WO2024050923A1

WO2024050923A1 - Neuronal transfection method based on adipose-derived exosomes and use thereof

Info

Publication number: WO2024050923A1
Application number: PCT/CN2022/125542
Authority: WO
Inventors: 毕艳; 王进
Original assignee: 南京鼓楼医院
Priority date: 2022-09-05
Filing date: 2022-10-17
Publication date: 2024-03-14
Also published as: CN115141804A; CN115141804B; LU504447B1

Abstract

Provided is a neuronal transfection method, comprising the following steps: 1) culturing adipocytes to obtain standardized adipose-derived exosomes; 2) packaging genetic material into isolated adipose-derived exosomes; and 3) co-culturing primary neurons with the adipose-derived exosomes packaged with the genetic material for transfection, wherein the standardized adipose-derived exosomes are obtained by means of orthotopic injection of adeno-associated viruses into adipose and culturing the adipose afterwards. Further provided is use of the adipose-derived exosomes in neuronal transfection.

Description

A neuron transfection method based on fat exosomes and its application

Technical field

The invention belongs to the fields of biotechnology and medicine. Specifically, it relates to a method for in vivo and in vitro transfection of neurons based on fat exosomes, and further relates to the application of this method.

Background technique

Transfection is the process of transferring genetic material into cells and is an important means to effectively change gene expression in cells. Its ability to silence endogenous protein expression/drive exogenous gene expression and protein modification makes transfection an important method for studying key molecules that regulate neuronal function. However, neurons are highly differentiated cells that are different from other cells. Due to the fragile nature of mature neurons, transfection of neurons is usually inefficient and difficult to survive, making their transfection very challenging.

The current methods for neuron transfection mainly include: nucleofection, gene gun and viral transfection. However, these three methods currently have obvious shortcomings: nucleofection has a high cell lethality rate, a large demand for cells, and can only be operated on cells in vitro; although gene gun has low damage to cells, its transfection efficiency is low. It is not high and can only be operated on cells in vitro; although viral transfection has a high transfection efficiency, its preparation procedures are complex, the virus construction cycle is long, there are many links, it is error-prone and it is expensive. The most widely used liposome-mediated method has extremely low transfection efficiency for neurons, and the transfection reagent is highly cytotoxic. Currently, there is no method that can achieve efficient transfection of neurons both in vivo and in vitro.

Exosomes are plasma membrane vesicles secreted by different cell types in the body (Ibrahim and Marbán (2016) Annu Rev Physiol. 78:67 83); reflects the contents of its source cell, its normal state or pathophysiological state. Exosomes are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal particles and are released into the extracellular matrix through the fusion of the outer membrane and the cell membrane; they are found in many biological fluids, such as blood, urine, Saliva, cerebrospinal fluid, lymph fluid, bile, alveolar lavage fluid, semen, synovial fluid, amniotic fluid, breast milk, etc. It can transmit a variety of signal transduction molecules, such as functional proteins, nucleic acids (such as DNA, mRNA, microRNA, siRNA), lipids, long non-coding RNA (lncRNA), etc., and enter recipient cells and exert biological functions. Thus participating in the transmission of information between cells (Valadi et al. (2007) Nat Cell Biol., 9(6):654 9; Li et al. (2016) Nat Commun. 7:10872; Liu et al. (2015) Cell Metab. 22(4):606 18). At the same time, exosomes can pass through strict biological barriers, such as blood-brain barrier and placental barrier (Alvarez Erviti et al. (2011) Nat Biotechnol. 29(4):341 5; Holder et al. (2016) Traffic 17(2): 168 78; Shi et al. (2017) Biochem. Biophys. Res Commun. 483(1):602 8); therefore, its It is expected to become an effective carrier for substance delivery.

In view of this, the present invention hopes to provide an efficient neuron transfection method based on fat exosomes, which can effectively achieve neuron transfection in vivo and in vitro, while having low cytotoxicity, low immunogenicity and high transfection efficiency. .

technical problem

Technical solutions

In order to solve the above-mentioned technical defects in neuron transfection, the present invention first provides a method for efficient transfection of neurons based on fat exosomes, which can achieve efficient transfection of neurons both in vivo and in vitro. Dyeing, high safety and simple operation.

First, in a first aspect, the present invention relates to an in vivo or in vitro transfection method of neurons, wherein the method is implemented based on fat exosomes. Specifically, the method includes the following steps:

1) Adipocyte culture to obtain standardized adipose exosomes;

2) Packaging of genetic material into isolated fat exosomes;

3) Co-culture fat exosomes packaging genetic material with primary neurons to achieve neuronal transfection.

In one embodiment, the standardized adipose exosomes are obtained by in situ injection of adeno-associated virus through fat and cultured in fat; in a preferred embodiment, the adeno-associated virus is rAAV-siDicer.

In one embodiment, the transfection is performed in vivo or in vitro; in a preferred embodiment, the transfection is performed in vivo.

In one embodiment, the in vivo transfection is achieved by stereotactic injection of fat exosomes encapsulating genetic material into the brain.

Preferably, the genetic material is selected from one or more of DNA, mRNA, dsRNA, siRNA, shRNA, miRNA or plasmid; more preferably, it is siRNA or plasmid; further preferably, it is siBACE1 or mCherry plasmid.

In one embodiment, the fat in situ injection refers to in situ injecting the rAAV-siDicer virus into the visceral adipose tissue of infected mice; optionally, 2 weeks after the injection infection, the visceral fat of the mice is taken for Culture and isolate exosomes.

In one embodiment, the exosome extraction method is: cut the adipose tissue of the infected mice into small pieces of adipose tissue, culture it in DMEM complete medium and collect the supernatant; centrifuge to extract the adipose tissue exosomes ;

Preferably, the adipose tissue is chopped into small fat pieces of 1 mm3; the DMEM complete medium contains: 1% 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2% exosome-depleted fetal bovine. serum. In a preferred embodiment, adipose tissue exosomes are extracted using ultracentrifugation.

In one embodiment, after step 1), it also includes:

1a) Identification of standardized fat exosomes; wherein the identification process includes one or more of electron microscopy, particle size molecules, and RNA content detection methods.

In one embodiment, the genetic material is packaged into isolated adipose exosomes by chemical transfection, electroporation, heat shock, viral infection or microinjection; in a preferred embodiment, the method is electroporation .

In one embodiment, in step 3) of the method, adipose exosomes are co-cultured with neurons for 24-48 h for transfection. In a preferred embodiment, the neurons are co-cultured for about 48 h.

In specific embodiments, the neuronal transfection method includes:

1. Preparation of standardized adipose exosomes: Preparation of rAAV-SiDicer adeno-associated virus: rAAV-U6-shRNA(Dicer)-CMV-mCherry-SV40 pA.

2. Inject the constructed rAAV-siDicer virus in situ into the visceral adipose tissue of infected mice. After 2 weeks, the visceral fat of the mice was harvested, and the adipose tissue of the rAAV-siDicer-infected mice was cut into small pieces, cultured in DMEM complete medium for 24h to 48h, and then the supernatant was collected. Exosomes from adipose tissue were extracted by ultracentrifugation.

3. Identification of standardized fat exosomes: Use electron microscopy, particle size analysis and/or RNA content detection to identify the morphology of exosomes, analyze the particle size of exosomes, extract total RNA from exosomes and detect changes in RNA concentration.

4. Encapsulating target genetic material such as siRNA and/or plasmids: The target genetic material is packaged into standardized fat exosomes through electroporation, and the efficiency of packaging is detected by qRT-PCR.

5. Neuron transfection: Co-culture the standardized fat exosomes loaded with target genetic material with primary neurons for 24-48 hours to perform neuron transfection; optionally, use fat wrapped with genetic material such as siRNA/plasmid Exosomes are injected stereotaxically into the brain to achieve in vivo transfection of neurons.

In another aspect of the invention, the invention also relates to the use of adipose exosomes in neuron transfection, wherein the standardized adipose exosomes are obtained by injecting adeno-associated virus in situ through fat and performing fat culture. . In a preferred embodiment, the adeno-associated virus is rAAV-siDicer.

The following definitions and descriptions are explanations of terms used in the present invention; when describing the present invention, unless otherwise stated, technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. , the publications and documents mentioned herein are incorporated herein by reference.

As used herein, the term "comprises" or "comprises" means that the method, structure, or composition includes any of the steps/operations, components, components, etc. of the examples, but does not exclude any other steps/operations. , components, components.

In the context of this invention, when referring to a numerical range, it is to be understood that the upper and lower limits of the range, and every intervening value therebetween, as well as any other specified value or intervening value within the range, are included in the invention. The upper and lower limits of such smaller ranges, which may independently be included within the range, are also included within the invention, subject to any express exclusion within the specified range. When the specified range includes one or both of the limiting values, ranges excluding either of the limiting values are also within the invention.

The present invention involves growing cells, isolated cells and related clones, DNA isolation, amplification and purification, standard technologies for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., and various separation technologies are It is well known and commonly used by those skilled in the art.

The term "neuron" includes a neuron and one or more of its component parts (e.g., neuronal cell body, axon, or dendrite). "Neuron" means a nervous system cell that contains a central cell body and two types of extensions, or projections: dendrites, through which most neuronal signals are transmitted to the cell body; and axons, through which most neuronal signals are usually transmitted Axons travel from the cell body to effector cells, such as target neurons or muscles.

As used herein, the term "transfection" means that exogenous nucleic acid is transferred or introduced into a host cell. A "transfected cell" is a cell that has been transfected with an exogenous nucleic acid, including primary subject cells and their progeny; preferably, as used herein, the cells are neuronal cells.

The term "Exosome" refers to the nanovesicle component secreted by cells in biological fluids. It is a membranous lipid vesicle with a lipid bilayer structure and a surface including proteins and sugars. ; According to the different particle sizes of exosomes, their diameter is usually 30-200nm, specifically 40-150nm, more specifically 50nm-120nm, even more specifically 50-100nm. Preferably, the exosomes are fat exosomes.

As used herein, the terms "extracellular vesicles" or "exosomes" are used interchangeably and are understood to refer to any type of vesicles that may be obtained from cells in any form, such as microvesicles (e.g., from cellular plasma any vesicle shed from the membrane), exosomes (e.g. any vesicles derived from the intralysosomal pathway), apoptotic bodies (e.g. obtained from apoptotic cells), particles (e.g. derived from platelets), nuclei Extragranular bodies (for example, they can be derived from neutrophils or monocytes in serum), prostatic bodies (for example, they can be obtained from prostate cancer cells), or cardiac bodies (for example, they can be derived from heart cells). Furthermore, the terms "exosomes" and/or "microvesicles" are also to be understood as referring to extracellular vesicle mimics, cell membrane-based vesicles obtained by, for example, membrane extrusion, sonication or other techniques.

The term "genetic material" refers to genes, nucleic acids, DNA and/or RNA; other examples of nucleic acid molecules include, but are not limited to, oligonucleotides, such as interfering ribonucleic acids (iRNA), including but not limited to small interfering RNA (siRNA). ), microRNA (miRNA), short hairpin RNA (shRNA). The term "siRNA" refers to short double-stranded RNA or RNA analogs composed of about ten to dozens of nucleotides that induce RNA interference (RNAi). "shRNA" refers to a single molecule of RNA capable of performing RNAi and having a follower strand, a loop, and a guide strand, wherein the follower strand and the guide strand may be substantially complementary to each other. miRNAs are generally single-stranded molecules of approximately 20 nucleic acids on average.

beneficial effects

Beneficial effects of the present invention: The method of the present invention has a high transfection efficiency of more than 80%, low cytotoxicity, can realize the transfection of neurons in vivo and in vitro at the same time, and has the characteristics of low immunogenicity and high safety. ; The method of the present invention is flexible, can realize the delivery of various substances such as miRNA, siRNA, mRNA and plasmid, and is easy to operate.

In order to clearly explain the technical solutions of the embodiments of the present invention, the steps involved in the embodiments and the corresponding drawings will be introduced and explained below. It is obvious that the drawings or embodiments described below are only illustrative of part of the invention.

Description of the drawings

Figure 1: (A) Electron microscope photo of exosomes; (B) Nanosight particle size analysis results of exosomes; (C) Comparison of RNA content in fat exosomes;

Figure 2: (A) Relative concentration of siBACE1 after adipose exosomes package siBACE1-cy3; (B) Fluorescence micrograph after exosomes package fluorescently labeled siBACE1-cy3; (C) Standardized adipose exosomes loaded with siBACE1-cy3 Western-blot results after co-culture of secretosomes and primary neurons;

Figure 3: (A) Relative protein levels of BACE1 after exosome transfection of primary neurons; (B) Percentage of primary neurons transfected with exosomes packaging siBACE1-cy3;

Figure 4: (A) Fluorescence micrograph of primary neurons transfected with exosomes containing the packaging plasmid; (B) Percentage of primary neurons transfected with exosomes containing the packaging plasmid;

Figure 5: Neuronal cell activity after transfection of primary neurons with exosomes packaging mCherry plasmid;

Figure 6: Fluorescence micrograph of mouse hippocampus after exosomes packaging mCherry plasmid are transfected into neurons in mice;

Figure 7: (A) Comparison of IBA-1 density after transfection in mice with exosomes packaging mCherry plasmid; (B) Comparison of pro-inflammatory cytokine concentrations.

Best Mode of Carrying Out the Invention

The invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the invention and are not intended to limit the scope of the invention.

Example 1: Preparation of standardized adipose exosomes

(1) Prepare rAAV-siDicer adeno-associated virus and control virus, rAAV-siDicer adeno-associated virus: rAAV-U6-shRNA(Dicer)-CMV-mCherry-SV40 pA, control virus: rAAV-U6-shRNA(scramble)-CMV -mCherry-SV40 pA, constructed by Wuhan Primus Brain Science and Technology Co., Ltd.

(2) Inject the constructed rAAV-siDicer virus and control virus in situ into the visceral adipose tissue of infected mice. After 2 weeks, mice (8E+12 vg/ml, 2 μL) visceral fat was cultured, and exosomes were isolated and extracted. The method was as follows: cut the adipose tissue of virus-infected mice into small pieces of 1 mm3, and place them in DMEM complete medium (containing 1% 100 μg/mL penicillin + 100 μg/mL streptomycin + 2% exosome-removed fetal bovine serum), and the supernatant was collected after 24 h of culture. Exosomes from adipose tissue were extracted by ultracentrifugation.

Example 2: Identification of standardized adipose exosomes

Identification of standardized adipose exosomes includes: electron microscopy, particle size analysis, and RNA content detection. The specific steps are as follows:

(1) Electron microscopy detects the morphology of exosomes (Figure 1A), and Nanosight analyzes the particle size of exosomes (Figure 1B).

(2) Extract total exosome RNA and detect changes in RNA concentration: homogenize the sample with TRIzol reagent, add chloroform, centrifuge the homogenate, take the upper water layer, and add isopropanol to precipitate RNA from the water layer. The precipitated RNA was washed with 75% ethanol to remove impurities and then resuspended in RNase-free ddH2O. The RNA concentration was measured with a spectrophotometer, and the comparison found that compared with the adipose tissue treated with the control virus, the RNA content of exosomes extracted from adipose tissue with rAAV-siDicer was significantly reduced (Figure 1C).

Example 3: Standardized adipose exosomes encapsulate siBACE1, enter primary neurons and reduce BACE1 expression

(1) Package fluorescently labeled siBACE1-cy3 into standardized adipose exosomes by electroporation method (400V, 125μF) (ratio: 5nmol siRNA: 5μg standardized exosomes), and qRT-PCR detects the packaging efficiency (As shown in Figure 2A), the results show that siBACE1-cy3 has been effectively packaged into fat exosomes, with a relative concentration of 81.65%;

(2) Standardized adipose exosomes (5 μg) loaded with siBACE1-cy3 were co-cultured with primary neurons. After 6 hours, the siBACE-cy3 signal in primary neurons was observed under a fluorescence microscope. The results showed that the siBACE-cy3 signal in primary neurons appeared A large amount of cy3 signal (Figure 2B);

(3) Standardized adipose exosomes (5 μg) loaded with siBACE1-cy3 were co-cultured with primary neurons for 48 hours, and Western-blot detected the BACE1 protein expression level of primary neurons. The results showed that the BACE1 expression level was significantly reduced (Figure 2C , Figure 3), indicating that standardized adipose exosomes packaging siBACE1-cy3 have been successfully transfected into primary neurons, and the expression of BACE1 was reduced, and the neuron transfection percentage reached 93.85%.

Example 4: Plasmid transfection of primary neurons using standardized adipose exosomes

(1) Refer to the electroporation method in Example 3 to package the mCherry-expressing plasmid into standardized fat exosomes.

(2) The standardized adipose exosomes loaded with mCherry plasmid were co-cultured with primary neurons for 48 hours, and the transfection efficiency was detected by fluorescence. The results showed that the plasmid had been successfully transfected into neurons (as shown in Figure 4(A)). The dyeing efficiency is about 80% (as shown in Figure 4(B)). That is to say, standardized adipose exosomes can achieve plasmid transfection of primary neurons, which is significantly higher than traditional transfection methods.

Example 5: Neurotoxicity assay of standardized adipose exosome transfection

Standardized adipose exosomes loaded with mCherry plasmid were co-cultured with primary neurons for 48 hours, and the cell activity of neurons was detected.

The results showed that the cell activity of neurons after transfection with standardized adipose exosomes did not change significantly compared with the control, that is, the transfection of adipose exosomes did not reduce the cell activity of primary neurons (Figure 5).

The above results of Examples 3-5 show that standardized adipose exosomes can achieve effective transfection of siRNA/plasmid in neurons without obvious neurotoxicity.

Example 6: Standardized adipose exosomes can target centrally transfected neurons

Standardized fat exosomes loaded with mCherry plasmid were stereotaxically injected into the hippocampus of mice. After 5 days, mouse brain tissue was taken, and the expression of mCherry in the mouse hippocampus was detected by fluorescence.

The test found that obvious mCherry signals appeared in the hippocampus of mice (Figure 6).

Example 7: Safety verification of standardized adipose exosome brain stereotaxic injection

Standardized adipose exosomes loaded with mCherry plasmid and an equal volume of normal saline were injected into the lateral ventricle of mice. The mice were sacrificed after 3 days, and the brain tissue was taken and immunofluorescence stained microglia. The results showed that compared with the control, there was no significant change in IBA-1 density in the brain tissue transfected with mCherry-packaged standardized adipose exosomes. (As shown in Figure 7(A)); The expression of inflammatory factors was detected by qRT-PCR. The results showed that compared with the control, in the brain tissue of mice transfected with standardized adipose exosomes loaded with mCherry plasmid, pro-inflammatory cytokines There was no significant difference in the concentrations (TNFα, IL-6 and IL-1β), suggesting that in vivo transfection of adipose exosomes did not cause central inflammation (Figure 7). This shows that the method of the present invention has the characteristics of low immunogenicity and high safety.

In summary, through the present invention, efficient transfection of neurons can be achieved based on standardized adipose exosomes, including transfection of siRNA/plasmid, etc.

The above descriptions are only exemplary embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

A neuron transfection method, which includes the following steps:

1) Adipocyte culture to obtain standardized adipose exosomes;

2) Packaging of genetic material into isolated fat exosomes;

3) Co-culture and transfect the fat exosomes packaging genetic material with primary neurons;

Wherein, the standardized adipose exosomes are obtained by in situ injection of adeno-associated virus through fat and cultured in fat; preferably, the adeno-associated virus is rAAV-siDicer.
The method of claim 1, wherein said transfection is performed in vivo or in vitro; preferably, in vivo.
The method of claim 2, wherein the in vivo transfection is achieved by stereotactic injection of fat exosomes encapsulating genetic material into the brain.
The method according to any one of claims 1-3, wherein the genetic material is selected from DNA, mRNA, dsRNA, siRNA, shRNA, miRNA or plasmid; preferably, it is siRNA or plasmid; further preferably, it is siBACE1 or mCherry plasmid.
The method according to claim 1, wherein the fat in situ injection is to in situ inject the rAAV-siDicer virus into the visceral adipose tissue of infected mice; optionally, 2 weeks after the injection infection, the viscera of the mice is taken Fat was cultured and exosomes were isolated.
The method according to claim 5, wherein the exosome extraction method is: cut the adipose tissue of the infected mice into small pieces of adipose tissue, place it in DMEM complete medium and culture it and collect the supernatant; centrifuge to extract the adipose tissue. exosomes;

Preferably, the adipose tissue is cut into small pieces of 1 mm3 ; the DMEM complete medium contains: 1% 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2% exosome-removed fetuses. bovine serum;

Preferably, ultracentrifugation is used to extract adipose tissue exosomes.
The method according to claim 6, further comprising:

1a) Identification of standardized adipose exosomes; wherein the identification process includes one or more of electron microscopy, particle size molecule, and RNA content detection methods.
The method according to any one of claims 1-4, wherein the method of packaging the genetic material into isolated fat exosomes is selected from chemical transfection, electroporation, heat shock, viral infection or microinjection; preferably for electroporation.
The method according to any one of claims 1 to 4, wherein in step 3), fat exosomes and neurons are co-cultured for 24-48 hours for transfection; preferably, the co-culture is performed for 48 hours.
The use of adipose exosomes in neuron transfection, wherein the standardized adipose exosomes are obtained by in situ injection of adeno-associated virus through fat and performing fat culture; preferably, the adeno-associated virus is rAAV-siDicer .