WO2022228516A1

WO2022228516A1 - Nasal drops containing stem cell-derived extracellular vesicles and use thereof in treatment of cerebral neurovascular diseases

Info

Publication number: WO2022228516A1
Application number: PCT/CN2022/089901
Authority: WO
Inventors: 戴成祥; 雷继刚; 李苏克; 常晶; 李萍; 王静; 李蒙; 陈应炉; 盛一凡
Original assignee: 西比曼生物科技（上海）有限公司
Priority date: 2021-04-28
Filing date: 2022-04-28
Publication date: 2022-11-03
Also published as: CN115245521A

Abstract

Provided are a nasal drop preparation containing genetically engineered stem cell-derived vesicles and a use thereof in treatment of cerebral neurovascular diseases. Specifically, provided are a nasal drop preparation which takes an active substance in stem cell-derived vesicle contents having nerve cell targeting capabilities as a main active ingredient and a preparation method therefor, and a use of the preparation in treatment of Alzheimer's disease. After the preparation is administered by dropping the nasal cavity, the main ingredient of the preparation can effectively penetrate the blood brain barrier and is taken up by microglia and nerve cells, so that the main active ingredient in the stem cell-derived vesicles is released, and a significant inflammation inhibition effect is generated; moreover, the expression level of amyloid precursor protein hydrolase BACE1 is reduced, the expression level of ADAM10 is maintained, and generation of amyloid Aβ is inhibited, thereby effectively preventing or treating neurodegenerative diseases such as the Alzheimer's disease.

Description

Nasal drops containing stem cell extracellular vesicles and its application in the treatment of cerebral neurovascular diseases

technical field

The invention relates to the field of stem cell therapeutic drugs, in particular to a nasal-drop preparation of extracellular vesicles secreted by stem cells and its application in the treatment of cerebral neurovascular diseases.

Background technique

Alzheimer's disease (AD), commonly known as senile dementia, is a severe age-related neurodegenerative disease characterized by the accumulation of amyloid beta protein (Aβ) to form plaques, hyperphosphorylation of Tau protein. It induces the formation and accumulation of neurofibrillary tangles, the loss of synapses and neurons, and the decline of cognitive function (Zhang et al., 2016). Studies have shown that amyloid precursor protein (APP) forms Aβ under the action of BACE1 hydrolase, and Aβ can induce intracellular IκB phosphorylation and subsequent degradation, enabling NF-κB to activate and enter the nucleus to regulate inflammation-related gene transcription ( Li et al., 2018). Therefore, the development of AD is accompanied by chronic inflammation.

With an aging population and increased life expectancy, Alzheimer's disease will become the most common neurological disorder in the world. In 2019, an estimated 44 million AD cases have been reported worldwide (Chopra et al., 2020). The prevalence of AD is expected to double every 20 years due to an increase in life expectancy of the global population, implying that 131 million people will suffer from the disease by 2050 (Shaik et al., 2018).

Currently approved drugs for AD treatment mainly include cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists. The use of these drugs can cause patients to experience varying degrees of side effects or lower efficacy (Briggs et al., 2016; Shaik et al., 2018). In addition, many preclinical studies have shown that reduced expression or activity of BACE1 can reverse the pathology of AD mice (Hu et al., 2018), but clinical trials have not been successful. In fact, most anti-AD drug candidates were BACE1 inhibitors but were withdrawn due to their toxicity or lack of efficacy (Chopra et al., 2020). Because these drugs reduce the expression level or activity of BACE1 systemically, thereby weakening the protein's important role in liver metabolism, they are prone to abnormal liver function (Kitazume et al., 2005; Lahiri et al., 2014; Shaik et al. al., 2018). Therefore, in order to avoid the above-mentioned side effects caused by the use of conventional AD drugs, reduce the uncontrollable liver tissue toxicity caused by systemic administration, control the course of the disease, and improve the quality of life of patients, it is urgent to find a new AD treatment strategies and treatments.

A large number of studies have shown that many miRNAs play an important role in the regulation of BACE1 expression and the inhibition of inflammation-related NF-κB signaling pathway in the development of AD (Shaik et al., 2018). Therefore, these miRNAs can be potential molecular targets for the treatment of AD. Extracellular vesicles or exosomes produced by mesenchymal stem cells can penetrate the blood-brain barrier (Morales-Prieto et al., 2009) and are rich in miRNAs related to tissue repair and anti-inflammatory effects (

et al., 2020). Therefore, stem cell extracellular vesicles or exosomes have great application potential in the prevention and treatment of nervous system-related diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a nasal-drop preparation and a preparation method thereof using the active ingredients that are abundant in the genetically engineered stem cell extracellular vesicles that regulate targets related to inflammation and amyloid β formation as effective ingredients, and The application of the preparation in the treatment of neurodegenerative diseases such as Alzheimer's disease.

The first aspect of the present invention provides a preparation containing stem cell extracellular vesicles, wherein the stem cell extracellular vesicles of the preparation contain active ingredients for regulating targets related to inflammation and amyloid β formation; The preparation is used for:

(a) Inhibit the inflammatory response of brain nerve tissue;

(b) inhibiting the production of amyloid Aβ; and

(c) preventing and/or treating neurodegenerative diseases.

In another preferred embodiment, the preparation contains 1×10 ⁵ to 9×10 ⁸ stem cell extracellular vesicles/ml.

In another preferred embodiment, the stem cell extracellular vesicles are derived from the supernatant collected during the in vitro culture of genetically engineered human stem cells.

In another preferred embodiment, the human stem cells are selected from the group consisting of human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, and human adipose-derived mesenchymal stem cells , human bone marrow-derived stem cells, or a combination thereof.

In another preferred embodiment, the human stem cells are human adipose-derived mesenchymal stem cells.

In another preferred embodiment, the stem cell extracellular vesicle contains a complete lipid bilayer structure, the membrane surface of the structure contains specifically expressed CD9, CD63, and CD81, and the membrane contains TSG101 and HSP70.

In another preferred embodiment, the membrane of the stem cell extracellular vesicle also contains DNA and RNA with certain non-coding functions.

In another preferred embodiment, the DNA and RNA with non-coding functions include DNA and RNA molecules with non-coding functions produced by stem cells cultured in a natural state, and DNA and RNA with non-coding functions produced by the expression of genetically engineered stem cells Molecules, non-coding functional DNA and RNA molecules, or combinations thereof, that are chemically synthesized and transduced into stem cells.

In another preferred embodiment, the RNA includes miRNA, tRNA, rRNA, snoRNA and snRNA.

In another preferred embodiment, the RNA includes a miRNA or its precursor miRNA with a length of 17-100 nucleotides.

In another preferred example, the cells used to produce the stem cell extracellular vesicles include cells from the following sources: specific gene modification, specific gene editing, specific gene transduction, and specific microRNA miRNA introduction in a GMP laboratory obtained cells.

In another preferred embodiment, the stem cell extracellular vesicles have the following characteristics: they are prepared by a polyethylene glycol (PEG) precipitation method, and the diameters of the extracellular vesicles are concentrated between 80-200 nm, and the particle diameters are uniform.

In another preferred embodiment, the polyethylene glycol (PEG) precipitation includes the steps of: using PEG of 3000-9000 PEG, using PBS to prepare a 8%-20% PEG stock solution, and sterilizing by filtration (for example, using 0.22 μm PEG). filter), added to the treated conditioned medium in a certain proportion (such as about 1:1 volume ratio) (the conditioned medium is processed by differential centrifugation and filtration to treat the culture supernatant of human-derived adipose-derived mesenchymal stem cells). solution), incubate at 4°C overnight; then centrifuge at 3,000-5000g for 30-60 minutes at 4°C, discard the supernatant, and add pre-cooled PBS to resuspend the pellet; 4°C, 100000-120000g ultracentrifugation for 60-120 minutes, discard the supernatant to obtain the stem cell extracellular vesicles.

In another preferred embodiment, the stem cell extracellular vesicles have the following characteristics: (a) their particle size is small, and they can penetrate the blood-brain barrier and enter the brain nerve tissue; (b) the small miRNA molecules in them can be expressed in the stem cell cells. The outer vesicles enter the body and are released to reach the brain.

In another preferred example, the preparation has the following characteristics: the stem cell extracellular vesicles in the preparation are genetically engineered to have brain nerve tissue-specific targeting ability, which can convert the activity of stem cell extracellular vesicles into the preparation. The components are targeted for delivery into brain nerve tissue for uptake by nerve cells and microglia.

In another preferred embodiment, the active ingredient in the stem cell extracellular vesicle can simultaneously exert an inhibitory effect on inflammatory response and reduce the expression of amyloid precursor proteolytic enzyme BACE1 and maintain the expression level of ADAM10.

In another preferred embodiment, the active ingredients in the stem cell extracellular vesicles are selected from the group consisting of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24 -3p, hsa-let-7b-3p and their precursor forms, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa- Antisense oligonucleotides of miR-1246 and its precursor forms, and any combination of the foregoing.

In another preferred example, the active ingredients include hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides.

In another preferred embodiment, the preparation comprises a pharmaceutically acceptable carrier or adjuvant.

In another preferred embodiment, the pharmaceutically acceptable carrier or adjuvant is selected from the group consisting of sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, clodronate, Heparin, or a combination thereof.

In another preferred embodiment, the preparation is a cell-free and cell-debris-free preparation.

In another preferred embodiment, the "cell-free" means that the preparation does not contain live cells and dead cells.

In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, cerebral ischemia, stroke, or a combination thereof.

A second aspect of the present invention provides a pharmaceutical composition comprising: (a) the preparation containing stem cell extracellular vesicles according to the first aspect of the present invention, and (b) pharmaceutically acceptable Carrier.

In another preferred embodiment, the pharmaceutical composition is a cell-free and cell-debris-free pharmaceutical composition.

In another preferred embodiment, the "cell-free" means that the pharmaceutical composition does not contain live cells and dead cells.

In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, clodronate, heparin, or a combination thereof.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid dosage form, solid dosage form (eg, freeze-dried dosage form).

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of nasal drops, aerosol inhalation, eye drops, and injection.

In another preferred embodiment, the dosage form of the pharmaceutical composition is nasal drops.

In another preferred embodiment, the pharmaceutical composition has the following characteristics: the stem cell extracellular vesicles in the pharmaceutical composition are genetically engineered to have brain nerve tissue-specific targeting ability, which can make stem cell extracellular vesicles The active ingredients in the vesicles are targeted for delivery into the brain nerve tissue for uptake by nerve cells and microglia.

In another preferred embodiment, the pharmaceutical composition has the following characteristics: the active ingredient in the stem cell extracellular vesicles in the pharmaceutical composition can reach the brain through the nose.

In another preferred embodiment, the active ingredient is selected from the group consisting of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p, hsa-miR-24-3p, hsa-let- 7b-3p and its precursor forms, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR-151a-3p, hsa-miR-1246 and its precursors forms of antisense oligonucleotides, and any combination of the above.

In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating neurodegenerative diseases.

In another preferred embodiment, the neurodegenerative disease is Alzheimer's disease.

A third aspect of the present invention provides a method for preparing a pharmaceutical composition containing stem cell extracellular vesicles, the method comprising the steps of:

(S1) culturing the genetically engineered human stem cells to reach a predetermined confluency (eg, 75-90%);

(S2) continuing to culture the cells for a period of time T1 under conditions suitable for producing extracellular vesicles; wherein, the T1 is usually 24-72 hours, preferably 30-60 hours;

(S3) removing cells from the culture system, thereby separating and obtaining a culture medium containing stem cell extracellular vesicles, which is a "conditioned medium";

(S4) mixing the conditioned medium (conditioned medium) with polyethylene glycol (PEG) to form a first mixture, and placing it for a period of time T2, thereby forming PEG-modified stem cell extracellular vesicles; wherein, Described T2 is usually 6-60 hours, preferably 12-48 hours;

(S5) centrifuging the first mixture in the previous step to precipitate the PEG-modified stem cell extracellular vesicles, and discard the supernatant to obtain the PEG-modified stem cell extracellular vesicle precipitate;

(S6) resuspending the PEG-modified stem cell extracellular vesicle precipitate obtained in the previous step to obtain a first resuspension mixture;

(S7) centrifuging the first resuspension mixture to precipitate the PEG-modified stem cell extracellular vesicles, and discard the supernatant to obtain a PEG-modified stem cell extracellular vesicle precipitate;

(S8) Resuspend the PEG-modified stem cell extracellular vesicle pellet obtained in the previous step, thereby obtaining a medically acceptable stem cell extracellular vesicle preparation.

In another preferred embodiment, the method further includes:

(S9) Mixing a medically acceptable stem cell extracellular vesicle preparation (or active substance) with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.

In another preferred embodiment, the method further comprises preparing the pharmaceutical composition into nasal drops, aerosol inhalation preparations, eye drops, injections, or freeze-dried preparations.

In another preferred embodiment, the genetically engineered human stem cells refer to cells obtained by transducing the active ingredient into human stem cells.

In another preferred example, the human stem cells are human adipose-derived mesenchymal stem cells.

In another preferred embodiment, the active ingredients hsa-miR-1290 and hsa-miR-126-5p antisense oligonucleotides are introduced into human adipose-derived mesenchymal stem cells (that is, overexpressed in human adipose-derived mesenchymal stem cells). ), the combination ratio of the number of moles (pmol) is 1:(1~6) or (1~6):1, including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and 2:1, 3:1, 4:1, 5:1, and 6:1.

The fourth aspect of the present invention provides the use of the preparation containing stem cell extracellular vesicles according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention, for the preparation of prophylaxis and/or Drugs or preparations for the treatment of neurodegenerative diseases.

The fifth aspect of the present invention provides a method for treating neurodegenerative diseases, comprising the steps of: administering the preparation of the first aspect of the present invention, or the pharmaceutical composition of the second aspect to a subject in need.

In another preferred embodiment, the subject in need is a human or a non-human mammal.

In another preferred embodiment, the required object is a human being.

In another preferred embodiment, the subject in need suffers from a neurodegenerative disease.

The present invention provides pharmaceutical compositions comprising extracellular vesicles (eg, exosomes). The extracellular vesicles (eg, exosomes) can be derived from engineered stem cells.

Stem cells can be engineered to express or overexpress antisense oligonucleotides targeting one or more miRNAs or miRNA precursors, which can be selected from the group consisting of: miR-1290 (eg, hsa-miR-1290), miR-126-5p (eg, hsa-miR-126-5p), miR-130a-3p (eg, hsa-miR-130a-3p), miR-24-3p (eg, hsa -miR-24-3p), let-7b-3p (eg, hsa-let-7b-3p), let-7b-5p (eg, hsa-let-7b-5p), let-7a-5p (eg, hsa-let-7b-5p) hsa-let-7a-5p), miR-92a-3p (eg, hsa-miR-92a-3p), miR-151a-3p (eg, hsa-miR-151a-3p), miR-1246 (eg, hsa -miR-1246), and their precursors.

The present invention provides in vitro methods for generating extracellular vesicles (eg, exosomes) derived from human cells. Specifically, stem cells (eg, engineered stem cells) are cultured, and extracellular vesicles are isolated from the cell culture medium, wherein the extracellular vesicles express or overexpress an antisense oligonucleotide of interest.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the human adipose-derived mesenchymal stem cell extracellular vesicles prepared by the present invention and the identification of their biological characteristics.

Figure 2 shows the detection of the main effective components hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the drug nasal drop preparation of the modified human adipose-derived mesenchymal stem cell extracellular vesicles.

Figure 3 shows that the drug intranasal formulation of human adipose-derived mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides is specifically taken up by nerve cells.

Figure 4 shows the specific uptake of hsa-miR-1290 and hsa-let-7b-5p by neural cells and microglia after the drug intranasal preparation of genetically engineered human adipose-derived mesenchymal stem cell extracellular vesicles Sense oligonucleotide level detection.

Figure 5 shows a schematic diagram of the treatment of Aβ42-induced AD mice with a drug nasal preparation of human adipose-derived mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides.

Detailed ways

Through extensive and in-depth research, the inventors unexpectedly developed a preparation containing genetically engineered stem cell extracellular vesicles for the first time. In the present invention, human adipose-derived mesenchymal stem cells produced under GMP conditions are preferably used as parent cells for producing stem cell extracellular vesicles, and through a series of genetic engineering transformations, stem cells with brain nerve tissue-specific targeted delivery function are obtained The vesicle drug nasal drop preparation is used for the treatment of neurodegenerative diseases such as Alzheimer's disease through the intranasal route of administration, in order to provide a new effective treatment method for it. On this basis, the present invention has been completed.

the term

antisense oligonucleotide

Antisense oligonucleotides can comprise ribonucleotides and/or deoxyribonucleotides (e.g., oligoribonucleotides, RNA, oligodeoxyribonucleotides, DNA, etc.). In some embodiments, the antisense oligonucleotide targets one or more miRNAs or miRNA precursors, which can be selected from the group consisting of: miR-1290 (eg, hsa-miR-1290 ), miR-126-5p (eg, hsa-miR-126-5p), miR-130a-3p (eg, hsa-miR-130a-3p), miR-24-3p (eg, hsa-miR-24- 3p), let-7b-3p (eg, hsa-let-7b-3p), let-7b-5p (eg, hsa-let-7b-5p), let-7a-5p (eg, hsa-let-7a) -5p), miR-92a-3p (eg, hsa-miR-92a-3p), miR-151a-3p (eg, hsa-miR-151a-3p), miR-1246 (eg, hsa-miR-1246) , and their precursors.

Antisense oligonucleotides can be single-stranded or double-stranded. An antisense oligonucleotide can have at least one chemical modification (ie, the oligonucleotide is chemically modified).

Antisense oligonucleotides can be about 5 to about 50 nucleotides in length, about 10 to about 30 nucleotides in length, about 8 to about 18 nucleotides, about 12 to 16 nucleotides in length , about 8 nucleotides or longer, or about 20 to about 25 nucleotides in length.

In certain embodiments, an antisense oligonucleotide may comprise at least partial complementarity to a mature miRNA sequence or miRNA precursor, eg, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary sequences. In some embodiments, an antisense oligonucleotide can be substantially complementary to a mature miRNA sequence or miRNA precursor, ie, at least about 95%, 96%, 97%, 98%, or 99% to a target miRNA sequence or miRNA precursor % Complementary. In one embodiment, the antisense oligonucleotide comprises a sequence that is 100% complementary to a mature miRNA sequence or miRNA precursor.

neurodegenerative disease

As used herein, the term "cerebral neurovascular disease" primarily includes neurodegenerative diseases.

Neurodegenerative diseases are caused by the loss of neurons and/or their myelin sheaths, which worsen and become dysfunctional over time. It can be divided into acute neurodegenerative diseases and chronic neurodegenerative diseases. The former mainly includes cerebral ischemia (CI), brain injury (BI), and epilepsy; the latter includes Alzheimer's disease (AD), Parkinson's disease ( PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.

Stem cell extracellular vesicles of the present invention

There are three main types of stem cell extracellular vesicles (EVs), namely exosomes, microvesicles and apoptotic bodies. All three major types of EVs are encapsulated by lipid bilayers with diameters ranging from 30–2000 nm.

The term exosomes refers to a subclass of endosome-derived EVs with a diameter of 50-100 nm, which are the main source of paracrine secretions from a variety of cell types, including mesenchymal stem cells (MSCs). component.

MSCs exosomes (Exosomes) are a class of MSCs-derived EVs with a diameter in the range of 50-100 nm and a complete lipid bilayer membrane structure.

Large extracellular vesicles can range in diameter from about 5 μm to about 12 μm. The diameter of apoptotic bodies can range from about 1 μm to about 5 μm. Microvesicles can range in diameter from about 100 nm to about 1 μm.

Exosomes can have about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 80 nm to about 200 nm, about 80 nm to about 150 nm, about 80 nm to about 100 nm, about 100 nm to about 100 nm to about 200 nm, about 100 nm to about 180 nm, about 100 nm to about 150 nm, about 100 nm to about 120 nm, about 120 nm to about 200 nm, about 120 nm to about 180 nm, about 120 nm to about 150 nm, about 120 nm to about 140 nm, about 140 nm to about 200 nm , about 140 nm to about 180 nm, about 140 nm to about 160 nm, about 150 nm to about 200 nm, about 150 nm to about 180 nm, about 150 nm to about 160 nm, about 180 nm to about 200 nm, or about 50 nm to about 200 nm in diameter.

The present invention provides a human stem cell-derived extracellular vesicle, the stem cells include but are not limited to human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, Human adipose-derived mesenchymal stem cells, human bone marrow-derived stem cells, etc. In a preferred embodiment of the present invention, the extracellular vesicles are derived from the supernatant collected during the in vitro culture of genetically engineered human stem cells.

In certain embodiments, the formulations of the invention are derived from stem or progenitor cells. In certain embodiments, the methods of the invention culture stem or progenitor cells. Stem cells are undifferentiated cells with the ability to self-renew and produce differentiated progeny (see Morrison et al. (1997) Cell 88:287-298). In mammals, there are two main types of stem cells: embryonic stem cells and adult stem cells, which are found in a variety of tissues.

The stem cells can be bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs), neural stem cells (NSCs), blood stem cells or hematopoietic stem cells. Stem cells can also be derived from cord blood. Stem cells can be generated by somatic cell nuclear transfer or dedifferentiation.

Stem cells include, but are not limited to, blood stem cells, adipose stem cells, bone marrow mesenchymal stem cells, mesenchymal stem cells, neural stem cells (NSC), skin stem cells, endothelial stem cells, liver stem cells, pancreatic stem cells, intestinal epithelial stem cells, or germinal stem cells. In certain embodiments, mesenchymal stem cells are isolated from mesodermal organs, such as bone marrow, umbilical cord blood, and adipose tissue.

In certain embodiments, the stem cells are induced pluripotent stem cells (iPS cells or iPSCs). iPSC refers to a class of pluripotent stem cells artificially generated from non-pluripotent cells, usually adult somatic or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells Wait.

Cells may include autologous cells and/or biocompatible allogeneic cells or syngeneic cells harvested from the subject being treated, such as autologous cells, allogeneic cells or syngeneic cells Genetic cells (eg, mesenchymal stem cells), progenitor cells (eg, connective tissue progenitor cells) or multipotent adult progenitor cells) and/or other further differentiated cells.

Any stem cell with differentiation potential, including but not limited to embryonic stem cells, induced high-potency stem cells, cancer stem cells, and tissue stem cells, can be used to generate extracellular vesicles (eg, exosomes). Tissue stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, breast stem cells, neural stem cells, intestinal stem cells, skin stem cells, umbilical cord blood stem cells, limbal stem cells, hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells, and ovary stem cell. The stem cells used to generate exosomes can be selected from embryonic stem cells, induced pluripotent stem cells, cancer stem cells, mesenchymal stem cells, hematopoietic stem cells, breast stem cells, neural stem cells, intestinal stem cells, skin stem cells, umbilical cord blood stem cells, and limbal stem cells , hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells and ovarian stem cells.

The stem cell extracellular vesicles of the present invention contain active ingredients that regulate targets related to inflammation and amyloid β formation, which can simultaneously exert an inhibitory effect on inflammation, reduce the expression of amyloid precursor proteolytic enzyme BACE1 and maintain the expression level of ADAM10 . In a preferred example of the present invention, the active ingredients mainly include: miR-1290, miR-126-5p, miR-130a-3p, miR-24-3p, let-7b-3p and their precursor forms, and also Antisense oligonucleotides in the form of let-7b-5p, let-7a-5p, miR-92a-3p, miR-151a-3p, miR-1246, and precursors thereof, and any combination of the foregoing, may be included.

The term "hsa" preceding each miRNA name indicates that the miRNA is a human sequence.

In addition, the stem cell extracellular vesicles of the present invention also have the following characteristics: the extracellular vesicles are prepared by the polyethylene glycol (PEG) precipitation method, and the diameters of the extracellular vesicles are concentrated between 80-200 nm, and the particle diameters are uniform.

The preparation method of the preparation containing stem cell extracellular vesicles of the present invention

The present invention provides a preparation method of a nasal drop preparation using the active ingredient of the genetically engineered-improved stem cell extracellular vesicle that regulates targets related to inflammation and amyloid β formation as an effective ingredient. The specific technical scheme is as follows:

In a preferred embodiment, GMP grade human adipose-derived mesenchymal stem cells are mainly used as parent cells for producing stem cell extracellular vesicles.

The preparation operation of the nasal drop preparation of stem cell vesicles involved in the present invention mainly includes:

1) construct a backbone vector containing RVG polypeptide sequence and Lamp2b sequence fusion, and then construct the target sequence hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide sequence on this backbone plasmid;

2) The above-mentioned preferred plasmids overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides were transfected into human adipose mesenchymal stem cells by liposome 3000 (Lipofectamine 3000), and stable A cell clone expressing the target sequence is prepared into a genetically engineered human adipose mesenchymal stem cell; in another preferred embodiment, the active components hsa-miR-1290 and hsa-miR-126-5p antisense oligo When the nucleotides are introduced into human adipose-derived mesenchymal stem cells (that is, overexpressed in human adipose-derived mesenchymal stem cells), the combination ratio of the number of moles (pmol) is 1:(1-6) or (1-6):1, Including but not limited to 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and 2:1, 3:1, 4:1, 5:1 and 6:1.

3) Collect the culture supernatant of the above-mentioned transformed human adipose-derived mesenchymal stem cells produced on a large scale by GMP with effective conditioned treatment, obtain conditioned medium by differential centrifugation and filtration treatment, and then combine the polymer precipitation method to carry out vesicles. Separation; wherein, the polymer includes, but is not limited to, polyethylene glycols (PEG3000-9000) of different molecular weights and compositions in different ratios (1:1 to 1:8);

4) Use PBS to prepare a PEG stock solution of an appropriate concentration (8%-20% g/ml) and filter it. According to the volume ratio of 1:1, mix it with the above-treated conditioned medium. Under low temperature conditions (4-8 ℃) incubate overnight;

5) After centrifugation (4°C, 3000～5000g, 45～60min), discard the supernatant, add pre-cooled PBS to resuspend the pellet, ultracentrifuge (4°C, 100000～120000g, 1～2h) and discard the supernatant ;

6) Add an appropriate volume (3-10ml) of medical physiological saline, resuspend the pellet, and then identify the modified human adipose-derived mesenchymal stem cell extracellular vesicles by particle size analysis, scanning electron microscope observation and immunoblotting techniques. biological characteristics;

7) Detecting the expression of miRNAs, the main effective components in the stem cell extracellular vesicles, by real-time quantitative PCR (qRT-PCR);

8) The stem cell extracellular vesicles obtained above can be diluted to an appropriate concentration with 3-10 ml of a pharmaceutically acceptable carrier or excipient.

In a preferred embodiment of the invention, the preparation of the intranasal preparation of stem cell extracellular vesicles preferably uses allogeneic human adipose-derived mesenchymal stem cells as the parent cells for producing extracellular vesicles after genetic engineering.

In a preferred embodiment of the invention, the intranasal preparation of stem cell extracellular vesicles is preferably a pharmaceutically acceptable carrier or excipient, including but not limited to sodium chloride, sodium phosphate, polyethylene glycol, chitosan, glass sodium, trehalose, clodronate, heparin, etc., and any combination of the above.

In addition, real-time quantitative PCR was performed on the expression levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the pharmaceutical nasal preparations of human adipose-derived mesenchymal stem cell extracellular vesicles that were preferably produced The results showed that the sequence was expressed at a high level. At the same time, the above-mentioned vesicles were also used as an uptake assay by nerve cells in vitro. The modified human adipose-derived mesenchymal stem cell extracellular vesicles (1×10 ⁵ ～9×10 ⁸ extracellular vesicles) were labeled with lipophilic fluorescent dye PKH26, added to the cells and cultured for 24 to 48 hours, and observed under an inverted fluorescence microscope , the expected results indicate that the vesicles in this pharmaceutical preparation can be specifically taken up by the above-mentioned cells.

Process-scale production of stem cell extracellular vesicles under GMP conditions

One HyperFlask cell factory can produce 500ml of conditioned medium for stem cell extracellular vesicles, and it is estimated that 5 x 10 ¹⁰ -10 x 10 ¹⁰ total stem cell extracellular vesicles will be isolated.

At present, one production operation unit in the laboratory can isolate stem cell extracellular vesicles with a total of 800-1200ml of conditioned medium from two cell factories. It is estimated that 2-5×10 ¹¹ stem cell extracellular vesicles can be isolated. The usage volume of ⁹ , one production operation unit can meet the usage volume of 100-250 patients.

Application of the preparation containing stem cell extracellular vesicles of the present invention

On the other hand, the present invention also provides a nasal-drop preparation using the genetically engineered stem cell extracellular vesicles to regulate targets related to inflammation and amyloid β formation as an active ingredient in a nasal preparation for Alzheimer's disease, etc. Applications in the treatment of neurodegenerative diseases. The neurodegenerative disease includes, but is not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, or stroke.

In a preferred embodiment, 4 groups of experiments (Table 1) are set up, which are the PBS-induced control group (PBS), the Aβ42 model and the saline-treated group (Aβ42+saline), the Aβ42 model and the dimethylacetate The amantadine treatment group (Aβ42+memantine) and the Aβ42 model and the treatment group administered with stem cell extracellular vesicle nasal drops (Aβ42+modified haMSC-Exos). First, 2 to 5 days before the preparation of the AD animal model (D-2-D-5), the above-mentioned four groups of mice were given normal saline, dimamantadine (1 mg/kg mouse body weight) and the modified stem cell capsules respectively. The intranasal vesicles were treated with intranasal vesicles, once a day; the dosage of intranasal vesicles was 1×10 ⁵ to 1×10 ⁸ extracellular vesicles/mouse, and they were treated continuously for 2 to 5 days; then, Aβ42 (5～20μg) was used to induce AD mouse model. Except for the control group, the mice in the other experimental groups were given normal saline nasal instillation treatment and the above-mentioned stem cell extracellular vesicle drug nasal instillation preparation (1×10 ⁵ ～1× ), respectively. 10 ⁸ extracellular vesicles/mouse), administered once a day for 5 to 10 days. The cognitive function of the mouse brain was tested, and then the mice were euthanized. The mouse brain tissue was removed to prepare paraffin sections, and then the levels of Aβ42 in the hippocampus and cortex and the activation level of microglia were detected. The results showed that AD symptoms in mice were significantly improved.

Table 1 The drug nasal preparation of human adipose-derived mesenchymal stem cell extracellular vesicles treats Aβ42AD mice grouping

Pharmaceutical composition and application

The present invention provides a pharmaceutical composition comprising: (a) the preparation containing stem cell extracellular vesicles according to the first aspect of the present invention, and (b) a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention is a cell-free and cell-debris-free pharmaceutical composition, which contains a therapeutically effective amount of the stem cell extracellular vesicles of the present invention. The term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats, ameliorates, or prevents a target disease or condition, or an amount that exhibits a measurable therapeutic or prophylactic effect. The precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as the stem cell extracellular vesicles of the invention. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in the compositions can include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, the compositions can be prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid vehicles prior to injection can also be prepared. Liposomes are also included in the definition of pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from the group consisting of sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, clodronate, Heparin, or a combination thereof.

The pharmaceutical composition of the present invention can be prepared into various conventional dosage forms, such as liquid dosage form, solid dosage form (such as freeze-dried dosage form), and the dosage form of the pharmaceutical composition is selected from the following group: nasal drops, aerosol inhalation, eye drops preparations, injections, preferably nasal drops.

Routes of administration of the pharmaceutical compositions include inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular administration.

The present invention also provides a method of treating a disease in a mammal, comprising the step of delivering to the mammal a therapeutically effective amount of a pharmaceutical composition of the present invention, the pharmaceutical composition comprising extracellular vesicles, such as exosomes. Extracellular vesicles (eg, exosomes) can be derived from engineered stem cells.

The compositions of the present invention may be administered by any method known in the art, including but not limited to intranasal, oral, transdermal, ocular, intraperitoneal, inhalation, intravenous, ICV, intracisternal injection or infusion, subcutaneous, implantable Intravenous, sublingual, urethral (eg, urethral suppositories), subcutaneous, intramuscular, intravenous, rectal, sublingual, mucosal, ophthalmic, spinal, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial and lymphatic administration. Topical formulations can be in the form of gels, ointments, creams, aerosols, etc.; intranasal formulations can be delivered as sprays or drops; transdermal formulations can be administered by transdermal patches or iontophoresis; inhaled formulations can be used Nebulizer or similar device delivery. The compositions may also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable composition.

The present invention also provides compositions for intranasal administration as described above. Compositions can be administered intranasally in liquid form such as solutions, emulsions, suspensions, drops or in solid forms such as powders, gels or ointments. Devices for delivering intranasal drugs are well known in the art. Intranasal drug delivery can be performed using devices including, but not limited to, intranasal inhalers, intranasal spray devices, nebulizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, nebulizers, metered Inhalers (MDIs), pressurized dose inhalers, insufflators and bidirectional devices. The nasal delivery device can be metered to administer the exact effective dose to the nasal cavity. Nasal delivery devices can be used for single unit delivery or for multiple unit delivery. Compositions can also be delivered by tube, catheter, syringe, Packtail, cotton swab, nasal plug, or by submucosal infusion.

The composition can be formulated into an aerosol using standard procedures. Extracellular vesicles can be formulated with or without solvents, with or without carriers. The formulation can be a solution, or it can be an aqueous emulsion with one or more surfactants. For example, an aerosol spray can be generated from a pressurized container with a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbon compounds, compressed air, nitrogen, carbon dioxide or other suitable gas. As used herein, the term "aerosol" refers to the suspension of fine solid particles or droplets of liquid solutions in a gas. In particular, aerosols include airborne suspensions of droplets, which can be produced in any suitable device, such as MDIs, nebulizers, or nebulizers. Aerosols also include dry powder compositions of compositions suspended in air or other carrier gas.

The composition can be delivered to the nasal cavity as a powder in the form of microspheres such as delivered by a nasal insufflator. The composition can be adsorbed to the surface of a solid such as a carrier. The powder or microspheres can be applied in a dry, air-dispensable form. The powder or microspheres can be stored in the container of the insufflator. Alternatively, the powder or microspheres can be filled into capsules, such as gelatin capsules, or other single dosage units suitable for nasal administration.

Pharmaceutical compositions can be delivered to the nasal cavity by placing the composition directly in the nasal cavity, eg, in the form of a gel, ointment, nasal cream, lotion, cream, nasal plug, nasal drop, or biostrip. In certain embodiments, it may be desirable to prolong the residence time of the pharmaceutical composition in the nasal cavity, eg, to enhance absorption. Thus, the pharmaceutical composition can optionally be combined with bioadhesive polymers, gums (eg, xanthan gum), chitosan (eg, highly purified cationic polysaccharides), pectin (or when applied to the nasal mucosa, such as gel-like thickening or emulsification of any carbohydrate), microspheres (e.g. starch, albumin, dextran, cyclodextrin), gelatin, liposomes, carbomer, polyvinyl alcohol, alginate, gum arabic , chitosan and/or cellulose (eg methyl or propyl cellulose; hydroxy or carboxy cellulose; carboxymethyl or hydroxypropyl cellulose).

The composition can be administered to the respiratory tract, ie, the lungs, by oral inhalation.

Typical delivery systems for inhalables include nebulizing devices (eg, nebulizers), dry powder inhalers (DPIs), and metered dose inhalers (MDIs).

Once formulated into a composition of the present invention, it can be administered directly to a subject. The subject to be treated can be a mammal, especially a human. The subject to be treated suffers from a neurodegenerative disease, representative diseases including, but not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, or stroke.

In addition, the pharmaceutical compositions of the present invention may be administered in combination with or with other therapeutic agents for neurodegenerative diseases.

The main advantages of the present invention include:

(1) The genetically engineered stem cell extracellular vesicles of the present invention are rich in specific miRNAs that simultaneously exert anti-inflammatory, inhibit the amyloid precursor proteolytic enzyme BACE1 and maintain the expression of ADAM10.

(2) The stem cell extracellular vesicles of the present invention are prepared by the polyethylene glycol (PEG) precipitation method, and the diameters of the extracellular vesicles are concentrated in the range of 80-200 nm, and the particle diameters are uniform; After crossing the blood-brain barrier and entering the brain nerve tissue, the small miRNA molecules in it can be released after the stem cell extracellular vesicles enter the body, thereby reaching the brain.

(3) Compared with conventional drug treatment, the nasal preparation of stem cell extracellular vesicles of the present invention can more effectively prevent or treat neurodegenerative diseases such as Alzheimer's disease.

The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacture conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Test materials and instruments

Human adipose-derived mesenchymal stem cells, DMEM medium, 5% serum replacement, Lipofectamine 3000 (L3000015, Invitrogen), wild-type BALB/c mice (7 weeks old, female, SPF grade), anesthetics (50 mg/kg ketamine and 30mg/kg xylazine), neuron Neuro2A, microglia BV-2, Aβ42 (AG968, Sigma-Aldrich), dimethylamantadine (#187836, Sigma-Aldrich), medical saline, PEG with different molecular weights , sterile phosphate buffered saline (PBS), 1ml sterile syringe and 20G needle, PKH26 dye, paraffin block, H&E staining solution, PAS staining solution, Trizol kit (#12183555, Invitrogen),

III RT SuperMix (R323-01, Novozymes), chloroform, 10% neutral formaldehyde fixative, OCT embedding medium, liquid nitrogen, mouse IL-1β (PI301, Biyuntian) and Aβ (#KMB3441, Thermo Scientific ) ELISA detection kit, centrifuge tube, 12-well cell culture plate, centrifuge, paraffin embedding machine, paraffin microtome, PCR instrument, real-time fluorescence quantitative PCR instrument, Nanodrop2000 instrument, ordinary brightfield microscope (40× objective), Upright fluorescence microscope (40× objective), inverted fluorescence microscope (20× objective), cell incubator.

Data Statistical Analysis Methods

All experimental data were statistically analyzed using GraphPad Prism 6.0 software, and were expressed as mean ± standard error (Mean ± SEM). When comparing two groups of data, Mann-Whitney U test was used for data comparative analysis; when comparing multiple groups of data, One-way ANOVA and Tukey were used for data comparative analysis. P values less than 0.05 were considered significant differences.

Example 1

Preparation of drug nasal drop preparations of human adipose-derived mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides

In all the examples of the present invention, the human adipose-derived mesenchymal stem cells overexpressing the target sequence produced under GMP conditions are preferably used as the parent cells for further production of stem cell extracellular vesicles. It should be emphasized that the parent cells that produce vesicles also Stem cells can be derived from umbilical cord blood, umbilical cord, placenta, bone marrow and other tissue sources. In the present invention, the main process of preparing the pharmaceutical nasal drop preparation of human adipose-derived mesenchymal stem cell extracellular vesicles that preferably overexpress hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides is as follows:

(1) In the early stage, a backbone vector containing the RVG polypeptide sequence and Lamp2b sequence was constructed to express the fusion protein and containing a GFP tag, and then hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides The sequence is constructed on the plasmid backbone;

(2) The above-mentioned preferred plasmids overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides were transfected into human adipose-derived mesenchymal stem cells through Lipofectamine 3000, and selected Cell clones that stably express the target sequence, and prepared into human adipose-derived mesenchymal stem cells transformed by genetic engineering;

(3) Under GMP conditions, human adipose-derived mesenchymal stem cells overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides were prepared and cultured;

(4) collecting the culture supernatant of the above-mentioned GMP large-scale production of human-derived adipose-derived mesenchymal stem cells treated with effective conditions, removing cell debris by differential centrifugation and filtration to obtain a conditioned medium, and separating in conjunction with the polymer precipitation method, The polymer is preferably PEG9000;

(5) Prepare a 20% g/ml PEG9000 stock solution with PBS, filter it with a 0.22 μm filter, add it to the treated conditioned medium at a volume ratio of 1:1, incubate at 4°C overnight, and then pass Centrifuge at 4000g for 50min at 4℃, discard the supernatant and keep the precipitate;

(6) Add pre-cooled PBS to resuspend the pellet, centrifuge at 120,000g for 1 h at 4°C, remove the supernatant, and add 5ml of medical carrier solution to resuspend the pellet;

(7) The stem cell extracellular vesicles obtained above are diluted to an appropriate concentration with 3 ml of medical carrier solution.

The results showed that a drug nasal drop preparation of human adipose-derived mesenchymal stem cell extracellular vesicles with uniformly dispersed vesicle particles and transparent vesicles was obtained (Fig. 1).

Example 2

The content of the main active ingredients hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides in the drug nasal drop preparation of genetically engineered human adipose-derived mesenchymal stem cell extracellular vesicles is increased

In order to determine the expression level of the target sequence in the extracellular vesicles of human adipose-derived mesenchymal stem cells after the above transformation, real-time quantitative PCR (qRT-PCR) was used to analyze the hsa-miR-1290 and hsa-let-7b- To detect the relative expression level of 5p antisense oligonucleotide, the main steps are as follows:

(1) In a fume hood, add 0.5ml Trizol reagent, let stand for 5min at room temperature, and centrifuge (4°C, 12000g, 5min);

(2) Pipet the supernatant into a new RNase-free EP tube, add 100 μl of chloroform, shake and mix by hand, let stand at room temperature for 5 min, and centrifuge (4°C, 12000 g, 15 min);

(3) Slowly pipette the colorless supernatant of the uppermost layer into a new RNase-free EP tube;

(4) Add an equal volume of isopropanol, mix gently, stand at room temperature for 10 min, and centrifuge (4°C, 12000g, 10min);

(5) Gently pour the supernatant, put it on absorbent paper to dry the residue, add 500 μl of DEPC-water prepared 75% ethanol to resuspend the precipitate, and centrifuge (4°C, 7500g, 5min);

(6) Gently pour the supernatant, put it upside down on absorbent paper at room temperature to dry the residual liquid, and add 10 μl DEPC-treated ddH ₂ O to dissolve the RNA;

(7) Pipette 2 μl to measure the RNA concentration in Nanodrop2000 instrument, and dilute it to 1 μg/μl;

(8) According to cDNA synthesis kit

III RT SuperMix instruction manual for reverse transcription of cDNA;

(9) The expression levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotide sequences were detected by real-time fluorescent quantitative PCR.

The results showed that compared with unengineered human adipose-derived mesenchymal stem cell extracellular vesicles, hsa-miR-1290 and hsa-let-7b- The content of 5p antisense oligonucleotides was significantly increased (Figure 2).

Example 3

Drug intranasal preparations of human adipose-derived mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides are specifically taken up by nerve cells

In order to prove that the RVG-Lamp2b fusion protein-expressing human adipose-derived mesenchymal stem cell extracellular vesicles can be taken up by nerve cells, an in vitro experiment was designed. Co-incubated, and then detected its uptake using fluorescence microscopy, the specific steps are as follows:

(1) Use PKH26 dye to incubate the above-mentioned stem cell extracellular vesicles with or without modification for 12 hours to label the vesicles on the membrane; then use 120,000g ultracentrifugation for 1 hour to precipitate the vesicles, and after resuspending, use molecular The free dye and vesicles remaining in the solution were separated by resistance chromatography.

(2) Neuro2A cells were seeded in a 12-well plate at a density of 5×10 ⁴ cells per well. After culturing in a cell incubator (37°C, 5%) for 24 hours, 5 μg/ml of the above-mentioned pre- and post-transformation capsules were added. Bubble.

(3) After co-incubating for 3 hours, the uptake of vesicles by cells was observed under a fluorescence microscope.

The results showed that the number of cells with red fluorescent signal increased significantly in Neuro2A cells incubated with the above-mentioned human adipose-derived mesenchymal stem cell extracellular vesicles transfected with RVG-Lamp2b fusion protein particles (Figure 3), indicating that the fusion protein transfected vesicles The vesicles are more readily taken up by nerve cells.

Example 4

Levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides following uptake of genetically engineered human adipose-derived mesenchymal stem cell extracellular vesicles by neuronal and microglia drug-delivered nasal preparations Increase

To determine whether neuronal Neuro2A and microglia BV-2 uptake of these vesicles increased the levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides, real-time fluorescence quantification was performed. PCR detection, the main operations are as follows:

(1) According to the operation method of Example 2, neuron Neuro2A cells and microglia BV-2 were seeded in a 12-well plate at a density of 5×10 ⁴ cells per well, respectively, in a cell culture incubator (37°C, 5%) cultured for 24h;

(2) Adding or not adding 5 μg/ml of the above labeled PKH26 vesicles to the above cell culture dish, placing the cells in a cell culture incubator (37°C, 5%) and continuing to culture for 3 hours;

(3) After observing the increase in intracellular green fluorescence after ingesting vesicles under a fluorescence microscope, remove the cells and place them on ice;

(4) RNA extraction, purification and cDNA synthesis were carried out according to the operation method of Example 2, as well as real-time quantitative PCR detection of the levels of hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides.

The results showed that compared with the control, after uptake of genetically engineered human adipose-derived mesenchymal stem cell extracellular vesicles by neuro2A cells and microglia BV-2, hsa-miR-1290 and hsa-let The content of both -7b-5p antisense oligonucleotides was significantly increased (Fig. 4).

Example 5

Drug intranasal preparation of human adipose-derived mesenchymal stem cell extracellular vesicles overexpressing hsa-miR-1290 and hsa-let-7b-5p antisense oligonucleotides to prevent or treat Aβ42-induced AD mice

In order to explore the cognitive function of the modified human adipose-derived mesenchymal stem cell extracellular vesicles in the intranasal drug nasal administration to treat AD model mice, the Aβ42-induced AD mouse disease model was first established, and then the Treatment (Figure 5), to detect the relevant indicators of brain cognitive function, the main operations are as follows:

(1) First, 4 groups of experiments were set up, namely the PBS-induced control group (PBS), the Aβ42 model and the saline-treated group (Aβ42+saline), and the Aβ42 model and the mintamine-treated group (Aβ42+memantine) ) and Aβ42 modeling and administration of stem cell extracellular vesicle nasal drops (Aβ42+modified haMSC-Exos);

(2) Two days before the preparation of the AD animal model (D-2), the above four groups of mice were given saline, dimethylamantine (1 mg/kg mouse body weight) and drug drops of the modified stem cell vesicles, respectively. Nasal preparation treatment, once a day; wherein, each drug dosage of nasal preparation is 1 × 10 ⁶ particles/mouse, and it is treated continuously for 2 days;

(3) AD mouse model was induced by 10 μg Aβ42. Except for the control group, the mice in the other experimental groups were given normal saline nasal instillation treatment and the above-mentioned stem cell extracellular vesicle drug nasal instillation preparation (1×10 ⁶ particles/mouse). ) nasal administration treatment, administration once a day, continuous treatment for 5 days;

(4) The Barnes maze was used to test the cognitive function of the mice; after that, the mice were euthanized, and the mouse brain tissue was taken out to prepare paraffin sections, and then the levels of Aβ42 and microglia in the hippocampus and cortex were measured. Cell activation levels were detected.

After the above-mentioned modified human adipose-derived mesenchymal stem cell extracellular vesicles were administered nasally to AD mice, the production level of Aβ42 in the hippocampus and cortex was also significantly reduced, and the activation level of microglia decreased. , and the cognitive level of mice was significantly improved.

references

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All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A preparation containing stem cell extracellular vesicles, characterized in that the stem cell extracellular vesicles of the preparation contain active ingredients for regulating targets related to inflammation and amyloid beta formation; and the preparation is used for:

(a) Inhibit the inflammatory response of brain nerve tissue;

(b) inhibiting the production of amyloid Aβ; and

(c) preventing and/or treating neurodegenerative diseases.
The preparation of claim 1, wherein the stem cell extracellular vesicles are derived from the supernatant collected during the in vitro culture of genetically engineered human stem cells.
The preparation of claim 2, wherein the human stem cells are selected from the group consisting of stem cells derived from human umbilical cord blood, stem cells derived from human peripheral blood, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, Human adipose-derived mesenchymal stem cells, human bone marrow-derived stem cells, or a combination thereof.
The preparation according to claim 1, wherein the stem cell extracellular vesicles have the following characteristics: they are prepared by a polyethylene glycol (PEG) precipitation method, and the extracellular vesicles are concentrated in a diameter of 80-200 nm. The particle size is uniform.
The preparation according to claim 1, wherein the stem cell extracellular vesicles have the following characteristics: (a) their particle size is small, and they can penetrate the blood-brain barrier and enter the brain nerve tissue; (b) the miRNA in them Small molecules can be released after stem cell extracellular vesicles enter the body, thereby reaching the brain.
The preparation according to claims 1-5, wherein the preparation has the following characteristics: the stem cell extracellular vesicles in the preparation are genetically engineered to have brain nerve tissue-specific targeting ability, which can Active ingredients in stem cell extracellular vesicles are targeted for delivery to brain nerve tissue for uptake by nerve cells and microglia.
The preparation of claim 6, wherein the active ingredient in the stem cell extracellular vesicles is selected from the group consisting of hsa-miR-1290, hsa-miR-126-5p, hsa-miR-130a-3p , hsa-miR-24-3p, hsa-let-7b-3p and their precursor forms, hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-92a-3p, hsa-miR- Antisense oligonucleotides of 151a-3p, hsa-miR-1246 and their precursor forms, and any combination of the foregoing.
A pharmaceutical composition, characterized in that the pharmaceutical composition contains: (a) the preparation containing stem cell extracellular vesicles according to any one of claims 1-7, and (b) a pharmaceutically acceptable vector.
The pharmaceutical composition according to claim 8, wherein the dosage form of the pharmaceutical composition is selected from the group consisting of nasal drops, aerosol inhalation, eye drops, and injection.
A preparation containing stem cell extracellular vesicles as claimed in claims 1-7 or the purposes of the pharmaceutical composition as claimed in claims 8-9, characterized in that, for the preparation of prevention and/or treatment of neurodegeneration Medicines or preparations for diseases.