WO2024239571A1 - Polynucleotide for inhibiting syn2a, exosome containing same, and use thereof - Google Patents

Abstract

The present application discloses a polynucleotide for inhibiting syn2a, an exosome containing same, and a use thereof. The present application provides a syn2a siRNA sequence, which can be used as a syn2a inhibitor for preventing/treating/assisting in treating post-traumatic stress disorder (PTSD). The present application further provides an exosome loaded with a syn2a siRNA sequence, and the exosome can efficiently deliver the syn2a siRNA sequence to a target gene in a tissue-specific manner, inhibit the expression of the target gene or an encoded protein of the target gene, have a significant treatment effect on fear memory refractory model mice, and can obviously reduce the average stiffness of the mice.

Description

Polynucleotide for inhibiting syn2a, exosome containing the same and use thereof

Cross-reference to related applications

This patent application claims priority to the Chinese patent application filed on May 22, 2023, with application number 2023105755825 and invention name “Polynucleotides for inhibiting syn2a, exosomes containing the same and uses thereof”. The full text of the above application is incorporated herein by reference.

Technical Field

The embodiments of the present invention relate to the field of biomedicine, and in particular to polynucleotides for inhibiting syn2a, exosomes containing the same, and uses thereof.

Background Art

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that is mainly caused by intense trauma in individuals, and the symptoms can last for months or decades. Moreover, PTSD often coexists with other psychiatric disorders, such as depression and suicidal thoughts. Therefore, the treatment of PTSD is more urgent. However, the most commonly used method in clinical practice is exposure therapy under the guidance of psychologists or psychiatrists, but this process is inefficient and it may take a long treatment process to overcome this painful memory. Of course, there are some drug therapies, such as antidepressants, such as SSRI drugs fluoxetine (Prozac) and paroxetine (Paxil), which are effective in treating some symptoms of PTSD, but such drugs have potential psychological dependence and addiction. Therefore, it is crucial to develop new and safe drugs for the treatment of post-traumatic stress disorder.

Summary of the invention

The object of the present invention is to provide a polynucleotide for inhibiting syn2a.

Another object of the present invention is to provide exosomes.

Another object of the present invention is to provide a pharmaceutical composition.

Another object of the present invention is to provide a method for preventing, treating and/or assisting in treating post-traumatic stress disorder.

In order to solve the above technical problems, the present invention provides a first aspect of an isolated polynucleotide. The polynucleotide is selected from any one of the following:

(i) the polynucleotide shown in SEQ ID NO: 1;

(ii) a polynucleotide having a homology greater than 80% with the sequence shown in SEQ ID NO:1; and (iii) a polynucleotide formed by substitution, deletion or addition of one or several nucleotides in the sequence shown in SEQ ID NO:1.

The second aspect of the present invention provides a small nucleic acid molecule containing the polynucleotide described in the first aspect of the present invention, wherein the small nucleic acid molecule is siRNA, siRNA precursor, dsRNA, shRNA, miRNA or small interfering RNA.

The third aspect of the present invention provides an exosome containing the polynucleotide described in the first aspect of the present invention or the small nucleic acid molecule described in the second aspect of the present invention.

The fourth aspect of the present invention provides a pharmaceutical composition, comprising: the polynucleotide described in the first aspect of the present invention/the small nucleic acid molecule described in the second aspect of the present invention/the exosomes described in the third aspect of the present invention, and a pharmaceutically acceptable carrier.

The fifth aspect of the present invention provides a method for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder, the method comprising the steps of: applying a therapeutically effective amount of the polynucleotide described in the first aspect of the present invention to the subject, or applying a therapeutically effective amount of the small nucleic acid molecule described in the second aspect of the present invention to the subject, or applying a therapeutically effective amount of the exosomes described in the third aspect of the present invention to the subject, or applying a therapeutically effective amount of the pharmaceutical composition described in the fourth aspect of the present invention to the subject.

In some preferred embodiments, the administration is by intravenous injection.

The sixth aspect of the present invention provides the use of the polynucleotide according to the first aspect of the present invention, for:

(i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder;

(ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder;

(iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or

(iv) Use as a syn2a inhibitor.

The seventh aspect of the present invention provides the use of the small nucleic acid molecule described in the second aspect of the present invention for:

(i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder;

(ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder;

(iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or

(iv) Use as a syn2a inhibitor.

In an eighth aspect, the present invention provides the use of the exosomes described in the third aspect of the present invention for:

(i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder;

(ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder;

(iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or

(iv) Use as a syn2a inhibitor.

The ninth aspect of the present invention provides a method for preparing exosomes containing the polynucleotide according to the first aspect of the present invention, the method comprising the steps of:

Original exosomes are obtained, and then the polynucleotides described in the first aspect of the present invention are introduced into the original exosomes to obtain exosomes containing the polynucleotides described in the third aspect of the present invention.

In some preferred embodiments, the polynucleotide described in the first aspect of the present invention is introduced into the original exosomes by electrotransduction or chemical transduction.

In some preferred embodiments, the raw exosomes are obtained by treating cells with an ECS reagent.

Compared with the prior art, the present invention has at least the following advantages:

(1) The present invention provides a syn2a siRNA sequence, which can be used as a syn2a inhibitor for the prevention/treatment/adjuvant treatment of post-traumatic stress disorder (PTSD);

(2) The present invention also provides an exosome loaded with a syn2a siRNA sequence, which can efficiently and tissue-specifically deliver the syn2a siRNA sequence to the target gene, inhibiting the expression of the target gene or its encoded protein. In a mouse animal model, tail vein injection of the exosome-encapsulated syn2a siRNA can significantly reduce the average rigidity rate of ordinary C57 mice and fear memory refractory model mice. Therefore, the exosome drug is therapeutic and can effectively prevent and/or treat and/or assist in the treatment of PTSD.

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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

Figure 1 shows the metabolic status of DiD-labeled exosomes in vivo after injection of DiD dye into the tail vein of mice according to an embodiment of the present invention, at 0, 2, and 8 hours;

Figure 2 is a comparison of Western blot validation results of syn2a/b protein in the cerebral cortex after the mouse tail vein was injected with control exosomes and si-syn2a exosomes according to an embodiment of the present invention, wherein Con is the control group, Si-syn2a is the experimental group, GAPDH is the internal reference, and the t-test was used for analysis. The data are mean ± standard error, **P<0.01;

FIG3 is a two-way ANOVA of the changes in freezing rate of the control group and the si-syn2a exosome group during the extinction of fear memory after the tail vein injection of control RNA exosomes and si-syn2a exosomes in common C57BL/6J mice in an embodiment of the present invention, the data are mean ± standard error, *P<0.05;

Figure 4 is a t-test analysis of the control group and the si-syn2a exosome group after tail vein injection of control RNA exosomes and si-syn2a exosomes in common C57BL/6J mice according to an embodiment of the present invention. There is no significant difference in the freezing rate between the control group and the si-syn2a exosome group during the fear memory acquisition stage, while there is a significant change in the freezing rate of the fear memory extinction test (n=5), and the data are mean ± standard error, *P<0.05.

5 is a two-way ANOVA of the changes in freezing rate of the control group and the si-syn2a exosome group during the extinction of fear memory after the AtLAS-/- mice were injected with control RNA exosomes and si-syn2a exosomes through the tail vein according to an embodiment of the present invention;

Figure 6 is a t-test analysis according to an embodiment of the present invention, showing that there was no significant difference in the freezing rate between the control group and the si-syn2a exosome group during the fear memory acquisition stage, but there was a significant change in the freezing rate during the fear memory extinction test (n=7), data are mean ± standard error, *P<0.05, **P<0.01.

DETAILED DESCRIPTION

There is no safer and effective drug for the treatment of PTSD in the existing technology. RNA interference (RNAi) is a new gene therapy technology with antiviral, anti-tumor and anti-inflammatory effects. The application prospects in the medical field of diseases and so on are broad and have been rapidly developed. Some RNA drugs have entered the clinical trial stage, opening up a new treatment approach for difficult and complicated diseases, especially multifactorial diseases such as cancer and viral infection. However, small interfering RNA must be intracellular to play a role. RNA molecules are negatively charged and sensitive to the nucleases widely present in the body, so it is not easy to be delivered to the target gene to achieve therapeutic effects.

After extensive and in-depth research, the inventors have designed a nucleic acid drug that targets the syn2a gene and inhibits its expression. In addition, the inventors have found that loading it into exosomes has low immunogenicity, low toxicity, and no induced mutation in the body, and is extremely effective in delivering small interfering RNA to treat post-traumatic stress disorder. Therefore, exosomes containing the polynucleotide of the present invention as a drug for treating PTSD is a very novel, feasible and effective method.

Polynucleotide

The present invention relates to an isolated polynucleotide for inhibiting the expression of syn2a and its encoded protein. As a nucleic acid drug, it can bind to the target gene syn2a to inhibit syn2a expression. As used in the present invention, the term "polynucleotide" or "nucleic acid" refers to a nucleotide or nucleotide monomer sequence composed of naturally occurring bases, sugars and sugars (hubs). The term also includes a limited or replacement sequence containing a monomer or a portion thereof that does not exist in nature. The polynucleotide sequence of the present invention can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and can contain natural bases of adenine, guanine, cytosine and uracil. In one embodiment of the present invention, the polynucleotide is a polynucleotide whose ribonucleic acid sequence is a polynucleotide as shown in SEQ ID NO:1. In one embodiment of the present invention, the polynucleotide is a polynucleotide whose ribonucleic acid sequence is a polynucleotide with a homology greater than 80% (preferably greater than 90%, further preferably greater than 95%, further preferably greater than 98%, and further preferably greater than 99%) compared to the sequence shown in SEQ ID NO:1. In one embodiment of the present invention, the polynucleotide is a polynucleotide whose ribonucleic acid sequence is a polynucleotide formed by replacing, deleting or adding one or more nucleotides of the sequence shown in SEQ ID NO: 1. For the target gene syn2a, the inventors designed a plurality of polynucleotide molecules, and after screening and verification, obtained the above polynucleotide sequence, which has the strongest ability to inhibit syn2a expression at the same dose. The aforementioned SEQ ID NO: 1: CCCAGAAATGACCATGTGATA.

As used herein, the terms "homology", "sequence identity" and "percentage of identity" are used interchangeably and refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between peptides. The nucleotide or amino acid sequences of polynucleotides or polypeptides are arranged, and the number of positions containing the same nucleotide or amino acid residue in the arranged polynucleotides or polypeptides is scored and compared with the number of positions containing different nucleotides or amino acid residues in the arranged polynucleotides or polypeptides. Polynucleotides can be different at one position, for example, according to the presence of different nucleotides (i.e., substitutions or variations) or the absence of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide). Polypeptides can be different at one position, for example, by containing amino acids (i.e., substitutions or variations) or the absence of amino acids (i.e., insertion of amino acids or the absence of amino acids in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

Small nucleic acid molecules

The present invention also relates to a small nucleic acid molecule containing the above polynucleotide, and the small nucleic acid molecule can be at least one of siRNA, siRNA precursor, dsRNA, shRNA, miRNA or small interfering RNA.

As used herein, the term "RNAi" (RNA interference) refers to the phenomenon of highly conserved evolutionary process, induced by double-stranded RNA (dsRNA), and efficient and specific degradation of RNA with complementary paired sequences. Since the use of RNAi technology can specifically shut down the expression of specific genes, this technology has been widely used in the fields of exploring gene function and gene therapy for infectious diseases and tumors. The dsRNA-mediated RNAi phenomenon has been found in many eukaryotic organisms such as fungi, fruit flies, Arabidopsis, trypanosomes, hydras, planarians, zebrafish, etc., and posttranscriptional gene silencing (PTGS), cosuppression, RNA-mediated virus resistance, and fungal inhibition (quelling) in plants are also manifestations of RNAi in different species.

As used herein, the term "siRNA" (Small interfering RNA, siRNA) refers to a small RNA molecule (about 21-25 nucleotides), which can be processed by Dicer (an enzyme in the RNase III family that is specific for double-stranded RNA) from its precursor (such as dsRNA, shRNA, etc.), or can be synthesized by chemical methods or produced by other protein processing. siRNA is a major member of siRISC, which stimulates the rapid cleavage and degradation of target RNAs with complementary sequences, resulting in the silencing of target genes, and thus becomes a key component of RNAi. Key functional molecules: Given a specific gene target sequence, those skilled in the art can design and obtain siRNA targeting the target sequence by conventional methods.

As used herein, the term "siRNA precursor" refers to an RNA molecule that can be processed in mammalian cells to produce siRNA, specifically, it is selectively processed by Dicer or other similar proteins to produce mature siRNA, thereby implementing RNAi.

As used herein, the term "miRNA" (microRNA) is a class of non-coding single-stranded RNA molecules with a length of about 20-24 nucleotides encoded by endogenous genes, which participate in the expression regulation of a large number of genes in animals and plants. So far, more than 4,000 miRNA molecules have been found in animals, plants and viruses. Most miRNA genes exist in the genome in the form of single copies, multiple copies or gene clusters. Each miRNA can regulate multiple target genes, and several miRNAs can also participate in the regulation of the same gene together to form a complex regulatory network. It is speculated that miRNA regulates the expression of more than half of human genes. There are many forms of miRNA, the most primitive of which is pri-miRNA; after pri-miRNA is processed by Drosha, it becomes pre-miRNA, i.e., miRNA precursor, with a length of about 50-90 nucleotides; after pre-miRNA is cleaved by Dicer enzyme, it becomes a mature miRNA of about 20-24 nucleotides. MiRNA mainly inhibits the expression of target genes by inhibiting translation and accelerating the deadenylation of mRNA, and its mechanism is different from siRNA-mediated mRNA degradation.

One way to produce "small interfering RNA" (siRNA) in vivo is to clone the siRNA sequence into a plasmid vector as part of a "short hairpin". When delivered into an animal, the hairpin sequence is expressed to form a "double-stranded RNA" (shRNA) with a top loop structure, which is recognized and processed by the Dicer protein in the cell to produce a functional siRNA.

As used herein, the terms "shRNA" and "shRNA" are used interchangeably and are a special shRNA constructed with the precursor of human miR-26b as the backbone. The shRNA comprises, from the 5' end to the 3' end, the following: (a) a 5' end flanking sequence region; (b) a 5' end pairing siRNA region; (c) a top loop region; (d) a 3' end pairing siRNA region, and the 5' end pairing siRNA region and the 3' end pairing siRNA region form a double-stranded region; (e) a 3' end flanking sequence region; the shRNA produces siRNA, and the nucleotide sequence of the siRNA corresponds to the 3' end pairing siRNA region or the 5' end pairing siRNA region.

In a broad sense, shRNA is the abbreviation of short hairpin RNA. shRNA consists of two short reverse complementary sequences separated by a top loop sequence, forming a hairpin structure. The shRNA sequence is usually transcribed by the endogenous RNA polymerase III promoter, and 5-6 Ts are connected to the end of the shRNA sequence as the transcription terminator of RNA polymerase III. shRNA can also be transcribed by the promoter of other RNA polymerases.

Exosomes

The present invention also relates to exosomes containing the above-mentioned polynucleotides. As used in the present invention, the terms "exosome" and "exosome" are used interchangeably and refer to vesicles with a diameter of 30 to 100 nm produced by cells and having a lipid bilayer membrane structure. Exosomes are secreted by living cells and naturally exist in body fluids, including blood, saliva, urine, cerebrospinal fluid and milk. They are released from cells by fusion of late endosomes with cell membranes in the cell endocytic system or directly through the cell membrane, and play an important role in intercellular signaling. In one embodiment of the present invention, the exosomes contain a polynucleotide as shown in SEQ ID NO: 1. In one embodiment of the present invention, the exosomes contain a polynucleotide with a homology greater than 80% (preferably greater than 90%, further preferably greater than 95%, further preferably greater than 98%, and further preferably greater than 99%) compared to the sequence shown in SEQ ID NO: 1. In one embodiment of the present invention, the exosomes contain a polynucleotide formed by substitution, deletion or addition of one or more nucleotides of the sequence shown in SEQ ID NO: 1.

In a preferred embodiment, the exosomes may also be engineered, such as targeted modification, loading modification. In one embodiment of the present invention, the purified exosomes are loaded with a polynucleotide of the present invention.

Method for preparing exosomes

The present invention also relates to a method for preparing exosomes containing polynucleotides, comprising the steps of:

(1) Obtaining raw exosomes;

(2) Introducing the polynucleotide into the original exosomes to obtain exosomes containing the polynucleotide.

In the present invention, raw exosomes refer to exosomes obtained directly from cells without loading exogenous target sequences. The method of obtaining raw exosomes can be carried out in a conventional manner in the art, such as using a commercially available exosome extraction kit and obtaining exosomes according to the instructions of the kit. In a preferred embodiment of the present invention, ECS (Exosomoe Concentration Solution) is used to test The original exosomes were obtained by treating cells with the agent.

The polynucleotides of the present invention can be introduced into exosomes using conventional nucleic acid delivery methods in the art, that is, the target polynucleotide sequence is engineered and loaded into the exosomes. Depending on whether the operation of the load occurs before the exosome purification step or after the exosome purification step, the loading is divided into exogenous loading or endogenous loading. Exogenous loading is to first purify the acquired exosomes, and then introduce the target polynucleotides into the purified exosomes by electrotransduction methods such as electroporation or chemical transduction. Endogenous loading is to first transform the source cells, for example, by directly transfecting or co-incubating the target polynucleotides into the source cells, and then causing the source cells to produce exosomes to obtain exosomes containing the target polynucleotides. In a preferred embodiment of the present invention, the target polynucleotides are introduced into exosomes using an exogenous loading method to obtain exosomes containing the target polynucleotides.

The method for purifying exosomes is carried out in accordance with conventional methods in the art. For example, the crude exosome particles are purified using an Exosomoe Purification Filter (EPF column), centrifuged at 3000 g for 10 min at 4°C, and the liquid at the bottom of the EPF column is collected after centrifugation to obtain purified exosome particles.

Pharmaceutical composition

The present invention also relates to a pharmaceutical composition, comprising the polynucleotide of the present invention or the exosome of the present invention, and a pharmaceutically acceptable carrier.

As used in the present invention, the term "pharmaceutically acceptable carrier" may include one or more of a pharmaceutically acceptable excipient, a buffer, a stabilizer, or other substances known to those skilled in the art. Examples of pharmaceutically acceptable carriers include, but are not limited to, one or more of water, saline, a buffer, an isotonic agent such as a sugar, a polyol, an auxiliary substance such as a wetting agent or an emulsifier, a preservative, and a combination thereof. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient at the dosage and concentration employed. The exact nature of the carrier or other substance may depend on the route of administration, such as intramuscular, subcutaneous, oral, intravenous, skin, mucosal (e.g., intestinal), intranasal or intraperitoneal routes. In some embodiments of the present invention, the pharmaceutical composition comprises a polynucleotide of the present invention and a nucleic acid delivery vector as a pharmaceutically acceptable carrier, examples of which include liposomes, biocompatible polymers (including natural polymers and synthetic polymers), lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, viruses (such as baculovirus, adenovirus and retrovirus), bacteriophages, cosmids, plasmids, fungal vectors and vectors commonly used in the art and described Other recombinant vectors for expression in various eukaryotic hosts.

Treatment

The present invention also relates to a method for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder, comprising the steps of: applying a therapeutically effective amount of the above-mentioned polynucleotide to a subject, or applying a therapeutically effective amount of exosomes to a subject, or applying a therapeutically effective amount of a pharmaceutical composition to a subject.

As used herein, the term "treat" refers to eradicating or ameliorating a disease or condition, or one or more symptoms associated with the disease or condition. In one embodiment, such symptoms are known to those skilled in the art to be associated with the disease or condition to be treated. In a specific embodiment, the term refers to minimizing the spread or worsening of a disease or condition by administering one or more preventive or therapeutic agents to a subject suffering from the disease or condition. In some embodiments, the term refers to administering a compound of the present invention after the onset of symptoms of a particular disease, with or without other additional active agents.

As used in the present invention, the term "prevention" refers to preventing the onset, recurrence or spread of a disease or condition, or one or more symptoms associated with the disease or condition. In one embodiment, the symptom is known to those skilled in the art to be associated with the disease or condition to be prevented. In a specific embodiment, the term refers to the administration of the polynucleotides or exosomes of the present invention in combination with or without other additional active agents before the onset of symptoms to patients at risk of suffering from the diseases or disorders described herein. The term includes the inhibition and reduction of symptoms of a particular disease. In a specific embodiment, patients with a family history of a disease are specifically considered candidates. In addition, patients with a history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" can be used interchangeably with the term "prophylactic treatment".

In the present invention, a "therapeutically effective amount" of a polynucleotide or exosome refers to an amount of the drug sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent that can provide a therapeutic effect in the treatment or management of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

As used herein, the term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

As the mode of administration of the polynucleotide, exosome or pharmaceutical composition in the present invention, oral, parenteral, inhalation spray, topical, rectal, nasal, oral, vaginal or via implanted reservoir administration can be selected. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally or intravenously. In a more preferred embodiment of the present invention, the mode of administration is intravenous injection.

use

The present invention also relates to the use of the above-mentioned polynucleotide, exosome or pharmaceutical composition for

(i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder;

(ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder;

(iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or

(iv) Use as a syn2a inhibitor.

As used in the present invention, the term "and/or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Therefore, the term "and/or" used in phrases such as "A and/or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and/or" used in the term "A, B and/or C" is intended to cover the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

Example 1

In this example, exosomes were extracted and purified. The specific steps are as follows:

For routine mouse cell culture, the supernatant was first pretreated. Exosomoe Concentration Solution (ECS reagent) was added to the centrifugal supernatant after impurities were removed. After adding the ECS reagent, the centrifuge tube was tightly capped and mixed by vortex oscillator for 1 min, and then placed at 4°C for 2 h; the centrifuge tube containing the mixed solution was taken out and centrifuged at 10000g for 60 min at 4°C, and the supernatant was discarded. The precipitate was rich in exosome particles; 100μL 1×PBS was taken to evenly blow the centrifuged precipitate, and after it was evenly suspended in PBS, the resuspension was transferred to a new 1.5mL centrifuge tube; the 1.5mL centrifuge tube containing the resuspension was centrifuged at 12000g for 2 min at 4°C, and the supernatant was retained. The supernatant was rich in exosome particles.

The harvested crude exosome particles were transferred into the upper chamber of Exosomoe Purification Filter (EPF column), centrifuged at 3000 g for 10 min at 4°C, and the liquid at the bottom of the EPF column was collected after centrifugation. This liquid was the purified exosome particles.

Example 2

In this example, in order to better determine the localization of our exosomes, the purified exosomes obtained in Example 1 were labeled with fluorescent dyes. The specific steps are as follows:

Preparation of dye working solution: dilute the storage solution with Diluent C to prepare a dye working solution with a concentration of 100 μM (protect from light);

Exosome staining: Add the dye working solution to the exosomes, cover the centrifuge tube tightly after adding the dye working solution, mix by vortexing for 1 min, and then incubate for 10 min. Add 1 mL of 1X PBS to the incubated exosome-dye complex and mix well. Extract the exosomes again according to the exosome extraction method to remove excess dye, take 100 μL of 1×PBS to resuspend the precipitate, and the precipitate is the labeled exosomes.

Example 3

In this example, siRNA is electroporated into exosomes labeled with fluorescent dyes. The specific steps are as follows:

The fluorescent dye-labeled exosomes obtained in Example 2 were taken, and 100 μg of si-syn2a was electroporated into the exosomes using an electroporation procedure.

The exosomes labeled with fluorescent dye obtained in Example 2 were taken, and 100 μg of other siRNA sequences designed by the inventors were sequentially electroporated into the exosomes using an electroporation procedure to obtain a control group. For example, the siRNA NC sequence is electroporated into exosomes to obtain a control group.

syn2a siRNA(SEQ ID NO:1):CCCAGAAATGACCATGTGATA;

siRNA NC sequence (SEQ ID NO:2): TTCTCCGAACGTGTCACGT.

Example 4

In this example, exosomes were injected into the brains of experimental animals to conduct behavioral experiments on cued fear conditioning memory extinction.

The exosomes obtained in Example 3 were injected into mice by tail vein injection, and the distribution of exosomes in the body was observed at 0 hours, 2 hours and 8 hours by small animal in vivo imaging technology (Figure 1). The inventors found that the exosomes had entered the brain at 2 hours and could last up to 8 hours.

The inventors tested the effect of si-syn2a exosome drugs through classic cue-based fear memory extinction behavior. First, 10 male 8-10 week wild-type mice were selected. Before the experiment, exosomes were injected into the tail vein 6 hours in advance (5 experimental groups and 5 control groups). On the first day of the experiment, the mice were placed in the electric shock box. At this time, the electric shock box scene was A, and the smell was low-concentration ethanol. After 3 minutes, the first foot shock (0.8mA, 1s) and sound (80db, 4000hz, 30s) were paired, that is, 1s electric shock was performed 29s after the sound appeared, and the sound and electric shock ended at the same time. After 5 such pairing exercises, the interval was 20s. The mice were taken out and put back into the cage 60s after the last training, and the stiffness rate of each mouse was recorded. The mice were conditioned for fear memory on the second day. On the third day of the experiment, exosomes were injected into the tail vein 6 hours in advance, and then fear memory extinction training was performed. The mice were placed in the experimental box, and the scene of the experimental box was changed to scene B. The odor was low-concentration acetic acid. After 3 minutes of adaptation, 14 cycles of sound stimulation (80dB, 4000Hz, 20s) were presented, without electric shock. The interval between two adjacent sounds was 20s. 60s after the last training, the mice were taken out of the experimental box and put back into the mouse cage, and the stiffness rate was recorded. On the fourth day of the experiment, the same operation was performed as on the third day, and the stiffness rate was finally counted. The experiment achieved the expected effect. Through the statistics of the stiffness rate, it was found that the stiffness rate of the group injected with si-syn2a exosomes was significantly reduced (Figure 3-4). At the same time, the mice were killed and brain tissue was taken. Through the protein immunoblotting experiment, it was proved that the group injected with si-syn2a exosomes did reduce the protein level of syn2a in the brain compared with the control group (Figure 2).

At the same time, referring to the methods in the prior art, a fear memory-difficult extinction mouse AtLAS-/- mouse model was prepared. It is a good PTSD model mouse. The same behavioral experiment was used to verify it again. Through statistics, it was found that the stiffness rate of the AtLAS-/- mouse group injected with si-syn2a exosomes was significantly lower than that of the AtLAS-/- mouse group. AtLAS-/- mice were injected with a control exosome group (Figures 5-6).

Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims (10)

An isolated polynucleotide, characterized in that the polynucleotide is used as a syn2a inhibitor, and the polynucleotide is selected from any one of the following: (i) the polynucleotide shown in SEQ ID NO: 1; (ii) a polynucleotide having a homology greater than 80% with the sequence shown in SEQ ID NO:1; and (iii) A polynucleotide formed by substitution, deletion or addition of one or several nucleotides in the sequence shown in SEQ ID NO:1. A small nucleic acid molecule containing the polynucleotide according to claim 1, wherein the small nucleic acid molecule is siRNA, siRNA precursor, dsRNA, shRNA, miRNA or small interfering RNA. An exosome containing the polynucleotide according to claim 1 or the small nucleic acid molecule according to claim 2. A pharmaceutical composition, characterized in that it comprises: the polynucleotide according to claim 1 or the small nucleic acid molecule according to claim 2, and a pharmaceutically acceptable carrier. A method for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder, characterized in that the method comprises the steps of: applying a therapeutically effective amount of the polynucleotide according to claim 1 to a subject; or Applying a therapeutically effective amount of the small nucleic acid molecule of claim 2 to a subject; or Applying a therapeutically effective amount of the exosomes according to claim 3 to a subject; or A therapeutically effective amount of the pharmaceutical composition of claim 4 is administered to the subject. Use of the polynucleotide according to claim 1, for: (i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder; (ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder; (iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or (iv) Use as a syn2a inhibitor. Use of the small nucleic acid molecule according to claim 2, for: (i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder; (ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder; (iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or (iv) Use as a syn2a inhibitor. The use of the exosomes according to claim 3, for: (i) prevention, treatment and/or auxiliary treatment of post-traumatic stress disorder; (ii) preparing a medicament for preventing, treating and/or assisting in the treatment of post-traumatic stress disorder; (iii) non-therapeutically reducing the expression level of the syn2a gene or its encoded protein in vitro; and/or (iv) Use as a syn2a inhibitor. The method for preparing exosomes according to claim 3, characterized in that the method comprises the steps of: The original exosomes are obtained, and then the polynucleotide according to claim 1 is introduced into the original exosomes. The method according to claim 8, characterized in that the original exosomes are obtained by treating cells with an ECS reagent.