (control cells) after 24 hours was 2.56 ± 0.4 p.u., after 2 days - 5.87 ± 0.4 p.u., after 4 days - 3.47 ± 0.5 p.u. The mitochondrial potential of MSC recipients 24 hours after treatment with their MV-CV MSCs (experiment) was 3.59 ± 0.5 p.u., after 2 days - 7.39 ± 0.3 p.u., after 4 days - 6.77 ± 1.5 p.u.

The mitochondrial potential of untreated Fb recipient cells (control cells) after 24 hours was 1.52 ± 0.6 p.u., after 2 days - 4.61 ± 0.2 p.u., after 4 days - 3.43 ± 0.4 p.u. The mitochondrial potential of Fb recipient cells 24 hours after treatment with their MV-CV MSC (experiment) was 2.69 ± 0.4 p.u., after 2 days - 7.49 ± 0.8 o.u., after 4 days - 6.91 ± 0.6 p.u.

The mitochondrial potential of untreated A549 recipient cells (control cells) after 24 hours was 0.81 ± 0.1 p.u., after 2 days - 1.49 ± 0.2 p.u., after 4 days - 2.02 ± 0.1 p.u. The mitochondrial potential of A549 recipient cells 24 hours after treatment with their MV-CV MSC (experiment) was 1.11 ± 0.1 p.u., after 2 days - 3.16 ± 0.2 p.u., after 4 days - 2.56 ± 0.2 p.u.

The increase in mitochondrial potential during culture growth, which was observed in all cultures of recipient cells over time (24 hours - 4 days) (Fig. 7-8), is probably associated with the logarithmic growth of the culture after subculture into a culture dish, accompanied by an increase in metabolic activity [11 - Miettinen, TP Cellular Allometry of Mitochondrial Functionality Establishes the Optimal Cell Size. Dev Cell, 2016. V. 39, P.370-382.]. Against the background of a natural increase in mitochondrial potential in recipient cell cultures, treatment with MV-CV MSCs led to a statistically significant increase in mitochondrial potential in recipient cells at all periods of observation (p <0.05) (Fig. 7).

Thus, the applicant has shown that MV-CV MSCs contain functionally active mitochondria that can be delivered to recipient cells. At the same time, delivery of mitochondria by MV-CV MSCs to recipient cells with equal and reduced mitochondrial potential leads to an increase in mitochondrial potential in recipient cells. At the same time, no tumor formation is observed, which indicates the safety of the use of MV-CV MSCs based on human cell vesicles.

The product obtained after the completion of the fourth stage is membrane vesicles induced by cytochalasin B, containing functionally active mitochondria, which have an increased (compared to analogs) effectiveness of action when used as directed, and also meet the biosafety criterion, as they are natural cell formations.

Thus, obtaining the claimed membrane vesicles induced by cytochalasin B, containing functionally active mitochondria, complete the fourth, final stage of the implementation of the claimed technical solution. The resulting vesicles were used by the applicant immediately after receipt.

Based on the foregoing, it is possible to conclude that, in general, experimental data have been obtained proving the effectiveness of the use of cytochalasin B-induced membrane vesicles of MSCs containing functionally active mitochondria, namely, the fact of an increase in the mitochondrial potential in recipient cells of MSCs, fibroblasts and A549 has been proved. in vitro (Fig. 7). The quantitative relationship is shown in Fig. 8.

Membrane vesicles of human MSCs are fundamentally incapable of division and tumor formation, which is irrefutable proof, according to the applicant, that the claimed technical solution meets the “non-obviousness” criterion for specialists in this field.

In addition, at the date of registration of the application, the membrane vesicles of multipotent stromal cells containing mitochondria are at the stage of further testing, however, already at this stage of research, it is possible to state the fact that membrane vesicles of multipotent stromal cells have the ability to increase mitochondrial potential in vitro (Fig. 7 ). At the same time, unlike known technical solutions, membrane vesicles of human cells obtained with the help of cytochalasin B contain bioactive molecules, are protected by the cytoplasmic membrane from the negative effects of extracellular enzymes, and their number significantly exceeds the number of vesicles naturally secreted by cells. At the same time, the membrane vesicles of MSCs do not carry the risk of tumor formation, which leads to an increase in the effectiveness of the application for the intended purpose of the claimed technical solution. In addition, human MV-CV MSCs are capable of targeted delivery of their contents to recipient cells, since their membrane contains specific receptors that provide recognition and targeted delivery.

The claimed technical solution seems to be possible to implement in another embodiment, namely, performing the above actions, the preparation of human cell vesicles is stored and used as needed. The human cell vesicle preparation retains its integrity and effectiveness during storage. Thus, the preparation of vesicles of human cells meets the criterion of "industrial applicability" presented to the invention.

Obtained according to the above example, MV-CV MCT can be used for the treatment of diseases in humans and animals. MV-CV MCT obtained using the claimed technical solution can be used for therapeutic purposes for a specific purpose, namely for the treatment of mitochondrial diseases or diseases, the consequence of which is a violation of mitochondrial function, which will provide a therapeutic effect, for example, by direct injection of membrane vesicles. human cells (transplantation) to the patient.

Cells with increased mitochondrial potential are used as cellular material for the production of human membrane vesicles. This condition is met by multipotent stromal cells. It is known that stromal cells have a positive effect on the regeneration processes with the replacement of lost body cells, as well as with paracrine stimulation of neighboring cells. The claimed technical solution for the use of MV-CV MSC combines all the positive properties of MSCs, is not capable of multiplication and tumor formation, and at the same time has sufficient effectiveness for the intended purpose, since the mitochondria enclosed inside the membrane vesicles are protected by the lipid bilayer of the membrane from the destructive effects of lytic enzymes , this fact is also not obvious to specialists and additionally is proof of the presence of an inventive step (non-obviousness).

Thus, from the above, we can conclude that the applicant has achieved all the goals and the claimed technical result , namely:

1 - used safe, effective in use, non-immunogenic membrane vesicles of multipotent stromal cells induced by cytochalasin B, which contain functionally active mitochondria and express little or no expression of molecules of the main histocompatibility complex;

2 - functionally active mitochondria are delivered, which are inside the MV-CV MCT, and are protected by the cytoplasmic membrane from external factors of the extracellular environment, which increases the life of mitochondria and, as a result, increases the effectiveness of the pharmaceutical composition as intended;

3 - the effectiveness of the intended action is increased, the speed of action and the dosing accuracy of MV-CV MCT are increased due to intravenous administration;

4 - membrane vesicles were obtained from multipotent stromal cells of mammals, which do not contain any toxins and components not typical for an animal cell;

5 - the replacement of cell therapy with vesicular therapy has been achieved, which will avoid the threat of unlimited division and tumor transformation;

6 - a highly effective, competitive, import-substituting new generation tool has been applied, which does not require a significant time interval, does not require the use of special expensive equipment;

7 - membrane vesicles of multipotent stromal cells containing functionally active mitochondria were obtained and their presence in the composition of membrane vesicles was confirmed.

The claimed technical solution meets the "novelty" criterion for inventions, since when determining the level of technology, no technical solutions were found that have features identical (that is, coinciding in the function they perform and the form of these features) to all the features listed in the independent paragraph claims.

The claimed method satisfies the criterion of "inventive step" for inventions on the above grounds, since no technical solutions have been identified from the studied prior art that have features that coincide with the essential features of the claimed technical solution and ensure the implementation of the set goals by applying the claimed set of features in according to the above formula. At the same time, according to the applicant, the claimed technical solution is not obvious to a specialist due to the fact that the claimed technical solution is not known to specialists in the studied field of technology.

The claimed technical solution meets the criterion "industrial applicability" for inventions, since it can be implemented in the industrial production of membrane vesicles isolated from human cells, and to apply the claimed technical solution in the activities of health and veterinary organizations through the use of well-known standard technical devices and equipment.

Sources of information

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2. Patent No. US2018036344.

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4. Patent No. IL247447.

5. Patent No. KR20190026045.

6. Patent No. SG11201901689Y.

7. Islam, M. N. et al., Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med, 2012. V. 18: p. 759-765.

8. Patent No. RU2605853.

9. Pick, H. et al., Investigating Cellular Signaling Reactions in Single Attoliter Vesicles. JACS, 2005. V.127: p. 2908-2912.

10. Fu, D. et al., Monitoring the Effects of Pharmacological Reagents on Mitochondrial Morphology. Current Protocols in Cell Biology, 2018. V.79: p. 34-37.

11. Miettinen, T.P. Cellular Allometry of Mitochondrial Functionality Establishes the Optimal Cell Size. Dev Cell, 2016. V.39, P.370-382.

12. Yumoto, K. et al., A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD. Cytometry A, 2014. V.85, no. 6: p. 548-555.

Claims (1)

The use of membrane vesicles of multipotent stromal cells induced by cytochalasin B, surrounded by a bilipid cytoplasmic membrane, carrying surface cytoplasmic receptors (CD90, CD29, CD44, CD73), having a diameter ranging from 100 to 2000 nm and containing functionally active mitochondria, to restore and increase mitochondrial functions by delivering functionally active mitochondria to mammalian cells for the therapy of mitochondrial dysfunction.

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