WO2021233346A1 - 一种被靶向修饰且装载有药物的外泌体及其制备方法和应用 - Google Patents

一种被靶向修饰且装载有药物的外泌体及其制备方法和应用 Download PDF

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WO2021233346A1
WO2021233346A1 PCT/CN2021/094662 CN2021094662W WO2021233346A1 WO 2021233346 A1 WO2021233346 A1 WO 2021233346A1 CN 2021094662 W CN2021094662 W CN 2021094662W WO 2021233346 A1 WO2021233346 A1 WO 2021233346A1
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exosomes
ngf
neurotrophic factor
disease
rvg
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French (fr)
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杨佳蕾
王拥军
杨国栋
吴诗坡
侯利华
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首都医科大学附属北京天坛医院
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Definitions

  • the present invention relates to the field of biotechnology, in particular to an exosome that is targeted to be modified and loaded with drugs, and a preparation method and application thereof.
  • the neurotrophic factor family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 4 (NT4), neurotrophic factor 5 (NT5) and so on.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT4 neurotrophic factor 4
  • NT5 neurotrophic factor 5
  • NGF is the first molecule discovered in the neurotrophic factor family, and it plays a key regulatory role in the physiological development of the nervous system. After the nervous system is injured, the neurotrophic factor family can play a role in reducing tissue damage and promoting tissue repair.
  • administration of NGF can reduce nerve cell death, regulate inflammation, promote cell polarization to anti-inflammatory, promote angiogenesis, promote nerve regeneration and improve nerve function, and has a broad-spectrum neuroprotective effect.
  • Previous studies have found that the expression level of endogenous NGF drops significantly after cerebral ischemic injury. Therefore, if neurotrophic factors can be effectively delivered to receptor cells in brain tissue, it may have a therapeutic effect on neurological diseases
  • neurotrophic factors to nerve tissue receptor cells has the following problems: (1) The neurotrophic factors given by conventional intravenous injection or subcutaneous injection cannot efficiently pass through the blood-brain barrier, although viral vectors and neurotrophic factor genes Modified cells, nanocapsules and hydrogels have been used as loading methods for neurotrophic factors. However, there is currently no efficient delivery method for loading neurotrophic factors into nerve tissues. (2) The half-life of neurotrophic factors is relatively short. For example, the half-life of NGF given exogenously by vein is only 2.3 hours in vivo, and the half-life of NGF given by subcutaneous injection is only 4.5 hours. (3) Non-specific delivery can cause unintended adverse effects on peripheral tissues. For example, intramuscular injection of NGF can cause local pain. The above-mentioned various factors hinder the application of NGF in the treatment of neurological diseases.
  • mRNA messenger RNA
  • the purpose of the present invention is to provide a targeted modified exosomes loaded with drugs and a preparation method and application thereof.
  • the exosomes are loaded with neurotrophic factors, neurotrophic factor mRNA, etc., which can be targeted to Nerve tissue receptor cells deliver neurotrophic factors, thereby reducing nerve tissue damage and promoting repair by means of anti-inflammatory, anti-apoptotic, promoting nerve cell survival, and promoting nerve regeneration.
  • embodiments of the present invention provide a rabies virus glycoprotein (RVG) modified exosomes loaded with one or more selected from the following: neurotrophic factors; Neurotrophic factor fragments with trophic factor activity; short peptides with neurotrophic factor activity; and mRNA encoding the same.
  • RVG rabies virus glycoprotein
  • the neurotrophic factor includes: one or more of nerve growth factor, brain-derived neurotrophic factor, neurotrophic factor 4, neurotrophic factor 5, neurotrophic factor 6, and neurotrophic factor 7. kind.
  • the rabies virus glycoprotein (RVG) modified exosomes loaded with neurotrophic factor and/or neurotrophic factor mRNA are freshly extracted or frozen.
  • the temperature condition of the cryopreservation is -80°C
  • the freezing time is 3 months or less.
  • the exosomes target nerve cells; alternatively, target nerve cells of brain tissue.
  • the exosomes can be taken up by neuronal cells, astrocytes, microglia, oligodendrocytes, and other nerve tissue cells.
  • the rabies virus glycoprotein includes the sequence shown in SEQ ID NO:1.
  • the rabies virus glycoprotein (RVG) and the exosomal protein lysosomal-associated membrane glycoprotein 2b (Lamp2b) form a fusion protein.
  • the fusion protein includes the sequence shown in SEQ ID NO: 2.
  • the embodiment of the present invention also provides a method for preparing the above exosomes, including the following steps: using a recombinant vector and clone expressed by the fusion of rabies virus glycoprotein (RVG) and exosomal protein lysosomal-associated membrane glycoprotein 2b (Lamp2b) Recombinant vectors containing neurotrophic factors are co-transfected into cells of the expression system, and after expression, the cell supernatant is collected and subjected to ultracentrifugation to extract exosomes.
  • RVG rabies virus glycoprotein
  • Lamp2b exosomal protein lysosomal-associated membrane glycoprotein 2b
  • the embodiment of the present invention also provides an expression system comprising a recombinant vector expressing fusion of rabies virus glycoprotein (RVG) and exosomal protein lysosomal-associated membrane glycoprotein 2b (Lamp2b) and a recombinant vector cloned with neurotrophic factors cell.
  • RVG rabies virus glycoprotein
  • Lamp2b exosomal protein lysosomal-associated membrane glycoprotein 2b
  • the neurotrophic factor includes: one or more of nerve growth factor, brain-derived neurotrophic factor, neurotrophic factor 4, neurotrophic factor 5, neurotrophic factor 6, and neurotrophic factor 7. kind.
  • the recombinant vector includes a recombinant plasmid.
  • the recombinant vector containing the fusion expression of rabies virus glycoprotein (RVG) and exosomal protein lysosomal-associated membrane glycoprotein 2b (Lamp2b) is pcDNA3.1(-)-RVG-Lamp2b recombinant vector; the recombinant vector cloned with neurotrophic factor is pCI-neo-NGF recombinant plasmid.
  • the cells of the expression system include: human embryonic kidney 293 cells, mammalian bone marrow mesenchymal stem cells, mammalian neural stem cells, mammalian umbilical cord mesenchymal stem cells, and mammalian placental mesenchyme One or more of stem cells and mammalian blood stem cells.
  • the preparation method further includes the following steps: freezing the extracted exosomes; optionally, the temperature condition of the freezing is -80°C, and the freezing time is 3 Month or less.
  • the embodiment of the present invention also provides a pharmaceutical composition, which includes the above-mentioned exosomes, and also includes one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition is a pharmaceutical composition for treating neurological diseases.
  • the pharmaceutical composition further includes other active pharmaceutical ingredients for the treatment of neurological diseases.
  • the embodiment of the present invention also provides an application of the above-mentioned exosomes in the preparation of drugs for treating neurological diseases.
  • the embodiment of the present invention also provides a method for treating a nervous system disease, including the following steps: administering an effective dose of the above-mentioned exosomes or pharmaceutical composition to a subject suffering from a nervous system disease.
  • the nervous system diseases include: ischemic stroke, hemorrhagic stroke, cerebral small vessel disease, vascular cognitive impairment and other cerebrovascular diseases, motor neuron disease, Alzheimer’s disease, etc.
  • Nervous system degenerative diseases such as Murray's disease and multiple system atrophy, central nervous system demyelinating diseases such as multiple sclerosis, neuromyelitis optica, and leukodystrophy, movement disorders such as Parkinson's disease and Huntington's disease, myelitis, spinal cord injury
  • Spinal cord diseases peripheral nerve diseases such as facial neuritis and polyneuropathy, autonomic nervous system diseases such as neurovascular edema, Raynaud’s disease, neuromuscular junction and muscle diseases such as myasthenia gravis, muscular dystrophy, and hereditary ataxia
  • Systemic hereditary diseases neurological developmental disorders such as cerebral palsy, neurological complications of medical system diseases such as diabetic nervous system complications, thyroid disease neurological complications, and neurotraumatic diseases such as brain injury and spinal cord injury.
  • the method for treating neurological diseases includes one or more of the following: anti-inflammatory, anti-apoptosis, promoting nerve cell survival, promoting nerve regeneration, and the like.
  • the anti-inflammatory method includes: promoting the polarization of type 1 pro-inflammatory microglia to type 2 anti-inflammatory microglia.
  • the subject includes: human, monkey, pig, rabbit, dog, rat, or mouse.
  • the method of administration includes: intravenous injection, arterial injection, tissue positioning injection, subcutaneous injection, and intramuscular injection.
  • the exosomes of the embodiments of the present invention are loaded with neurotrophic factors and/or neurotrophic factor mRNAs, and the exosomes can be targeted to cross the blood-brain barrier and receive neurotrophic factors through a single intravenous injection.
  • Somatic cells deliver neurotrophic factors to reduce nerve tissue damage and promote repair by anti-inflammatory, anti-apoptotic, promoting nerve cell survival, and promoting nerve regeneration.
  • exosomes of the examples of the present invention can be targeted to and taken up by nerve tissue receptor cells within 2 hours of a single intravenous injection, and a certain amount of neurotrophic factors carried by themselves can be directly released to the recipient cells In order to achieve rapid treatment of the recipient cells, and the neurotrophic factor mRNA loaded on it will be subsequently translated in the recipient cells until the mRNA is degraded, and the mRNA can stably exist in the exosomes for a relatively long time , Thereby prolonging the half-life of neurotrophic factors in the body.
  • the use of biologically targeted modified exosomes to deliver NGF protein and mRNA can achieve high-efficiency neural tissue targeted delivery and at the same time have a drug-loading slow-release effect.
  • NGF protein can have immediate therapeutic effect, and NGF mRNA can be further translated into NGF protein for therapeutic effect. Therefore, the efficacy of a single injection is effectively enhanced. If the exosomes provided herein are injected short-term (for example, preferably within 24 hours) after cerebral ischemia, the key pathological time point of inflammation and cell death is at this time, and the intervention can effectively target key pathological events at this time.
  • exosomes of the embodiments of the present invention can promote their concentration in the cerebral ischemic injury area, and relatively reduce the uptake of exosomes from other peripheral tissues and organs, thereby reducing the toxicity of peripheral tissues and other unexpected during treatment. effect.
  • the exosomes of the examples of the present invention can be stored stably for up to 3 months at -80°C, and during this period, the neurotrophic factors and their mRNA loaded in the exosomes can also be stored stably Further, the therapeutic effect of exosomes after long-term storage is not significantly lower than that of freshly harvested exosomes.
  • the single injection of exosomes in the embodiment of the present invention can also reduce the occurrence of side effects compared to repeated injections of NGF protein repeatedly or continuously.
  • exosomes are generated in cells using dual expression vectors, which can simultaneously perform biological targeted modification of exosomes and target protein/mRNA loading, without electroporation, and simplify preparation operations.
  • the expansion of cells modified with dual expression vectors can effectively increase the production of exosomes and is more suitable for large-scale production.
  • Figure 1 is a technical route diagram of plasmid recombination, co-transfection, and isolation and identification of exosomes in Example 1 of the present invention.
  • FIG. 2A shows the relative expression levels of NGF mRNA in HEK293 without transfection, HEK293 co-transfected with pcDNA3.1+pCI-neo, and co-transfected RVG-Lamp2b+NGF HEK293 in Example 2 of the present invention.
  • Figure 2B shows exosomes obtained from HEK293 that has never been transfected, HEK293 co-transfected with pcDNA3.1+pCI-neo, and co-transfected with RVG-Lamp2b+NGF HEK293 culture supernatant in Example 2 of the present invention The relative expression of NGF mRNA.
  • Figure 2C shows exosomes obtained from HEK293 that has never been transfected, HEK293 co-transfected with pcDNA3.1+pCI-neo, and co-transfected with RVG-Lamp2b+NGF HEK293 culture supernatant in Example 2 of the present invention
  • the data are expressed as the mean ⁇ SEM of 3 different experiments.
  • Figure 3A shows the expression of NGF mRNA in HEK293 cells treated with different doses of NGF@Exo RVG in Example 3 of the present invention, and HEK293 cells were harvested at 4 hours. * Indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
  • Fig. 3B shows the expression levels of NGF protein in HEK293 cells treated with 100 ⁇ g NGF@Exo RVG at different times in Example 3 of the present invention. ** means p ⁇ 0.01, **** means p ⁇ 0.0001. The data are expressed as the mean ⁇ SEM of 3 different experiments.
  • Figure 4A shows the expression levels of NGF mRNA in the following different exosomes in Example 4 of the present invention: freshly harvested exosomes in the culture supernatant of untransfected HEK293, freshly harvested NGF@Exo RVG , stored for 1 month or 3 Months of NGF@Exo RVG .
  • 4B is 100 ⁇ g NGF @ Exo RVG 4 with freshly harvested embodiment of the present invention, stored for 1 month or 3 months the amount of mRNA expression of NGF HEK293 cells were treated RVG NGF @ Exo, HEK293 cells were harvested at 4h , There is no significant difference between the 3 sets of data.
  • 4C is 100 ⁇ g NGF @ Exo RVG 4 with freshly harvested embodiment of the present invention, stored for 1 month or 3 months the amount of protein expressed RVG NGF HEK293 cells were treated NGF @ Exo, HEK293 cells were harvested at 4h , There is no significant between the 3 sets of data. The data are expressed as the mean ⁇ SEM of 3 different experiments.
  • Figure 5A is a technical roadmap of the test in Example 5 of the present invention.
  • Figure 5B is the in vivo tracking of DiI-labeled Ctrl Exo, Exo RVG and NGF@Exo RVG in Example 5 of the present invention.
  • the brain, liver, spleen, heart, lung, and kidney were harvested within 2 hours, and then frozen section was performed.
  • Figures 5C and 5D show the expression levels of NGF mRNA and protein in the contralateral (Contra) and ipsilateral (Ipsi) cortex of the ischemic injury area after the Ctrl Exo group and the NGF@Exo RVG group are treated with mice in Example 5 of the present invention , NGF mRNA and protein in the tissues of cerebral ischemia injury in NGF@Exo RVG group increased significantly.
  • Figures 5E and 5F show the changes of NGF mRNA and protein in the cortex of the contralateral (Contra) and ipsilateral (Ipsi) cortex of the ischemic injury area after the NGF@Exo Ctrl group and the NGF@Exo RVG group were treated in Example 5 of the present invention.
  • the expression level, NGF mRNA and protein in the tissues of cerebral ischemia injury in the NGF@Exo RVG group were significantly increased.
  • the data are expressed as the mean ⁇ SEM of 5 different experiments. ****, p ⁇ 0.0001.
  • 6A and 6B are the cell count values of the percentages of CD16/Iba1 and CD206/Iba1 in the ischemic area in Example 6 of the present invention.
  • the data is expressed as the mean ⁇ SEM of the data of 5 different mice. ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • Fig. 7 is the cell count value of the percentage of TUNEL-positive/DAPI-positive cells in the ischemic area in Example 6 of the present invention.
  • the data is expressed as the mean ⁇ SEM of the data of 5 different mice. ****, p ⁇ 0.0001.
  • Fig. 8 is the count value of DCX positive cells per mm 2 of ischemic injury area in quantitative cell quantification in Example 6 of the present invention.
  • the data is expressed as the mean ⁇ SEM of the data of 5 different mice. ****, p ⁇ 0.0001.
  • Rabies virus glycoprotein is the envelope protein of rabies virus, which constitutes the virus surface spike and is the main surface antigen of rabies virus.
  • RVG also known as G protein, is encoded by the G gene. The full-length sequence includes 524 amino acid residues (signal peptide containing 19 amino acid residues at the N-terminal).
  • RVG mediates the fusion of virus envelope and cell membrane, so that the virus can invade cells.
  • RVG is highly selective to nerve cells in animals, which is related to the fact that certain fragments contained therein can specifically bind to nerve cell surface receptors.
  • the fragment includes the sequence YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 1).
  • the neurophilicity of these fragments is used herein to allow the prepared exosomes to target nerve cells.
  • RVG when referring to RVG, it refers to such fragments with nerve cell targeting or polypeptides including these fragments.
  • Exosomes are tiny membrane vesicles that can be secreted by many cells. They have a lipid bilayer membrane structure with a diameter of about 10-300 nm (other sizes are also possible). Exosomes can contain specific proteins, lipids and nucleic acids secreted by their cells, which can be used as a medium for information and material exchange between cells. Exosomes play an important role in many physiology and pathology, such as antigen presentation in immunity, tumor growth and migration, and repair of tissue damage. The exosomes secreted by different cells have different components and functions, and can be used as biomarkers for disease diagnosis. Exosomes have a lipid bilayer membrane structure, which can well protect the materials they coat, and can be modified to target specific cells or tissues, so they can also be used as tools for targeted drug delivery.
  • RVG is fused with a membrane protein possessed by the exosome itself, so that the exosome has a nerve cell targeting function.
  • the membrane protein is an exosomal transmembrane protein, such as PGRL, Lamp1, Lamp2, CD13, and the like.
  • the membrane protein used is Lamp2b (lysosome associated membrane glycoprotein 2b).
  • the fusion protein formed by RVG and Lamp2b has the sequence shown in SEQ ID NO: 2.
  • RVG-modified exosomes are loaded with neurotrophic factors and their mRNA.
  • RVG-modified exosomes with human recombinant NGF protein and its mRNA is taken as an example to illustrate the preparation process.
  • NGF@Exo RVG Construction of NGF@Exo RVG : We used the plasmid pcDNA3.1(-)-RVG-Lamp2b (documented in "Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia.”, PMID: 28624203) and cloned Recombinant vector pCI-neo-NGF plasmid (CN 102898514A) with neurotrophic factor (the coding nucleotide sequence of which is shown in SEQ ID NO: 3) )
  • the human embryonic kidney 293 cells of the expression system were co-transfected, and the cell supernatant was collected and subjected to ultracentrifugation to extract exosomes, namely NGF@Exo RVG (see Figure 1).
  • control exosomes We used empty vectors pcDNA3.1(-) and pCI-neo (in this application, pcDNA3.1+pCI-neo will be used together to denote these two plasmids) to co-transfect human embryonic kidney 293 cells, Collect the cell supernatant and perform ultracentrifugation to extract exosomes, that is, control exosomes Ctrl Exo.
  • both Ctrl Exo and NGF@Exo RVG can significantly express the characteristic proteins CD63, Alix and Tsg101 of exosomes, but they do not express the characteristic Golgi protein GM130.
  • NGF@Exo RVG can express higher levels of Lamp2b.
  • ELISA was used to detect NGF protein in exosomes harvested from HEK293 cells that have never been transfected, HEK293 cells co-transfected with RVG-Lamp2b+NGF, and co-transfected with pcDNA3.1+pCI-neo HEK293 cells.
  • the results showed that the NGF@Exo RVG harvested from the culture supernatant of HEK293 cells co-transfected with RVG-Lamp2b+NGF significantly increased the content of NGF protein in the RVG (see Figure 2C).
  • NGF@Exo RVG can effectively load NGF mRNA and protein.
  • Example 3 NGF@Exo RVG can deliver NGF to recipient cells
  • NGF@Exo RVG can load NGF mRNA and protein.
  • exosomes NGF@Exo RVG can deliver NGF to recipient cells when it is taken up by recipient cells. For this reason, we did not add or added 20 ⁇ g, 50 ⁇ g, 100 ⁇ g, 200 ⁇ g or 300 ⁇ g NGF@Exo RVG to HEK293 cell culture medium and incubated for 4h (as shown in Figure 3A). It was found by qRT-PCR that HEK293 cells had no obvious expression of nerve growth factor when NGF@Exo RVG was not added; adding 100 ⁇ g NGF@Exo RVG could significantly increase the level of NGF mRNA in the recipient cells HEK293.
  • NGF@Exo RVG After incubating with 20 ⁇ g and 50 ⁇ g NGF@Exo RVG , although nerve growth factor has a rising trend, it does not significantly increase the expression of nerve growth factor mRNA; 100-300 ⁇ g NGF@Exo RVG can significantly increase the receptor cell HEK293 NGF mRNA and protein levels. When 200 ⁇ g and 300 ⁇ g NGF@Exo RVG are added, the NGF mRNA and protein levels in the recipient cells HEK293 increase relatively little, which may be related to the uptake ability of the recipient cells.
  • NGF@Exo RVG can effectively deliver NGF to recipient cells.
  • the elevated NGF protein level can be partially inhibited by the protein translation inhibitor CHX, which further verified that the protein in the recipient cell HEK293 is not only the protein directly delivered by NGF@Exo RVG , but also the delivered NGF mRNA translation Obtained later.
  • NGF@Exo RVG can be stored stably for a long time
  • NGF@Exo RVG is stable after long-term storage.
  • NGF@Exo RVG can deliver NGF to the area of cerebral ischemia injury
  • mice were sacrificed 2 hours after the injection and subjected to in vivo fluorescence exosomal tracing, 4 hours after the injection, the qRT-PCR analysis was performed, and 8 hours after the injection, the NGF ELISA analysis was performed (as shown in Figure 5A).
  • mice 8-9 weeks old, weighing 22-23g.
  • Cerebral ischemia model the mice were anesthetized with isoflurane, 25mg/kg body weight of amber red sodium salt was injected into the tail vein, the head skin was disinfected and the head skin was cut with ophthalmological scissors, 0.5 to 2.5 mm behind the Bregma point, beside the midline The cranial window was prepared by opening 0.5 to 2.5 mm, and the cold light source was irradiated with optical fiber for 8 minutes. After the operation, the skin was sutured and disinfected.
  • NGF@Exo RVG and Ctrl Exo is the same as the previous description.
  • pcDNA3.1(-)-RVG-Lamp2b and pCI-neo plasmid to co-transfect human embryonic kidney 293 cells, collected the cell supernatant and ultracentrifuged to extract exosomes, namely Exo RVG , obtained Exo RVG and NGF@Exo RVG
  • NGF is not loaded.
  • DiI dyes (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to label the above-mentioned NGF@Exo RVG , Ctrl Exo and Exo RVG . Exosomes.
  • mice The cerebral ischemia model mice were divided into groups, including: NGF@Exo RVG group, Ctrl Exo group and Exo RVG group.
  • the mice in different groups were injected with corresponding types of exosomes into the tail vein (the injection volume was all containing 200 ⁇ g exocytosis).
  • 200 ⁇ L of normal saline, ie, NGF@Exo RVG group mice were injected with NGF@Exo RVG exosomes
  • Ctrl Exo group mice were injected with Ctrl Exo exosomes
  • Exo RVG group mice were injected with Exo RVG exosomes).
  • mice in the Ctrl Exo group had only a small amount of fluorescence signal in the brain tissue ischemic injury area, and significant fluorescence signals in the liver, spleen, and lungs, and very significant fluorescence signals in the heart and kidneys.
  • mice The cerebral ischemia model mice were divided into groups, including: NGF@Exo RVG group, Ctrl Exo group, and NGF@Exo ctrl group.
  • the mice in different groups were injected with corresponding types of exosomes into the tail vein. After 4 hours of injection, the mice were sacrificed for qRT-PCR analysis, and 8 hours later for NGF ELISA analysis.
  • NGF@Exo RVG can significantly increase the area of cerebral ischemia injury
  • NGF@Exo ctrl cannot significantly increase the level of NGF mRNA and protein in the cerebral ischemic injury area.
  • Example 6 NGF@Exo RVG can reduce cerebral ischemic damage by anti-inflammatory, anti-apoptosis, and promoting nerve regeneration
  • mice were injected into the tail vein through the tail vein.
  • mice in different groups were injected with corresponding types of exosomes or saline through the tail vein (the injection volume of the three exosomes groups was 200 ⁇ L saline containing 200 ⁇ g exosomes, that is, mice in the NGF@Exo RVG group were injected with NGF @Exo RVG exosomes, Exo RVG group mice were injected with Exo RVG exosomes, Ctrl Exo group mice were injected with Ctrl Exo exosomes; normal saline group mice were injected with 200 ⁇ L of normal saline), 7 days after cerebral ischemia The mice were sacrificed, and the brain tissues were taken to make frozen sections and subjected to immunofluorescence staining.
  • microglia marker is Iba1; type 1 microglia (M1) has a pro-inflammatory effect, and its marker is CD16; type 2 microglia (M2) has an anti-inflammatory effect, and its marker is CD206. Because NGF has been proven to promote the conversion of type 1 microglia to type 2 microglia, we tested the microglia markers Iba1, CD16 (M1 type), and CD206 (M2 type) in the cerebral ischemic area. ) To explore whether a single injection of NGF@Exo RVG can promote the polarization of type 1 pro-inflammatory microglia to type 2 anti-inflammatory microglia. The double staining and cell quantification results shown in Figure 6A and Figure 6B show:
  • the ratios of CD16/Iba1 and CD206/Iba1 were 72.3% and 26.5%, respectively;
  • CD16/Iba1 and CD206/Iba1 are 70.6% and 28.0%, respectively;
  • the ratios of CD16/Iba1 and CD206/Iba1 were 39.4% and 67.5%, respectively.
  • NGF@Exo RVG can significantly promote the polarization of type 1 pro-inflammatory microglia to type 2 anti-inflammatory microglia to exert anti-inflammatory effects.
  • TUNEL staining method Seven days after cerebral ischemia, the apoptosis of cells in the cerebral ischemic injury area was evaluated by TUNEL staining method. TUNEL positive cells are apoptotic cells, DAPI is a nuclear staining marker, and the ratio of TUNEL+/DAPI+ cells is used to evaluate cerebral ischemia. The proportion of apoptotic cells in the injured area. Fluorescence images and cell quantification results (shown in Figure 7) showed that compared with the normal saline group, Exo RVG group and Ctrl Exo group, the apoptotic cells in the brain ischemic injury area of the NGF@Exo RVG group were significantly reduced. These results verify that NGF@Exo RVG can significantly reduce nerve cell apoptosis and promote survival.
  • Double adrenal cortex hormone is a marker of neuron precursors. Under normal circumstances, DCX is not expressed in the cortex of adult brain tissue. DCX-positive cells appear in the injured area after cerebral ischemia, suggesting nerve regeneration. We used DCX immunofluorescence staining and cell count (as shown in 8) to evaluate the nerve regeneration in the cerebral ischemic injury area. In the saline group, Exo RVG group and Ctrl Exo group, only a few DCX positive cells were scattered in the ischemic injury area. However, DCX positive cells increased significantly in the NGF@Exo RVG group of mice, suggesting that NGF@Exo RVG can significantly promote nerve regeneration in the area of cerebral ischemia.
  • NGF@Exo RVG a single injection of NGF@Exo RVG can effectively deliver NGF to the area of cerebral ischemia injury, reducing nerve tissue damage and promoting repair by reducing inflammation, reducing cell apoptosis, promoting cell survival, and promoting nerve regeneration.
  • this is the first study to deliver NGF mRNA and protein to cerebral ischemic injury areas through engineered exosomes.

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Abstract

一种被靶向修饰且装载有药物的外泌体及其制备方法和应用。该外泌体装载有选自以下的一种或几种：神经营养因子；具有神经营养因子活性的神经营养因子片段；具有神经营养因子活性的短肽；及编码其的mRNA，并可以靶向性地向神经组织受体细胞递送神经营养因子，从而通过抗炎、抗凋亡、促进神经细胞存活、促进神经再生的方式减轻神经组织损伤并促进修复。

Description

一种被靶向修饰且装载有药物的外泌体及其制备方法和应用
本申请要求于2020年5月19日提交至中国专利局、申请号为202010426058.8的专利申请的权益。
技术领域
本发明涉及生物技术领域，具体涉及一种被靶向修饰且装载有药物的外泌体及其制备方法和应用。
背景技术
缺血性脑卒中、出血性脑卒中、阿尔兹海默症、帕金森病等神经系统疾病发病率在现代社会中越来越高，且治疗比较困难。以急性缺血性脑卒中为例，经FDA批准的药物仅有rt-PA，但是其治疗时间窗仅有4.5小时，导致只有少部分患者能从中获益，所以亟待研发治疗药物。
神经营养因子家族包括神经生长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养因子4(NT4)、神经营养因子5(NT5)等。NGF是神经营养因子家族首个被发现的分子，在神经系统生理发育过程中起关键调控作用。在神经系统损伤后，神经营养因子家族可以起到减轻组织损伤、促进组织修复的作用。例如，给予NGF可以减少神经细胞死亡、调节炎症并促进细胞向抗炎型极化、促进血管新生、促进神经再生以及改善神经功能，具有广谱神经保护作用。既往研究发现脑缺血损伤后，内源性NGF的表达水平显著下降。因此，如果能将神经营养因子有效递送至脑组织受体细胞，将可能对神经系统疾病产生治疗作用。
但目前将神经营养因子递送至神经组织受体细胞存在以下问题：(1)常规静脉注射或皮下注射的方法给予的神经营养因子无法高效地穿过血脑屏障，虽然病毒载体、神经营养因子基因修饰的细胞、纳米胶囊和水凝胶等已经被用作神经营养因子的装载手段。但目前没有高效靶向神经组织装载神经营养因子的递送方式。(2)神经营养因子的半衰期较短，例如经静脉外源性给予的NGF在体内的半衰期只有2.3小时，皮下注射给予的NGF半衰期只有4.5h。(3)非特异性的递送会引起外周组织的非预期不良效应，如采用 肌肉注射NGF会引起局部疼痛。上述多种因素阻碍了NGF治疗神经系统疾病的应用。
即使采用静脉注射或皮下注射直接递送神经营养因子的信使RNA(mRNA)，由于mRNA极度不稳定、容易快速降解，导致无法通过直接递送神经营养因子的mRNA到达神经组织并翻译成神经营养因子。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本发明的目的在于提供一种被靶向修饰且装载有药物的外泌体及其制备方法和应用，该外泌体中装载有神经营养因子、神经营养因子mRNA等，可以靶向性地向神经组织受体细胞递送神经营养因子，从而通过抗炎、抗凋亡、促进神经细胞存活、促进神经再生的方式减轻神经组织损伤、促进修复。
解决方案
为实现本发明目的，本发明实施例提供了一种狂犬病病毒糖蛋白(RVG)修饰的外泌体，所述外泌体装载有选自以下的一种或几种：神经营养因子；具有神经营养因子活性的神经营养因子片段；具有神经营养因子活性的短肽；及编码其的mRNA。
在一种可能的实现方式中，神经营养因子包括：神经生长因子、脑源性神经营养因子、神经营养因子4、神经营养因子5、神经营养因子6、神经营养因子7中的一种或几种。
在一种可能的实现方式中，装载有神经营养因子和/或神经营养因子mRNA的狂犬病病毒糖蛋白(RVG)修饰的外泌体为新鲜提取的或冻存的。
在一种可能的实现方式中，当装载有神经营养因子和/或神经营养因子mRNA的狂犬病病毒糖蛋白(RVG)修饰的外泌体为冻存的时，冻存的温度条件为-80℃，冻存的时间为3个月或以内。
在一种可能的实现方式中，所述外泌体靶向神经组织细胞；可选地，靶向脑组织神经细胞。
在一种可能的实现方式中，所述外泌体能被神经元细胞、星形胶质细胞、小胶质细 胞、少突胶质细胞及其他神经组织细胞摄取。
在一种可能的实现方式中，所述狂犬病病毒糖蛋白(RVG)包括SEQ ID NO:1所示的序列。
在一种可能的实现方式中，所述狂犬病病毒糖蛋白(RVG)与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)形成融合蛋白。
在一种可能的实现方式中，所述融合蛋白包括SEQ ID NO：2所示的序列。
本发明实施例还提供了上述外泌体的制备方法，包括以下步骤：采用狂犬病病毒糖蛋白(RVG)与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)融合表达的重组载体与克隆有神经营养因子的重组载体共同转染表达系统的细胞，表达后收集细胞上清进行超速离心提取外泌体。
本发明实施例还提供了包含有狂犬病病毒糖蛋白(RVG)与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)融合表达的重组载体与克隆有神经营养因子的重组载体的表达系统的细胞。
在一种可能的实现方式中，神经营养因子包括：神经生长因子、脑源性神经营养因子、神经营养因子4、神经营养因子5、神经营养因子6、神经营养因子7中的一种或几种。
在一种可能的实现方式中，重组载体包括重组质粒，可选地，包含有狂犬病病毒糖蛋白(RVG)与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)融合表达的重组载体为pcDNA3.1(-)-RVG-Lamp2b重组载体；克隆有神经营养因子的重组载体为pCI-neo-NGF重组质粒。
在一种可能的实现方式中，所述表达系统的细胞包括：人胚肾293细胞、哺乳动物骨髓间充质干细胞、哺乳动物神经干细胞、哺乳动物脐带间充质干细胞、哺乳动物胎盘间充质干细胞、哺乳动物血液干细胞中的一种或多种。
在一种可能的实现方式中，所述制备方法还包括以下步骤：将提取获得的外泌体进行冻存；可选地，冻存的温度条件为-80℃，冻存的时间为3个月或以内。
本发明实施例还提供了一种药物组合物，包括上述外泌体，还包括一种或多种药学上可接受的辅料。
在一种可能的实现方式中，所述药物组合物为用于治疗神经系统疾病的药物组合物。
在一种可能的实现方式中，所述药物组合物还包括其他用于治疗神经系统疾病的药物活性成分。
本发明实施例还提供了一种上述外泌体在制备治疗神经系统疾病的药物中的应用。
本发明实施例还提供了一种用于治疗神经系统疾病的方法，包括以下步骤：向患有神经系统疾病的受试者施用有效剂量的上述外泌体或药物组合物。
在一种可能的实现方式中，所述神经系统疾病包括：缺血性脑卒中、出血性脑卒中、脑小血管病、血管性认知障碍等脑血管病，运动神经元病、阿尔兹海默病、多系统萎缩等神经系统变性疾病，多发性硬化、视神经脊髓炎、脑白质营养不良等中枢神经系统脱髓鞘疾病，帕金森病、亨廷顿病等运动障碍性疾病，脊髓炎、脊髓损伤等脊髓疾病，面神经炎、多发性神经病等周围神经疾病，神经血管性水肿、雷诺病等自主神经系统疾病，重症肌无力、肌营养不良等神经肌肉接头和肌肉疾病，遗传性共济失调等神经系统遗传性疾病，脑性瘫痪等神经系统发育异常性疾病，糖尿病神经系统并发症、甲状腺疾病神经系统并发症等内科系统疾病的神经系统并发症，脑外伤、脊髓外伤等神经创伤性疾病。
在一种可能的实现方式中，所述治疗神经系统疾病的方式包括以下的一种或几种：抗炎、抗凋亡、促进神经细胞存活、促进神经再生等。
在一种可能的实现方式中，抗炎的方式包括：通过促进1型促炎型小胶质细胞向2型抗炎型小胶质细胞极化。
在一种可能的实现方式中，所述受试者包括：人、猴、猪、兔、狗、大鼠或小鼠。
在一种可能的实现方式中，所述施用的方式包括：静脉注射、动脉注射、组织定位注射、皮下注射、肌肉注射。
有益效果
(1)本发明实施例的外泌体中装载有神经营养因子和/或神经营养因子mRNA，该外泌体仅通过单次静脉注射就可以靶向性地穿过血脑屏障向神经组织受体细胞递送神经营养因子，从而通过抗炎、抗凋亡、促进神经细胞存活、促进神经再生等方式来减轻神经组织损伤、促进修复。
(2)本发明实施例的外泌体在单次静脉注射2小时就能靶向神经组织受体细胞并被其摄取，其本身携带的一定量的神经营养因子就能直接释放到受体细胞中，从而实现对受体细胞的迅速治疗，并且其装载的神经营养因子mRNA后续也会在受体细胞中翻译，直至mRNA降解，而mRNA能相对较长时间的稳定存在于该外泌体中，从而延长了神经 营养因子在体内的半衰期。换言之，以生物靶向修饰的外泌体递送NGF蛋白及mRNA，在起到高效神经组织靶向递送的同时起到载药缓释效果。NGF蛋白可以起到即刻治疗效果，NGF mRNA可以进一步翻译为NGF蛋白起到治疗效果。因此，有效增强了单次注射药效。如果在脑缺血后短期(例如优选24小时内)注射本文提供的外泌体，此时正处于炎症和细胞死亡的关键病理时间点，此时干预可以有效靶向关键病理事件。
(3)本发明实施例的外泌体可以促进其在脑缺血损伤区的集中，并且相对减少其他外周组织器官对外泌体的摄取，从而在治疗时减少对外周组织的毒性和其他非预期效应。
(4)本发明实施例的外泌体可以在-80℃的条件下稳定地贮存长达3个月，并且在此期间内，外泌体中装载的神经营养因子及其mRNA也可以稳定贮存；进一步的，经过长期贮存的外泌体的治疗效果相对于新鲜收获的外泌体并没有显著下降。
(5)本发明实施例的外泌体的单次注射相对于直接将NGF蛋白质反复多次间断或持续的注射而言，也会减少副作用的产生。
(6)目前大多数制备外泌体的方式为先制备外泌体再进行内容物装载，该过程通常需要采用电穿孔仪，但每次电穿孔外泌体的数量有限，受到电穿孔仪通量的限制，增加了进行大规模产业化生产的难度与成本，难以满足临床需求。与此相比，本文提供的外泌体采用双表达载体方式在细胞内生成，可以同步进行外泌体生物靶向性修饰与目的蛋白/mRNA加载，无需进行电穿孔，简化制备操作。除此之外，将双表达载体修饰的细胞进行扩增可以有效增加外泌体产量，更适用于规模化生产。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1是本发明实施例1中质粒重组、共转染、外泌体分离鉴定的技术路线图。
图2A是本发明实施例2中未进行转染的HEK293、共转染有pcDNA3.1+pCI-neo的HEK293、共转染有RVG-Lamp2b+NGF HEK293中NGF mRNA的相对表达量。图2B是本发明实施例2中从未进行转染的HEK293、共转染有pcDNA3.1+pCI-neo的HEK293、共转染有RVG-Lamp2b+NGF HEK293培养上清中获得的外泌体中NGF mRNA的相对表达量。图2C是本发明实施例2中从未进行转染的HEK293、共转染有pcDNA3.1+pCI-neo 的HEK293、共转染有RVG-Lamp2b+NGF HEK293培养上清中获得的外泌体中的NGF蛋白的相对表达量。****表示p＜0.0001。数据以3次不同实验的平均值±SEM表示。
图3A是本发明实施例3中用不同剂量的NGF@Exo RVG处理的HEK293细胞中NGF mRNA的表达量，HEK293细胞在4h时收获。*表示p＜0.05，**表示p＜0.01，***表示p＜0.001。图3B是本发明实施例3中用100μg NGF@Exo RVG处理的HEK293细胞中的不同时间的NGF蛋白表达量。**表示p＜0.01，****表示p＜0.0001。数据以3次不同实验的平均值±SEM表示。
图4A是本发明实施例4中以下不同外泌体中NGF mRNA的表达量：未转染HEK293培养上清中新鲜收获的外泌体，新鲜收获的NGF@Exo RVG，保存1个月或3个月的NGF@Exo RVG。图4B是本发明实施例4中用新鲜收获的100μg NGF@Exo RVG，保存1个月或3个月的NGF@Exo RVG分别处理的HEK293细胞中NGF mRNA的表达量，HEK293细胞在4h时收获，3组数据之间没有显著性差异。图4C是本发明实施例4中用新鲜收获的100μg NGF@Exo RVG，保存1个月或3个月的NGF@Exo RVG分别处理的HEK293细胞中NGF蛋白质的表达量，HEK293细胞在4h时收获，3组数据之间没有显著性。数据以3次不同实验的平均值±SEM表示。
图5A是本发明实施例5中试验的技术路线图。图5B是本发明实施例5中DiI标记的Ctrl Exo、Exo RVG和NGF@Exo RVG的体内跟踪。尾静脉注射外泌体后于2h收获脑、肝、脾、心、肺、肾，然后进行冰冻切片。Ctrl Exo组在缺血损伤区的DiI荧光信号较少，而Exo RVG组和NGF@Exo RVG组的DiI荧光信号强度较Ctrl Exo组显著增加。Scale bar＝50μm。图5C和图5D是本发明实施例5中Ctrl Exo组和NGF@Exo RVG组处理小鼠后，NGF mRNA和蛋白质在缺血损伤区对侧(Contra)和同侧(Ipsi)皮层的表达水平，NGF@Exo RVG组脑缺血损伤组织中NGF mRNA和蛋白显著升高。图5E和图5F是本发明实施例5中NGF@Exo Ctrl组和NGF@Exo RVG组处理小鼠后，NGF mRNA和蛋白质在缺血损伤区对侧(Contra)和同侧(Ipsi)皮层的表达水平，NGF@Exo RVG组脑缺血损伤组织中NGF mRNA和蛋白显著升高。数据以5次不同实验的平均值±SEM表示。****，p<0.0001。
图6A和图6B是本发明实施例6中缺血区域CD16/Iba1和CD206/Iba1百分率的细胞计数值。数据以5只不同小鼠数据的平均值±SEM表示。***，p<0.001；****，p<0.0001。
图7是本发明实施例6中缺血区TUNEL阳性/DAPI阳性细胞百分比的细胞计数值。数据以5只不同小鼠数据的平均值±SEM表示。****，p<0.0001。
图8是本发明实施例6中细胞定量每mm 2缺血损伤区DCX阳性细胞计数值。数据以 5只不同小鼠数据的平均值±SEM表示。****，p<0.0001。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
狂犬病病毒糖蛋白(rabies virus glycoprotein,RVG)为狂犬病毒包膜蛋白，构成病毒表面刺突，是狂犬病毒的主要表面抗原。RVG也称为G蛋白，由G基因编码，全长序列包括524个氨基酸残基(含N末端19个氨基酸残基的信号肽)。RVG作为狂犬病毒与宿主细胞结合的配体，介导病毒包膜与细胞膜的融合，从而使病毒入侵细胞。RVG在动物体内对神经细胞具有高度选择性，这与其中包含的某些片段可以与神经细胞表面受体特异性结合有关。在一个具体实例中，该片段包括序列YTIWMPENPRPGTPCDIFTNSRGKRASNG(SEQ ID NO：1)。这些片段的嗜神经性在本文中被用来让制备的外泌体可靶向神经细胞。在本文中，提及RVG时指这样的具有神经细胞靶向的片段或包括这些片段的多肽。
外泌体(exosome)是一种能被众多细胞分泌的微小膜泡，具有脂质双层膜结构，直径大约10-300nm(其他尺寸也是可能的)。外泌体中可含有其分泌细胞特异的蛋白、脂质和核酸，能够作为细胞间信息和物质交流的媒介。外泌体在很多生理病理上起着重要的作用，如免疫中抗原呈递、肿瘤的生长与迁移、组织损伤的修复等。不同细胞分泌的外泌体具有不用的组成成分和功能，可作为疾病诊断的生物标志物。外泌体具有脂质双层膜结构，能很好的保护其包被的物质，且能经修饰而靶向特定细胞或组织，因此也可作为工具用于靶向给药。
在一些实施方案中，将RVG与外泌体自身具有的膜蛋白融合，以使得该外泌体具有神经细胞靶向功能。在一些具体实施方案中，该膜蛋白为外泌体跨膜蛋白，例如PGRL、Lamp1、Lamp2、CD13等。在一个具体实施方案中，所采用的膜蛋白为Lamp2b(lysosome associated membrane glycoprotein 2b)。在一个具体实施方案中，RVG与Lamp2b所形成的融合蛋白具有SEQ ID NO：2所示的序列。
Figure PCTCN2021094662-appb-000001
如无特殊记载，本发明实施例中的实验方法都是本领域常规方法，本发明实施例中的材料本领域技术人员都可以获得。
实施例1.NGF@Exo RVG的构建与鉴定
在本申请中我们采用狂犬病病毒糖蛋白(RVG)与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)融合表达的质粒对外泌体进行靶向修饰，制备RVG修饰的外泌体，并向该RVG修饰的外泌体装载神经营养因子及其mRNA。本实施例中以向该RVG修饰的外泌体装载人源重组NGF蛋白及其mRNA为例，说明制备过程。
NGF@Exo RVG的构建：我们采用RVG与Lamp2b融合表达的质粒pcDNA3.1(-)-RVG-Lamp2b(记载于《Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia.》，PMID:28624203)与克隆有神经营养因子(其编码核苷酸序列如SEQ ID NO：3所示)的重组载体pCI-neo-NGF质粒(CN 102898514A)(本申请中后续用RVG-Lamp2b+NGF共同表示这两个质粒)共同转染表达系统的人胚肾293细胞，收集细胞上清进行超速离心提取外泌体，即NGF@Exo RVG(见图1)。
Figure PCTCN2021094662-appb-000002
对照外泌体的构建：我们采用空载体pcDNA3.1(-)和pCI-neo(本申请中后续用pcDNA3.1+pCI-neo共同表示这两个质粒)共同转染人胚肾293细胞，收集细胞上清进行超速离心提取外泌体，即对照外泌体Ctrl Exo。
经Western-blot检测，Ctrl Exo与NGF@Exo RVG均可以显著表达外泌体的特征蛋白CD63,Alix和Tsg101，但是并不表达高尔基体特征蛋白GM130。与Ctrl Exo相比，NGF@Exo RVG可以表达更高水平的Lamp2b。
经透射电镜(TEM)及Nanosight颗粒跟踪分析显示，Ctrl Exo与NGF@Exo RVG的形态、粒径分布、产量没有显著差异。Ctrl Exo与NGF@Exo RVG的浓度没有显著差别，样品经1000倍稀释后，Ctrl Exo的浓度为(2.78±0.05)×10 8particles/ml(n＝4)，NGF@Exo RVG的浓度为(2.70±0.04)×10 8particles/ml(n＝4)。
实施例2.NGF@Exo RVG内含NGF的检测
采用qRT-PCR检测未进行转染的HEK293细胞、共转染有RVG-Lamp2b+NGF的HEK293细胞、共转染有pcDNA3.1+pCI-neo HEK293细胞中NGF mRNA的表达水平， 结果显示共转染有RVG-Lamp2b+NGF的HEK293细胞内NGF mRNA的含量水平显著升高(见图2A)。
采用qRT-PCR检测从未进行转染的HEK293细胞、共转染有RVG-Lamp2b+NGF的HEK293细胞、共转染有pcDNA3.1+pCI-neo HEK293细胞培养上清中收获的外泌体中NGF mRNA的表达水平，结果显示共转染有RVG-Lamp2b+NGF的HEK293细胞培养上清收获的NGF@Exo RVG内NGF mRNA的含量水平显著升高(见图2B)。
采用ELISA检测从未进行转染的HEK293细胞、共转染有RVG-Lamp2b+NGF的HEK293细胞、共转染有pcDNA3.1+pCI-neo HEK293细胞培养上清中收获的外泌体中NGF蛋白的表达水平，结果显示共转染有RVG-Lamp2b+NGF的HEK293细胞培养上清收获的NGF@Exo RVG内NGF蛋白的含量水平显著升高(见图2C)。
将图2A、2B和2C的结果横向比较，还可以发现通过双表达载体共转染方式获得的外泌体相对于细胞内目的mRNA和/或蛋白具有显著的富集效果，有效地增加了外泌体内目的mRNA和/或蛋白的含量。
以上内容说明NGF@Exo RVG可以有效装载NGF mRNA和蛋白质。
实施例3.NGF@Exo RVG可以向受体细胞递送NGF
前述结果证明NGF@Exo RVG可以装载NGF mRNA及蛋白质，我们还探索了外泌体NGF@Exo RVG在被受体细胞摄取时，是否能够向受体细胞传递NGF。为此，我们向HEK293细胞培养基中不加或加入20μg，50μg，100μg，200μg或300μg NGF@Exo RVG进行孵育4h(如图3A所示)。采用qRT-PCR检测发现，不加NGF@Exo RVG时，HEK293细胞没有明显的神经生长因子的表达；加入100μg NGF@Exo RVG可以显著增加受体细胞HEK293内的NGF mRNA水平。在与20μg和50μg NGF@Exo RVG进行孵育后，神经生长因子虽然有上涨的趋势，但并没有显著增加神经生长因子mRNA的表达；100-300μg NGF@Exo RVG可以显著增加受体细胞HEK293内的NGF mRNA及蛋白质水平，当加入200μg和300μg NGF@Exo RVG时受体细胞HEK293内的NGF mRNA及蛋白质水平增加相对较少，可能和受体细胞的摄取能力有关。
另外，为了进一步确认所递送的NGF mRNA是否可以在受体细胞中被翻译成蛋白，我们将HEK293细胞与100μg NGF@Exo RVG共孵育2h，4h，8h，12h，24h。在收集HEK293细胞并清洗三遍，然后进行ELISA检测。结果表明：受体细胞中的NGF浓度在2h到 4h内有上升，NGF表达量在8h时达到峰值，之后因为递送的NGF mRNA和翻译的NGF蛋白质有所降解，在12h到24h期间有缓慢下降，但是在24h和2h时NGF蛋白的量还是有显著差异的，表明NGF mRNA在其降解前会有持续的翻译(如图3B)。这些结果都表明NGF@Exo RVG能有效将NGF递送到受体细胞。另外，升高的NGF蛋白质水平可以被蛋白翻译抑制剂CHX部分抑制，也进一步验证了受体细胞HEK293内的蛋白不止是NGF@Exo RVG直接传递过来的蛋白，还包括由递送来的NGF mRNA翻译后获得的。
实施例4.NGF@Exo RVG可以稳定贮存较长时间
为了日后进行临床应用，我们对NGF@Exo RVG经过长时间贮存后是否稳定进行了检测。我们将提取好的NGF@Exo RVG冻存至-80℃，保存1个月或3个月。与未转染的HEK293细胞中获得的外泌体相比，转染有新鲜收获的、保存1个月、保存3个月的NGF@Exo RVG的HEK293细胞中，NGF mRNA的量都有显著增加(如图4A)。
另外，我们将新鲜收获的、保存1个月、保存3个月的NGF@Exo RVG分别与HEK293共孵育，并检测了受体细胞中NGF mRNA和NGF蛋白的表达量(如图4B和图4C)。与新鲜收获的NGF@Exo RVG相比，冻存了3个月的NGF@Exo RVG在向受体细胞HEK293递送NGF mRNA及蛋白质的能力方面没有显著变化，这表明包裹在NGF@Exo RVG中mRNA及蛋白质的稳定性。长期的保存并没有影响NGF mRNA及蛋白质的递送，为日后NGF@Exo RVG作为成品制剂提供了数据参考。
实施例5.NGF@Exo RVG可以递送NGF到达脑缺血损伤区
为了进一步确认NGF@Exo RVG向脑缺血损伤区域递送NGF的效率，我们建立了脑缺血模型，在脑缺血后24h经尾静脉向小鼠注射200μg外泌体。注射2小时后将小鼠处死并进行荧光外泌体在体示踪，注射4h后进行qRT-PCR分析，注射8h后进行NGF ELISA分析(如图5A所示)。
动物与脑缺血模型的建立如下：
采用C56BL/6雄性小鼠，8-9周龄，体重22-23g。
脑缺血模型：将小鼠采用异氟烷麻醉，尾静脉注射琥珀红钠盐25mg/kg体重，头部皮肤消毒并用眼科剪剪开头皮，在距Bregma点后侧0.5to 2.5mm、中线旁开0.5to 2.5mm制备颅窗，采用冷光源光纤照射8分钟，术后缝皮、消毒。
5.1荧光外泌体在体示踪检测
NGF@Exo RVG及Ctrl Exo的获取同前描述。我们采用pcDNA3.1(-)-RVG-Lamp2b与pCI-neo质粒共同转染人胚肾293细胞，收集细胞上清进行超速离心提取外泌体，即Exo RVG，所得Exo RVG与NGF@Exo RVG的区别在于没有装载NGF。
为了评估外泌体在体内的分布，我们采用DiI染料(1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate)标记上述NGF@Exo RVG、Ctrl Exo以及Exo RVG3种外泌体。
将脑缺血模型小鼠分组，包括：NGF@Exo RVG组、Ctrl Exo组以及Exo RVG组，向不同组的小鼠分别尾静脉注射相应种类的外泌体(注射量均为含有200μg外泌体的200μL生理盐水，即NGF@Exo RVG组小鼠注射NGF@Exo RVG外泌体、Ctrl Exo组小鼠注射Ctrl Exo外泌体以及Exo RVG组小鼠注射Exo RVG外泌体)后，取脑组织制作冰冻切片，经荧光显微镜观测：Ctrl Exo组小鼠在脑组织缺血损伤区仅有很少量荧光信号，在肝脏、脾脏、肺部有显著的荧光信号，在心脏、肾脏有很少量荧光信号；NGF@Exo RVG组小鼠与Exo RVG组小鼠的外泌体的分布没有显著差异，但是这两组小鼠相较于Ctrl Exo组小鼠，在脑组织缺血损伤区的荧光信号显著增加，在肝脏、脾脏、肺部的荧光信号有所减少，在心脏、肾脏有很少量荧光信号(如图5B所示)。上述结果提示，RVG-Lamp2b修饰外泌体可以显著增加其向脑组织缺血损伤区的靶向性。
我们还将冰冻切片与不同细胞类型的标志物进行染色，发现DiI标记NGF@Exo RVG的荧光信号可以与神经元标志物NeuN、星形胶质细胞标志物GFAP、小胶质细胞标志物Iba1共标，提示这些细胞都可以摄取外泌体。
5.2检测NGF@Exo RVG能否向脑缺血损伤区递送NGF
我们采用pcDNA3.1(-)与pCI-neo-NGF质粒共同转染人胚肾293细胞，收集细胞上清进行超速离心提取外泌体，即NGF@Exo ctrl(所得外泌体没有RVG靶向性修饰，但是装载了NGF)。
将脑缺血模型小鼠分组，包括：NGF@Exo RVG组、Ctrl Exo组以及NGF@Exo ctrl组，向不同组的小鼠分别尾静脉注射相应种类的外泌体。注射4h后将小鼠处死进行qRT-PCR分析，8h后进行NGF ELISA分析。qRT-PCR及ELISA检测的结果显示(如图5C，图5D，图5E和图5F所示)，相较于Ctrl Exo及NGF@Exo ctrl，NGF@Exo RVG可以显著增加脑缺血损伤区内的NGF mRNA及蛋白质水平，而NGF@Exo ctrl不能显著增加脑缺血损伤区内的NGF mRNA及蛋白质水平。
实施例6.NGF@Exo RVG可以通过抗炎、抗凋亡、促进神经再生减轻脑缺血损伤
如上所述，我们建立脑缺血模型，将脑缺血模型小鼠分组，包括：NGF@Exo RVG组、Exo RVG、Ctrl Exo组以及生理盐水组，在脑缺血后24h经尾静脉注射向不同组的小鼠分别尾静脉注射相应种类的外泌体或生理盐水(3个外泌体组的注射量均为含有200μg外泌体的200μL生理盐水，即NGF@Exo RVG组小鼠注射NGF@Exo RVG外泌体、Exo RVG组小鼠注射Exo RVG外泌体、Ctrl Exo组小鼠注射Ctrl Exo外泌体；生理盐水组小鼠注射200μL生理盐水)，在脑缺血后7天将小鼠处死，取脑组织制作冰冻切片并进行免疫荧光染色。
6.1抗炎
先前的研究已经强调了小胶质细胞极化在脑损伤和修复中的重要性。小胶质细胞标志物为Iba1；1型小胶质细胞(M1)具有促炎作用，其标志物为CD16；2型小胶质细胞(M2)具有抗炎作用，其标志物为CD206。因为NGF已经被证明具有促进1型小胶质细胞向2型小胶质细胞转变的作用，我们检测了脑缺血区域的小胶质细胞标志物Iba1、CD16(M1型)、CD206(M2型)以探索是否仅仅靠单次注射NGF@Exo RVG就能促进1型促炎型小胶质细胞向2型抗炎型小胶质细胞极化。如图6A、图6B的双染色和细胞定量结果显示：
在生理盐水组，CD16/Iba1和CD206/Iba1的比例分别是72.3％和26.5％；
在Ctrl Exo组，CD16/Iba1和CD206/Iba1的比例分别是70.6％和28.0％；
在Exo RVG组，CD16/Iba1和CD206/Iba1的比例分别是68.7％和27.5％
在NGF@Exo RVG组，CD16/Iba1和CD206/Iba1的比例分别是39.4％和67.5％。
由此可见，NGF@Exo RVG可以显著促进1型促炎型小胶质细胞向2型抗炎型小胶质细胞极化发挥抗炎作用。
6.2抗凋亡
在脑缺血后7天，通过TUNEL染色法评价脑缺血损伤区细胞的凋亡情况，TUNEL阳性细胞为凋亡细胞，DAPI为细胞核染色标志物，用TUNEL+/DAPI+细胞的比例评价脑缺血损伤区内凋亡细胞的比例。荧光图像和细胞定量结果(图7所示)显示：相比生理盐水组、Exo RVG组和Ctrl Exo组，NGF@Exo RVG组小鼠脑缺血损伤区内的凋亡细胞显 著减少。这些结果验证了NGF@Exo RVG可以显著减少神经细胞凋亡、促进存活。
6.3促神经再生
双肾上腺皮质激素(DCX)是神经元前体的标志物，正常情况下在成年脑组织皮层不表达DCX，脑缺血后损伤区出现DCX阳性细胞提示有神经再生现象。我们采用DCX免疫荧光染色和细胞计数(如8所示)评价脑缺血损伤区的神经再生情况。在生理盐水组、Exo RVG组和Ctrl Exo组，只有很少量的DCX阳性细胞散布在缺血损伤区。然而，NGF@Exo RVG组小鼠中DCX阳性细胞显著增加，提示NGF@Exo RVG可以显著促进脑缺血损伤区的神经再生。
6.4经长期低温贮存的NGF@Exo RVG对脑缺血损伤的治疗效果
为了进一步确认在-80℃长时间保存NGF@Exo RVG是否会影响其治疗效果，我们将提取好的NGF@Exo RVG冻存至-80摄氏度，保存1个月或3个月。重复上述检测炎症、凋亡、神经再生的实验发现，与新鲜提取的NGF@Exo RVG相比，冻存NGF@Exo RVG的治疗效果没有显著差异。
此外，在我们的研究中没有观察到不良的副作用，如体重减轻和身体活动的异常。总之，这些结果证明了NGF@Exo RVG的综合神经保护作用及其对脑缺血损伤的治疗效果。
综上，我们在本申请中已经证明了通过外泌体递送NGF可以治疗脑缺血性损伤。更具体地说，我们用装载有NGF的RVG-Lamp2b外泌体来生成NGF@Exo RVG。单次注射NGF@Exo RVG就能有效地将NGF递送到脑缺血损伤区，通过减轻炎症、减少细胞凋亡、促进细胞存活、促进神经再生来减轻神经组织损伤、促进修复。据我们所知，这是第一个通过工程外泌体将NGF mRNA和蛋白传递到脑缺血损伤区的研究。在我们的研究中，通过外泌体高效靶向递送NGF mRNA和蛋白，可以显著促进M1型小胶质细胞向M2型小胶质细胞极化，增强细胞对凋亡的抵抗并促进其存活，促进脑缺血损伤区的神经再生，这些结果直接证实了NGF@EXO RVG治疗脑缺血的疗效。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (15)

  1. 一种狂犬病病毒糖蛋白修饰的外泌体，其特征在于：所述外泌体装载有选自以下的一种或几种：神经营养因子；具有神经营养因子活性的神经营养因子片段；具有神经营养因子活性的短肽；及编码其的mRNA。
  2. 根据权利要求1所述的外泌体，其特征在于：所述神经营养因子包括：神经生长因子、脑源性神经营养因子、神经营养因子4、神经营养因子5、神经营养因子6、神经营养因子7中的一种或几种。
  3. 根据权利要求1所述的外泌体，其特征在于：装载有神经营养因子和/或神经营养因子mRNA的狂犬病病毒糖蛋白修饰的外泌体为新鲜提取的或冻存的；可选地，当装载有神经营养因子和/或神经营养因子mRNA的狂犬病病毒糖蛋白修饰的外泌体为冻存的时，冻存的温度条件为-80℃，冻存的时间为3个月或以内。
  4. 根据权利要求1所述的外泌体，其特征在于：所述外泌体是靶向神经组织细胞；
    和/或，所述外泌体能被神经元细胞、星形胶质细胞、小胶质细胞、少突胶质细胞及其他神经组织细胞摄取。
  5. 根据权利要求1-4之一所述的外泌体，其中所述狂犬病病毒糖蛋白包括SEQ ID NO：1所示的序列。
  6. 根据权利要求1-5之一所述的外泌体，其中所述狂犬病病毒糖蛋白与外泌体蛋白溶酶体相关膜糖蛋白2b(Lamp2b)形成融合蛋白。
  7. 根据权利要求1-6之一所述的外泌体，其中所述融合蛋白包括SEQ ID NO：2所示的序列。
  8. 一种权利要求1-7之一所述的外泌体的制备方法，其特征在于：包括以下步骤：采用狂犬病病毒糖蛋白与外泌体蛋白溶酶体相关膜糖蛋白2b融合表达的重组载体与克隆有神经营养因子的重组载体共同转染表达系统的细胞，表达后收集细胞上清进行超速离心提取外泌体。
  9. 根据权利要求8所述的制备方法，其特征在于：神经营养因子包括：神经生长因子、脑源性神经营养因子、神经营养因子4、神经营养因子5、神经营养因子6、神经营养因子7中的一种或几种；
    和/或，重组载体包括重组质粒，可选地，包含有狂犬病病毒糖蛋白与外泌体蛋白溶酶体相关膜糖蛋白2b融合表达的重组载体为pcDNA3.1(-)-RVG-Lamp2b重组载体；克隆 有神经营养因子的重组载体为pCI-neo-NGF重组质粒；
    和/或，所述表达系统的细胞包括：人胚肾293细胞、哺乳动物骨髓间充质干细胞、哺乳动物神经干细胞、哺乳动物脐带间充质干细胞、哺乳动物胎盘间充质干细胞、哺乳动物血液干细胞中的一种或多种。
  10. 根据权利要求8或9所述的制备方法，其特征在于：所述制备方法还包括以下步骤：将提取获得的外泌体进行冻存；可选地，冻存的温度条件为-80℃，冻存的时间为3个月或以内。
  11. 一种药物组合物，其特征在于：包括权利要求1-7之一所述的外泌体，还包括一种或多种药学上可接受的辅料；可选地，所述药物组合物为用于治疗神经系统疾病的药物组合物。
  12. 一种权利要求1-7之一所述的外泌体在制备治疗神经系统疾病的药物中的应用。
  13. 在患者中治疗神经系统疾病的方法，包括以权利要求1-7之一的外泌体或权利要求11所述的药物组合物向所述患者给药。
  14. 根据权利要求13所述的方法，其中所述给药采用静脉注射方式。
  15. 根据权利要求12所述的应用或权利要求13或14所述的方法，其特征在于：所述神经系统疾病选自以下的一种或几种：
    脑血管性疾病；可选地，所述脑血管性疾病为：缺血性脑卒中、出血性脑卒中、脑小血管病或血管性认知障碍；
    神经系统变性疾病；可选地，所述神经系统变性疾病为：运动神经元病、阿尔兹海默病或多系统萎缩；
    运动障碍性疾病；可选地，所述运动障碍性疾病为：帕金森病或亨廷顿病；
    神经系统脱髓鞘疾病；可选地，所述神经系统脱髓鞘疾病为：多发性硬化、视神经脊髓炎或脑白质营养不良；
    脊髓疾病；可选地，脊髓疾病为脊髓炎或脊髓损伤；
    周围神经疾病；可选地，周围神经疾病为面神经炎或多发性神经病；
    自主神经系统疾病；可选地，自主神经系统疾病为神经血管性水肿或雷诺病；
    神经肌肉接头和肌肉疾病；可选地，神经肌肉接头和肌肉疾病为：重症肌无力或肌 营养不良；
    神经系统遗传性疾病；可选地，神经系统遗传性疾病为遗传性共济失调；
    神经系统发育异常性疾病；可选地，神经系统发育异常性疾病为脑性瘫痪；
    神经系统并发症；可选地，神经系统并发症为：糖尿病神经系统并发症或甲状腺疾病神经系统并发症；
    神经创伤性疾病；可选地，神经创伤性疾病为：脑外伤或脊髓外伤；
    进一步可选地，所述治疗神经系统疾病包括以下方式的一种或几种：抗炎、抗凋亡、促进神经细胞存活、促进神经再生。
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