Patent Application
20230051925
The present disclosure belongs to the field of biomedicine, and relates to a vesicle and a use thereof.
Extracellular vesicles (EVs) are nanoscale carriers containing proteins, nucleic acids and various cytokines secreted by cells. Extracellular vesicles can act on target cells in an endocrine or paracrine manner, and play an important role in the process of substance transmission and information exchange between cells. It has been found that the information exchange mediated by extracellular vesicles is of great significance for regulation in the physiological or pathological process, involving immune regulation, tumor growth, angiogenesis, injury repair, etc. At present, studies in this field mainly focus on exosomes. Exosomes are extracellular vesicles with a diameter of about 30-150 nm, containing RNA, lipids, proteins, etc. Exosomes are widely involved in the various physiological/pathological regulations of the body, and can be used for diagnosis, treatment and prognosis assessment of a variety of diseases. So far, mesenchymal stem cells (MSCs) have been considered to be the most potent cells for producing exosomes. Numerous studies have found that MSCs-derived exosomes can mimic the biological function of MSCs, and play an important part in regulation in promoting cell growth and differentiation and repairing tissue defects. Therefore, cell vesicle therapy based on MSCs-derived exosomes has achieved remarkable development in recent years. However, current exosome-based cell vesicle therapies still have many problems, such as complex exosome extraction and purification, time-consuming, high requirements for equipment and reagents, low physiological exosome production, and so on, which limit the clinical transformation and application of exosome therapies.
Hemophilia is a series of inherited hemorrhagic disorders of coagulation dysfunction with the common feature of disorders in thromboplastin production, which prolongs the clotting time and causes a life-long tendency to bleed after a small injury, and even “spontaneous” bleeding in critically ill patients without apparent trauma. On May 11, 2018, the National Health Commission of the People’s Republic of China and other five departments jointly developed the First Catalog of Rare Diseases, with hemophilia included. There are mainly three forms of hemophilia, namely hemophilia A, hemophilia B and hemophilia C. Hemophilia A, namely factor VIII clotting factor (VIII: C) deficiency, is a sex-linked recessive genetic disease that is transmitted in women and occurs in men. Hemophilia B, namely factor IX (FIX) deficiency, is also sex-linked recessively inherited, and its incidence is less than that of hemophilia A. Hemophilia C, namely factor XI (FXI) deficiency, is a rare hemophilia with incomplete autosomal recessive inheritance. With respect to the incidence, hemophilia A is the most common one, accounting for 80%-85%, hemophilia B is 15%-20%, and hemophilia C is rare. Exogenous coagulation factor injection has long been used as the main intervention measure for treating hemophilia, but this method has many problems, such as high cost, short duration, and easy production of autoantibodies, and therefore cannot provide effective treatment.
In some embodiments, the present disclosure provides a vesicle derived from mesenchymal stem cells.
In some embodiments, the present disclosure provides a vesicle composition.
In some embodiments, the present disclosure provides a pharmaceutical composition including a vesicle and against hemophilia.
In some embodiments, the present disclosure provides a kit for screening or identifying or extracting a vesicle.
In some embodiments, the present disclosure provides a marker for a vesicle.
In some embodiments, the present disclosure provides a method for identifying or selecting vesicles using markers.
In some embodiments, the present disclosure provides a method for production of a vesicle.
In some embodiments, the present disclosure provides a vesicle derived from somatic cells or stem cells, wherein the vesicle is an included vesicle, and the vesicle has markers including Syntaxin 4.
In some embodiments, a method for treatment or prevention or amelioration of a disease or a complication of the disease in a subject, including administering to the subject an effective amount of the vesicle or the vesicle combination or the composition; the disease being a bleeding disorder. In some embodiments, the bleeding disorder includes bleeding due to deficiency of coagulation factors, decreased platelet count and/or functional defects. In some embodiments, the bleeding disorder includes hemophilia, lupus hemorrhage, or Chediak-Higashi syndrome. In some embodiments, hemophilia includes hemophilia A, hemophilia B, or hemophilia C. In some embodiments, the disease is hemophilia A.
In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells. In some embodiments, the stem cells include mesenchymal stem cells and induced pluripotent stem cells (IPSs).
In some embodiments, the somatic cells include osteoblast cell lines.
In some embodiments, the cells may be primary cultured cells, or may be existing or established cell lines.
In some embodiments, the cell line refers to an immortalized cell culture which is capable of propagating indefinitely in an appropriate fresh medium and space.
In some embodiments, the cells may be an established cell strain.
In some embodiments, the induced vesicle refers to a vesicle produced by inducing apoptosis of normally viable stem cells or somatic cells by external impacts.
In some embodiments, the induced vesicle is produced by inducing stem cells or apoptosis of stem cells by addition of Staurospora, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more thereof.
In some embodiments, the possessed markers of the vesicle further include one or more of Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5.
In some embodiments, the vesicle has a combination of the markers Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11, and Integrin alpha 5.
In some embodiments, the vesicle has a high expression of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4.
In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 in the vesicle are higher than that in MSCs or exosomes.
In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 in the vesicle are about 1-2 fold, 2-3 fold, 1-3 fold, 3-4 fold and 3-6 fold, respectively, of that in exosomes derived from mesenchymal stem cells.
In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 in the vesicle are about 1.5-2 fold, 2.5-3 fold, 1.5-2.5 fold, 3.5-4 fold and 3.5-5 fold, respectively, of that in exosomes derived from mesenchymal stem cells.
In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 in the vesicle are about 1.5-1.9 fold, 2.5-2.9 fold, 1.8-2.5 fold, 3.5-3.9 fold and 4-5 fold, respectively, of that in exosomes derived from mesenchymal stem cells.
In some embodiments, the expression levels of the markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 in the vesicle are 1.76 fold, 2.81 fold, 2.41 fold, 3.68 fold and 4.45 fold, respectively, of that in exosomes derived from mesenchymal stem cells.
In some embodiments, the exosome does not express Syntaxin 4, while the vesicle of the present disclosure expresses Syntaxin 4.
In some embodiments, the exosome does not simultaneously express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4, while the vesicle of the present disclosure simultaneously expresses Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4.
In some embodiments, the vesicle and the exosomes are derived from mesenchymal stem cells of the same source.
In some embodiments, the surface membrane proteins of IEVs are analyzed by flow cytometry, and the results show that IEVs derived from MSCs can express similar surface proteins as MSCs, i.e., CD29, CD44, CD73, CD166 positive, and CD34, CD45 negative; at the same time, IEVs can express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles.
In some embodiments, the induced vesicle is produced by inducing apoptosis of mesenchymal stem cells by the addition of staurosporine, ultraviolet irradiation, starvation, thermal stress, or a combination thereof.
In some embodiments, the vesicle is produced by inducing mesenchymal stem cells with staurosporine.
In some embodiments, the passage number of the mesenchymal stem cells may be about 2-5 passages, but is not limited thereto.
In some embodiments, the concentration of staurosporine is in a range of about 1-10000 nM. In some embodiments, the concentration of staurosporine is in a range of about 100-10000 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-10000 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-1000 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-900 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-800 nM.
In some embodiments, the vesicle has a diameter of about 0.03-6 µM. In some embodiments, the vesicle has a diameter of about 0.03-4.5 µM. In some embodiments, the vesicle has a diameter of about 0.03-1 µM. In some embodiments, the vesicle has a diameter of about 0.04-1 µM. In some embodiments, the vesicle has a diameter of about 0.05-1 µM. In some embodiments, the vesicle has a diameter of about 0.1-1 µM. In some embodiments, the vesicle has a diameter of about 0.15-1 µM.
In some embodiments, the present disclosure provides a vesicle combination including the vesicle described above.
In some embodiments, the vesicle combination further includes other prior art vesicles, including, but not limited to, exosomes, migrants, microbubbles, and ectosomes, for example.
In some embodiments, the proportion by count of the vesicle in the vesicle combination is about 65-100%.
In some embodiments, the proportion by count of the vesicle in the vesicle combination is about 75-98%.
In some embodiments, the proportion by count of the vesicle in the vesicle combination is about 80-96%.
In some embodiments, the present disclosure provides a composition including the vesicle described above, or the vesicle combination described above.
In some embodiments, the composition includes drugs, foods, health products, cosmetics, additives, or intermediates.
In some embodiments, the composition is a drug.
In some embodiments, the composition further includes a pharmaceutically or immunologically acceptable carrier.
In some embodiments, the composition is in a form selected from the group consisting of a lyophilized powder, an injection, a tablet, a capsule, a kit, or a patch.
In some embodiments, the vesicle is used as a drug carrier.
In some embodiments, the present disclosure provides a reagent or kit for screening or identification or extraction of the vesicle, including detection reagents for one or more of markers Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4.
In some embodiments, the detection reagents for the markers detect the expression levels of genes of the markers.
In some embodiments, the detection reagents for the markers detect the expression levels of mRNAs of the markers.
In some embodiments, the detection reagents for the markers detect the expression levels of proteins of the markers.
In some embodiments, the detection reagents for the markers are one or more of fluorescent quantitative PCR dyes, fluorescent quantitative PCR primers, fluorescent quantitative PCR probes, antibodies, antibody functional fragments, and conjugated antibodies.
In some embodiments, the kit is selected from one or more of a qPCR kit, an immunoblot assay kit, a flow cytometry assay kit, an immunohistochemical assay kit, and an ELISA kit.
In some embodiments, the kit is selected from a flow cytometry assay kit.
In some embodiments, the present disclosure also provides the use of the vesicle, or the vesicle composition, or the pharmaceutical composition in the preparation of a product for treatment or prevention or amelioration of a disease or a complication of the disease; such disease including liver diseases, and hemophilia.
In some embodiments, the disease is hemophilia, and the vesicle can play a procoagulant effect in vitro, can improve the bleeding tendency of mice with hemophilia after in vivo injection, and can be used for the treatment of improving the bleeding tendency of hemophilia, providing good application prospects.
In some embodiments, the disease is hemophilia A.
In some embodiments, the product includes drugs, foods, health products, cosmetics, additives, or intermediates.
In use of the vesicle for disease treatment, the vesicle may optionally be administered in a route selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection or infusion, and intraorgan infusion. For example, for intravenous injection, tail vein injection may be used. Intraorgan infusion includes infusion into anatomical spaces such as, by way of example, the gall bladder, gastrointestinal lumen, esophagus, pulmonary system (by inhalation), and/or bladder.
As an example, for intraperitoneal injection in gastrointestinal infusion, the same therapeutic effect can also be obtained by intraperitoneal injection as compared with tail vein injection. Intraperitoneal injection is superior to tail vein injection in safety and maneuverability.
In some embodiments, the present disclosure also provides a method for selecting or identifying the vesicle, including detecting one or more of the following markers: Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4.
When the detection result shows positive for the markers, the vesicle is identified.
In some embodiments, the expressions of the markers may be compared to a control, and the result is positive when the expression level is significantly higher than that of the control. The control may be other existing vesicles or exosomes (may include one or more of exosomes, migrants, microbubbles, and ectosomes); other vesicles or exosomes may be derived from mesenchymal stem cells.
Among the markers, the marker Syntaxin is particularly preferred. In some embodiments, the vesicle (e.g. an induced vesicle) is identified when the expression level of Syntaxin 4 in a test vesicle is greater than or equal to 2-6 fold (more preferably 4-5 fold) of that in an exosome (e.g. an exosome from the same cell source).
In some embodiments, the present disclosure provides use of a detection reagent for a marker in the preparation of a reagent or kit for detection or identification of a vesicle, in which the markers include one or more of Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4, and the reagent or kit further includes a control reagent which includes one or more of exosomes, migrants, microbubbles, and ectosomes, and when the expression levels of the markers in the test sample is higher than that in the control reagent, the result is positive.
In some embodiments, the control agent is an exosome.
In some embodiments, the vesicle (e.g. an induced vesicle) is identified when the expression level of Syntaxin 4 in the test sample is greater than or equal to 2-6 fold of that in the exosome.
In some embodiments, the vesicle (e.g. an induced vesicle) is identified when the expression level of Syntaxin 4 in the test sample is greater than or equal to 4-5 fold of that in the exosome.
In some embodiments, the present disclosure provides a method for production of the vesicle described above, including inducing stem cells or somatic cells to produce the vesicle with an apoptosis-inducing agent agent.
In some embodiments, the method includes following steps: (1) culturing mesenchymal stem cells; (2) collecting the culture supernatant of the mesenchymal stem cells; and (3) separating a vesicle from the culture supernatant in step (2).
In some embodiments, the step of culturing mesenchymal stem cells in step (1) includes: (4) separating mesenchymal stem cells from a tissue; and (5) adding a culture medium to culture the mesenchymal stem cells; and contacting the mesenchymal stem cells in the culture medium with the apoptosis inducing agent.
In some embodiments, the apoptosis-inducing agent includes staurosporine, ultraviolet irradiation, starvation, or thermal stress, or a combination of one or more thereof.
In some embodiments, the apoptosis-inducing agent is staurosporine.
In some embodiments, the concentration of staurosporine is in a range of about 500-1000 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-900 nM. In some embodiments, the concentration of staurosporine is in a range of about 500-800 nM.
In some embodiments, the cells are treated with the apoptosis inducing agent in step (5) for about 16-24 hours.
In some embodiments, in step (3), separating a vesicle includes separating a vesicle by ultracentrifugation.
In some embodiments, a single MSCs in the present disclosure is capable of yielding 300-2000 vesicles.
In some embodiments, the step of separating a vesicle by ultracentrifugation includes: (a) performing a first centrifugation on the collected culture supernatant, and taking the supernatant; (b) performing a second centrifugation on the supernatant collected in step (a), and taking the supernatant; (c) performing a third centrifugation on the supernatant collected in step (b), and taking the pellet; (d) performing a fourth centrifugation on the pellet collected in step (c), and taking the pellet; and (e) performing a fifth centrifugation on the pellet collected in step (c), and taking the pellet.
In some embodiments, the first centrifugation is performed at about 500-1500 g for 5-30 min. In some embodiments, the first centrifugation is performed at about 500-1000 g for 5-20 min. In some embodiments, the first centrifugation is performed at about 500-900 g for about 5-15 min. In some embodiments, the second centrifugation is performed at about 1000- 3000 g for 5-30 min. In some embodiments, the second centrifugation is performed at about 1500- 2500 g for 5-20 min. In some embodiments, the second centrifugation is performed at about 1500- 2200 g for about 5-15 min. In some embodiments, the third centrifugation is performed at about 10000-30000 g for 15-60 min. In some embodiments, the third centrifugation is performed at about 12000-25000 g for 20-60 min. In some embodiments, the third centrifugation is performed at about 12000-20000 g for 20-40 min. In some embodiments, the fourth centrifugation is performed at about 10000-30000 g for 15-60 min. In some embodiments, the fourth centrifugation is performed at about 12000-25000 g for 20-60 min. In some embodiments, the fourth centrifugation is performed at about 12000-20000 g for 20-40 min.
In some embodiments, a vesicle bearing specific markers may be enriched by enrichment methods for specific markers. After sufficient vesicles are obtained, the culture medium is collected, and a specific vesicle is purified and separated from the culture medium. This may be achieved by any suitable method known in the art. Such methods include, for example, original methods of separating exosomes by differential ultracentrifugation, as well as more recent methods such as polymer precipitation (ExoQuickTM from SBI, Palo Alto, CA), immunoaffinity capture (Greening et al. 2015, Methods in Molecular Biology), immunomagnetic capture (exo-FLOWTM, SBI) and the like.
Immunoaffinity purification is a method for selective capture of a specific vesicle based on surface markers. The efficient capture of a vesicle is achieved by high affinity coupling between streptavidin covalently coated magnetic beads and biotinylated capture antibodies. The captured vesicle, after elution, is structurally intact and biologically active. Based on the discovery of the present disclosure that the vesicle specifically express Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5, and Syntaxin 4 molecules at high levels, the method may be used in the present disclosure separate or purify or enrich the vesicle.
In some embodiments, the vesicle can also be enriched by a method of immunomagnetic beads, wherein the immunomagnetic beads are obtained by coupling an antibody to magnetic beads; the monoclonal antibody further includes one or more of an anti-Annexin V antibody, an anti-Flotillin-1 antibody, an anti-Cadherin 11 antibody, and an anti-Integrin alpha 5 antibody.
In some embodiments, the present disclosure further provides an induced vesicle derived from IPS cells. It is a subcellular product produced by intervening or inducing apoptosis of IPS cells when the IPS cells are normally viable.
When the term “enrich” is used in this application, it covers the separation of one or more vesicles from any other vesicles present in a sample or it means that a vesicle present in a composition including the vesicle with a content of a higher total percentage than when it is found to be present in a tissue of an organism.
In an embodiment, the enriched vesicle is not separated from the sample, but is subjected to any diagnosis while it is still present in the sample. The sample may then be presented on a slide, and may be diagnosed using a microscope, and in this embodiment, the vesicle is detected without being separated.
In another embodiment, the enriched vesicle is separated from the sample.
Immunomagnetic bead-based separation (IMS) is a new immunological technique developed in recent years. Immunomagnetic beads (IMB) can not only bind to active protein antibodies, but also can be attracted by magnets. After processing, an antibody may be bound to the magnetic beads such that the magnetic beads serve as a carrier of the antibody. After the antibody on the magnetic beads binds to a specific antigen, an antigen-antibody-magnetic bead immune complex is formed, and such complex mechanically moves under the action of magnetic force, so that the complex is separated from other substances, thus achieving the purpose of separating the specific antigen. Immunomagnetic beads (IMB) provide a platform which can be applied in all fields utilizing the principle of antigen-antibody binding, and its use has achieved remarkable results in the fields of medicine and biology for, such as, bone marrow transplantation, separation of stem cells, organelles, cancer cells, hormones, pathogens and toxins, etc. In recent years, IMB has been widely used in the separation and detection of mycotoxins in foods, water, biological samples and environmental samples due to its high sensitivity and specificity, showing good development and application prospects.
The method of immunomagnetic beads described in the present disclosure is a method in which magnetic beads, which are bound to a specific antibody, bind to a target vesicle with a specific surface antigen, and then are absorbed by a magnetic field adsorption for extraction of the target vesicle.
In some embodiments of the present disclosure, the vesicle is enriched specifically by adding immunomagnetic beads coated with one or more antibodies of an anti-Syntaxin 4 antibody, an anti-Annexin V antibody, an anti-Flotillin-1 antibody, an anti-Cadherin 11 antibody, and an anti-Integrin alpha 5 antibody to the cell culture supernatant containing the vesicle. In this way, the vesicle which specifically binds to one or more antibodies of the anti-Syntaxin 4 antibody, the anti-Annexin V antibody, the anti-Flotillin-1 antibody, the anti-Cadherin 11 antibody, and the anti-Integrin alpha 5 antibody can be separated, and the purpose of enriching a specific vesicle can be achieved. In some preferred embodiments, when immunomagnetic beads coated with all of the anti-Syntaxin 4 antibody, the anti-Annexin V antibody, the anti-Flotillin-1 antibody, the anti-Cadherin 11 antibody, and the anti-Integrin alpha 5 antibody are used to separate a vesicle, the vesicle can be separated with the highest purity, and enables the most effective treatment for diseases such as hemophilia A.
In some preferred embodiments, optimization is made by a combined method in which centrifugation is firstly performed to efficiently remove impurities such as cells and cell debris in the cell culture supernatant, and then immunomagnetic beads coated with one or more antibodies of an anti-Syntaxin 4 antibody, an anti-Annexin V antibody, an anti-Flotillin-1 antibody, an anti-Cadherin 11 antibody, and an anti-Integrin alpha 5 antibody are added to the cell culture supernatant containing the vesicle. In this way, the vesicle which specifically binds to the antibodies can be separated, and the purpose of enriching a specific vesicle can be achieved.
In some embodiments, the mesenchymal stem cells are derived from a human or a mouse, but are not limited thereto.
In some embodiments, the mesenchymal stem cells include, but are not limited to, bone marrow-derived mesenchymal stem cells, urine-derived mesenchymal stem cells, oral cavity-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, periosteum-derived stem cells, or a combination thereof.
In some embodiments, the mesenchymal stem cells are selected from the group consisting of bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and oral cavity-derived mesenchymal stem cells.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Note:
The embodiments of the present disclosure are further illustrated by the following specific examples, which are not intended to limit the scope of the disclosure. Certain insubstantial modifications and adaptations made by others in light of the disclosed concepts remain within the scope of the disclosure.
The IEVs in the examples of the present disclosure is an abbreviation for induced vesicles, and induced extracellular vesicles (IEVs). An induced extracellular vesicle refers to a subcellular product that is produced by intervening or inducing apoptosis of precursor cells (e.g. stem cells) which are normally viable. Such subcellular product generally has a membrane structure, expresses apoptotic markers, and partially contains the genetic material DNA. The inventors have found that the induced extracellular vesicle is a substance that distinguishes between cells and conventional extracellular vesicles (e.g. exosomes, etc.). In some embodiments, normal viable cells are, for example, non-apoptotic cells, non-senescent cells, non-senescent cells with arrested proliferation, non-post-cryopreservation cells, non-malignant cells with abnormal proliferation, or non-damaged cells, etc. In some embodiments, the normally viable cells are taken from cells at 80-100% confluence in the cell culture process. In some embodiments, the normally viable cells are taken from cells in the log phase. In some embodiments, the normally viable cells are taken from primary culture cells derived from a human or murine tissues and subculture cells thereof. In some embodiments, the normally viable cells are taken from an established cell line or cell strain. In some embodiments, the precursor cells are taken from early cells.
In the present disclosure, IEV is equivalent to IEVs. In the present disclosure, STS refers to staurosporine. In the present disclosure, exosomes refer to exosomes.
“Including” or “comprising” is intended to mean that the compositions (e.g. media) and methods include the recited elements, but not excluding others. When used to define compositions and methods, “consisting essentially of” means excluding other elements of any significance to the combination for the stated purpose. Thus, a composition substantially consisting of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel features of the claimed disclosure. “Consisting of” means excluding trace elements of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.
An “effective amount” is an amount sufficient to achieve a beneficial or desired result, e.g., an enhanced immune response, treatment, prevention, or amelioration of a medical condition (disease, infection, etc.), and so on. An effective amount may be applied in one or more administrations, applications or dosages. The appropriate dosage will vary depending on the body weight, age, health, the disease or condition to be treated, and the way of administration.
As used herein, the terms “high expression” and the like are intended to include increased expression of nucleic acids or proteins to levels higher than those contained in prior art vesicles (e.g. exosomes).
As used herein, the term “pharmaceutically acceptable carrier” refers to any of standard pharmaceutical carriers, such as lyophilized powders, injections, tablets, capsules, kits, or patches. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavoring and color additives, or other ingredients. Examples of pharmaceutically acceptable carriers include, but are not limited to, the following: water, saline, buffers, and inert non-toxic solids (e.g. mannitol and talc). Compositions including such carriers are formulated by well-known conventional methods. Depending on the intended mode of administration and intended use, the compositions may be in the form of solid, semi-solid or liquid dosage forms, such as powders, granules, crystals, liquids, suspensions, liposomes, pastes, creams, ointments, and the like, and may be in unit dosage forms suitable for administration of relatively precise dosages.
In the present disclosure, the components of the “composition” may be present in admixture or may be packaged separately. Separately packaged components may also contain their respective adjuvants. The adjuvant refers to a means used in pharmacy to aid the therapeutic efficacy of a drug. Where the components of the composition are packaged separately, the separately packaged components may be administered simultaneously or sequentially in any order where the patient is first treated with one drug and then administered with another drug. The patient refers to a mammalian subject, particularly a human subject.
In the present disclosure, the “composition” may also be present such that one component is encapsulated by another component. In some embodiments, in the composition, the induced vesicle serves as a drug carrier, and a drug for treating or preventing a disease is encapsulated in the induced vesicle.
In the present disclosure, the sources of corresponding reagents are as follows: Penicillin/Streptomycin Solution (BIOSOURCE; P303-100); Glutamine (BIOSOURCE; P300-100); Dexamethasone Sodium Phosphate (Sigma; D-8893); a-MEM (Gibco; 12571-063); 2-ME (GIBCO; 21985-023).
In accordance with the guidance of the Animal Ethics Committee, mice were sacrificed with excess CO2. Under sterile conditions, the tibia and femur were removed, and the attached muscle and connective tissue were stripped out. Then the metaphysis was further separated to expose the bone marrow cavity. PBS containing 10% by volume of fetal bovine serum was drawn using a 10 mL sterile syringe to repeatedly flush the bone marrow cavity, and was filtered with a 70 µm pore cell strainer, and centrifuged at 500 g for 5 min. After the supernatant was removed, the cell pellet at the bottom was collected, and then resuspend in PBS and centrifuged at 500 g for 5 min again to collect the final cell pellet. The cells were then subjected to flow cytometry sorting with CD34-and CD90+ as sorting criteria, such that BMMSCs were separated. Finally, the cells were resuspended in a Dex (-) culture solution, and inoculated to a 10 cm diameter cell culture dish and cultured at 37° C. in 5% CO2. After 24 h, non-adherent cells in the supernatant were removed by aspiration, washed with PBS, and added into the Dex (-) culture solution for further culture. After 1 week, an equal amount of Dex (+) culture solution was added, and after another week, dense primary BMMSCs colonies were observed. The BMMSCs were digested by trypsin incubation at 37° C., and passaged for amplification. Thereafter, the Dex (+) culture solution was changed every 3 days, and subcultured if confluent. The BMMSCs of second passage (P2) were taken for subsequent experiments.
The composition of the Dex (-) culture solution is shown in Table 1, and the composition of Dex (+) culture solution is shown in Table 2:
by flow cytometry analysis of surface markers. For surface marker identification, P2 BMMSCs were harvested by trypsinization, and then washed once with PBS containing 3% FBS to be resuspended in the PBS at a density of 5×105 /mL. Then, 1 µL of PE fluorescent conjugated CD29, CD44, CD90, CD45, and CD34 antibodies were added, with the blank group left. Incubation was carried out for 30 min in the dark at 4° C., followed by washing with PBS for 2 times, and then tests were carried out on the instrument. The results of flow cytometry are shown in
The MSCs cultured to the second passage in Example 1 (bone marrow-derived MSCs, BMMSCs) was further cultured with the medium (Dex (+) culture solution) in Example 1 until the cells were 80%-90% confluent, then washed twice with PBS, added with a serum-free medium (α-MEM medium) containing 500 nM STS to induce apoptosis, and incubated at 37° C. for 24 h, and the cell supernatant was collected for separation and extraction of IEVs.
The IEVs were separated and extracted from the collected cell supernatant. The operation process is shown in
The MSCs cultured to the second passage in Example 1 (bone marrow-derived MSCs, BMMSCs) was further cultured with the medium in Example 1 until the cells were 80%-90% confluent, then washed twice with PBS, added with a serum-free medium, and incubated at 37° C. for 48 h, and the cell supernatant was collected for separation and extraction of exosomes.
The extraction steps include: centrifuging at 800 g for 10 min — collecting supernatant — centrifuging at 2000 g for 10 min — collecting supernatant — centrifuging at 16000 g for 30 min — collecting supernatant — centrifuging at 120000 g for 90 min — removing supernatant and resuspending pellet in sterile PBS — centrifuging again at 120000 g for 90 min, removing supernatant, collecting bottom exosomes, and resuspending in sterile PBS.
The quantitative analysis of IEVs obtained in Example 2 was performed using flow cytometry, and the time points for measurement were 1 h, 4 h, 8 h, 16 h and 24 h. The results showed that 106 MSCs could produce 0.76×108, 1.29×108, 1.95×108, 2.48×108 and 3.14×108 IEVs after induction to 1 h, 4 h, 8 h, 16 h and 24 h, respectively. It can be seen that a single MSC could produce 300 IEVs after induction to 24 h (
In addition, the particle diameter distribution of IEVs found by flow cytometry was concentrated within less than 1 µm, accounting for 94.97% (
The surface membrane proteins of the IEVs extracted in Example 2 were analyzed by flow cytometry, and the results showed that the IEVs derived from MSCs expressed surface proteins similar to MSCs, i.e., CD29, CD44, CD73, CD166 positive, and CD34, CD45 negative. At the same time, IEVs could express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles (
Proteomic quantification of BMMSCs, MSCs-exosomes (extracted in Comparative Example 1), and MSCs-IEVs (obtained in Example 2) was performed using the protein DIA quantification technique. The results showed that the protein content expression of MSCs-exosomes and MSCs-IEVs had a high overlap with that of the parent cells, and 170 proteins were specifically highly expressed in IEVs (
Through bioinformatics analysis, IEVs-specific and highly expressed proteins were screened, and a heat map was drawn (
MSCs-Exosomes: exosomes derived from BMMSCs.
MSCs-IEVs: IEVs derived from BMMSCs.
The MSCs in the content analysis and the MSCs for extraction of the exosomes and IEVs are from the same BMMSCs cell strain.
The in vitro procoagulant effects of the IEVs obtained in Example 2 and the exosomes extracted in Comparative Example 1 were examined using an in vitro clotting assay. The results, as shown in Table 3, showed that the IEVs shortened the in vitro coagulation time of most plasma, and the procoagulant effect was better than exosomes. However, for the plasma deficient in factors II, V, and X, the IEVs failed to exert the in vitro procoagulant effect, indicating that the in vitro procoagulant effect of IEVs was more concentrated at the upstream of the common coagulation pathway.
Mice with hemophilia A (clotting factor VIII deficiency) were used as a model to observe the procoagulant effect of IEVs in vivo by tail vein injection of 9×108 IEVs. As shown in
The results showed that IEVs could significantly promote coagulation in vitro. Besides, it can significantly improve the bleeding tendency after in vivo injection, and can be used to improve the bleeding tendency caused by hemophilia A.
The levels of various coagulation factors in the plasma of mice were also tested, and it was found that there were no significant changes in the coagulation factor VIII, vWF factor, tissue factor (TF) and prothrombin (
In the hemophilia A mice model, normal IEVs, PS-negative IEVs and TF-negative IEVs were separately injected, with a tail-cutting test performed 7 days later. As shown in
In clinical practice, patients with systemic lupus erythematosus (SLE) often have a bleeding tendency, the specific mechanism is unclear at present, and according the existing literature reports, it is mostly believed to be related to the factors such as thrombocytopenia associated with SLE. In the treatment measures, blood transfusion or platelet transfusion methods are often used, but the effect is not ideal.
According to the present invention, 1pr mice were injected with IEVs (extracted in Example 2), and subjected to tail-cutting experiments 7 days later. The results showed that, the treatment with IEVs significantly improved the bleeding tendency of 1pr mice, and the treatment effect was stably maintained for 7 days (
The experimental results showed that IEVs could be used to improve the bleeding tendency of lupus erythematosus.
Chediak-Higashi syndrome (CHS) is an autosomal recessive genetic disease, most of which is found in the offspring of consanguineous marriage. The pathogenic gene is the lysosomal trafficking regulator (LYST) gene. Mutations in the LYST gene often result in abnormal LYST protein production, which leads to platelet dysfunction. Patients may present with a significant bleeding tendency in clinical practice, and currently there is no effective preventive and therapeutic measures.
According to the present invention, the CHS mice were injected with IEVs (obtained in Example 2), and subjected to tail-cutting experiments 10 days later. The results showed that, the treatment with IEVs significantly improved the bleeding tendency of CHS mice, and the treatment effect was stably maintained for 10 days (
The results showed that IEVs could be used to improve the bleeding tendency of mice with Chediak-Higashi syndrome.
In the hemophilia A mice model, the IEVs (obtained in Example 2) and exosomes (extracted in Comparative Example 1) derived from the same MSCs were separately used for injection treatments (9×108), and the results showed that the IEVs could significantly correct the bleeding tendency of mice, while the exosomes had no significant treatment effect (
The in vitro procoagulant effect of the IEVs obtained in Example 2 was compared with that of the exosomes prepared in Comparative Example 1:
the IEVs obtained in Example 2 had a diameter in the range of 0.03 µm - 0.2 µm and 0.2 µm - 1 µm, and expressed the markers Syntaxin 4, Annexin V, Flotillin-1, Cadherin 11 and Integrin alpha 5, with a strong in vitro procoagulant effect; the exosomes prepared in Comparative Example 1 had a diameter of 0.03 µm - 0.15 µm, and expressed the markers Complement C1q, Complement C3, Thrombospondin-1 and Thrombospondin-2, with a relatively weak in vitro procoagulant effect.
Purchased MC3T3-E1 Subclone 14 was thawed rapidly and centrifuged at 500 g for 5 min. The cell pellet at the bottom was collected after removing the supernatant. The cells were resuspended in a Dex (-) culture solution and inoculate on a 10 cm diameter cell culture dish at 37° C. in 5% CO2. After full growth, trypsin was used for incubation and digestion at 37° C. for subculture and amplification, and then the Dex (-) culture medium was changed every 3 days. The cells could be used for multiple passages. The composition of the Dex (-) culture solution is shown in Table 5:
IEVs derived from osteoblast MC3T3-E1, iPS cells and human bone marrow mesenchymal stem cells (hBMMSCs) were compared. The three cell-derived IEVs were obtained as described in Example 2.
As shown in
Flow cytometry was performed, and the results were as shown in
As shown in
4×106 IEVs prepared in Example 2 was labeled with DIR, and resuspended in 200 µL of PBS, and then systemically injected into nude mice BALB/c-nu/nu through the tail veins. The distribution of IEVs on the skin surface was observed after 1, 3 and 7 days using a living body imaging instrument, and the results were as shown in
The culture of hiPSCs is the same as that in Example 5, and hUCMSCs is also a conventional culture method in the art.
The hiPSCs can be of the 26th-29th passage, but is not limited thereto, and the 26th passage is specifically used in this example; the hUCMSCs may be of the 7th-9th passage, but is not limited thereto, and the 7th passage is the specific used in this example.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010066154.6 | Jan 2020 | CN | national |
| 202010066155.0 | Jan 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/072938 | 1/20/2021 | WO |
