Exosome small nucleic acid drug delivery system, preparation method thereof and application of exosome small nucleic acid drug delivery system in optic neurodegenerative diseases

Abstract

The invention discloses an application of MiR-29 in preparation of a drug for treating optic nerve injury and an application of an exosome loaded with agomiR-29b in preparation of a drug for treating optic nerve injury, miR-29b can significantly improve the survival rate of RGC after optic nerve injury and partially recover retina electrophysiological function display, can effectively improve the visual function level, and can be used for preparing a drug for treating optic nerve injury. Compared with treatment by purely applying agomiR-29b or neural progenitor cell exosome, the agomiR-29b loaded in the exosome derived from the neural progenitor cell has a more remarkable effect on improving the survival rate of RGC cells and the level of visual function recovery, and the invention further provides an engineering exosome and a construction method thereof. The problems that the cell biological activity is difficult to maintain, the cell phenotype is lost, the cost is high and the like in the conventional cell treatment process are effectively solved. In addition, compared with other carriers, the exosome is safer and more efficient, production and storage procedures are simpler, quality control is easier, the agomiR-29 loading rate is high, and the clinical transformation process of new drugs is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an exosome small nucleic acid drug delivery system and a preparation method thereof, and an application thereof in optic nerve degenerative diseases. Background Art

[0002] The retina is composed of neurovascular units composed of neurons, glial cells, and blood vessels, which participate in and maintain the normal physiological functions of neurons and the repair of damaged neurons. Glaucoma not only manifests RGC apoptosis, nerve fiber loss, and insufficient blood perfusion, but also involves pathological processes such as immune inflammatory response, oxidative stress, and fibrosis. Optic nerve degeneration is considered to be an important cause of permanent visual loss, and it is crucial to develop effective new technologies and methods for neuroprotection and regeneration and repair. In recent years, cell regenerative therapy has brought new hope, but it also faces many difficulties such as cell source problems and preparation, transplantation, survival, immune rejection, and tissue integration. Studies have shown that the therapeutic effect of cell transplantation is mainly through the factors secreted by it to nourish nerves or regulate microenvironmental homeostasis, while very few cells are integrated into local tissues, and it is not clear whether the integrated cells play normal physiological functions.

[0003] Gene therapy is another innovative treatment for neurodegenerative diseases that has attracted widespread attention. It has become a key area of ​​new drug research and development. Studies have shown that personalized RNA drugs may completely break the traditional thinking of drug research and development. At present, the research and development of small nucleic acid drugs such as siRNA, ASO, and miRNA is in full swing, and some drugs have been approved for marketing. However, the research and development of gene drugs still faces problems such as how to maintain their stability and effective delivery. Gene drug delivery methods include viral vectors, lipid nanoparticles (LNPs) and other non-viral types, but they all have their own limitations. Viral vectors need to overcome challenges such as load size limitations, possible immune responses, and cumbersome manufacturing processes, while LNPs are usually absorbed by the liver, and targeting tissues outside the liver is still a challenge that needs to be overcome. Although gene drug delivery based on adeno-associated virus (AAV) has made great progress, there are also reports of patients dying of sepsis due to large-dose injections. A 10-year follow-up evaluation of a large animal model based on AAV gene therapy found potential carcinogenicity. In a study of non-human primates, it was found that miRNA drugs delivered by AAV vectors were toxic in the cerebellum. This toxicity was not related to endogenous miRNA, but to transcription driven by the reverse terminal repeat sequence on AAV. Therefore, AAV vectors still face problems such as long-term safety and packaging capacity limitations, and the high cost of treatment is also prohibitive.

[0004] Exosomes have obvious advantages such as low immunogenicity, easy passage through biological barriers (such as blood-brain barrier, blood-eye barrier), and engineered exosomes carrying gene drugs. They are expected to be used as biomarkers, cell replacement therapies and new drug delivery systems, and have bright prospects in the development of precision medicine. A large number of studies have shown that exosomes have great application potential in both the diagnosis and treatment of diseases.

[0005] However, there are currently no reports on the use of MiR-29 in the treatment of optic neurodegenerative diseases, let alone the use of exosomes with high expression of MiR-29 in the treatment of optic neurodegenerative diseases. Summary of the invention

[0006] In view of the lack of research on the use of high expression of MiR-29 in the preparation of drugs for optic nerve degenerative diseases in the prior art, the present invention provides the use of MiR-29 in the preparation of drugs for optic nerve damage.

[0007] Preferably, the drug includes an agonist agomiR-29 or a miR-29 overexpression lentiviral plasmid, wherein the agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, and agomiR-29c-3p.

[0008] The effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1.

[0009] The stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.2.

[0010] The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.3.

[0011] The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5,

[0012] The agomiR-29c-3p effective working sequence is shown in SEQ ID NO.6, and the agomiR-29c-3p stem-loop sequence is shown in SEQ ID NO.7.

[0013] Preferably, the drug comprises the agonist agomiR-29b-3p or a miR-29b-3p overexpression lentiviral plasmid.

[0014] Preferably, the optic nerve damage drug is a drug for treating optic nerve damage caused by glaucoma, autoimmune, inflammatory, traumatic, ischemic or hereditary optic neuropathy.

[0015] Preferably, the optic nerve damage drug is a drug for treating chronic high intraocular pressure.

[0016] The present invention also provides the use of exosomes in preparing a drug for treating optic nerve damage, wherein the exosomes are derived from neural progenitor cells.

[0017] Preferably, the optic nerve damage drug is a drug for treating optic nerve damage caused by glaucoma, autoimmune, inflammatory, traumatic, ischemic or hereditary optic neuropathy.

[0018] Preferably, the optic nerve damage drug is a drug for treating chronic high intraocular pressure.

[0019] Preferably, the exosomes also carry drugs that are easily metabolized and degraded by human tissues or cells before exerting their therapeutic effects, including but not limited to drugs and molecules used for gene therapy.

[0020] The present invention also provides the use of exosomes loaded with agomiR-29-3p in preparing a drug for treating optic nerve damage, wherein the exosomes loaded with agomiR-29-3p are exosomes derived from neural progenitor cells;

[0021] The agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, agomiR-29c-3p,

[0022] The effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1,

[0023] The stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.2.

[0024] The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.3.

[0025] The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5,

[0026] The agomiR-29c-3p effective working sequence is shown in SEQ ID NO.6, and the agomiR-29c-3p stem-loop sequence is shown in SEQ ID NO.7.

[0027] Preferably, the method for preparing exosomes loaded with agomiR-29-3p comprises the following steps:

[0028] (1) Extraction of carrier exosomes: Use an exosome extraction kit to extract exosomes from the supernatant of neural progenitor cells, and resuspend them in PBS to obtain an exosome suspension;

[0029] (2) Acquisition of agomiR-29b-3p: Artificially synthesizing agomiR-29, wherein the agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, and agomiR-29c-3p, wherein the effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1, and the stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.2,

[0030] The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.3.

[0031] The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5,

[0032] The agomiR-29c-3p effective working sequence is shown in SEQ ID NO.6, and the agomiR-29c-3p stem-loop sequence is shown in SEQ ID NO.7.

[0033] Preferably, in step (3), 8 -1×10 9 1 μg of agomir-29b-3p and transfection reagent were added to each exosome to obtain a mixed solution, the mixed solution was added to an electrode cup for electrotransfection, and the exosome membrane structure was restored at 37°C to obtain engineered exosomes. The engineered exosomes were concentrated by nuclease treatment and ultrafiltration centrifugation to remove free agomir-29b-3p and other impurities.

[0034] Preferably, the parameters of the electroporation include: selecting an attenuation mode, adjusting the perforation electric field strength to 1000 V / cm, the electroporation duration to 10 ms, and the number of driving cycles to 1.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention has confirmed through a large number of studies that miR-29b-3p plays an important role in the repair of optic nerve damage. MiR-29b-3p can significantly improve the survival rate of RGCs after optic nerve damage and promote axon regeneration. At the same time, retinal electrophysiological functions including ERG and VEP, as well as visual physiological qMOR analysis, all show partial recovery, which can effectively improve the level of visual function.

[0037] Neural progenitor cell-derived exosomes showed significant RGC protective effects, and retinal electrophysiological functions including VEP were partially restored.

[0038] Loading agomiR-29b-3p into exosomes derived from neural progenitor cells has a more significant effect on improving the survival rate of RGC cells and the level of visual function recovery compared to the simple application of agomiR-29b-3p or neural progenitor cell exosomes.

[0039] The exosomes of the present invention are loaded with agomiR-29b-3p to construct engineered exosomes, which effectively solves the problems of difficulty in maintaining cell biological activity, loss of cell phenotype, and high cost in conventional cell therapy. In addition, compared with other carriers, the exosomes of the present invention are safer and more efficient, and the production and storage procedures are simpler, quality control is easier, and the agomiR-29b-3p loading rate is high, which is conducive to the clinical transformation process of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A is an OCT image of the anterior segment of the eye after silicone oil intracameral injection in the animal model; 1B is a diagram showing the intraocular pressure results of the COH model; 1C is a representative optical coherence tomography (OCT) image of the GCC complex cross section in the COH model experimental group and the control group; 1D is a statistical diagram of the thickness of the GCC complex cross section; 1E is a statistical diagram of the expression levels of retinal miR-29a-3p, miR-29b-3p, and miR-29c-3p in the COH model and the control group; 1F is the expression level of miR-29b-3p in the retina after the agonist agomiR-29b and the negative control vitreous cavity injection;

[0041] Figure 2 A is the ERG detection diagram of the experimental group and control group of the COH model treated with agomiR-29b, 2B is the quantitative analysis result diagram of the ERG detection of the experimental group and control group of the COH model treated with agomiR-29b, 2C is the VEP detection diagram of the experimental group and control group of the COH model treated with agomiR-29b, 2D is the quantitative analysis result diagram of the VEP detection of the experimental group and control group of the COH model treated with agomiR-29b, 2E is the qMOR analysis result diagram of the experimental group and control group of the COH model treated with agomiR-29b, 2F is the retinal flat mount RGCs image of the experimental group and control group of the COH model treated with agomiR-29b made with fluorescent labeled tool mouse Thy1-GFP, 2G is the quantitative analysis diagram of RGCs of the experimental group and control group of the COH model treated with agomiR-29b;

[0042] Figure 3A is a schematic diagram of the steps of directed differentiation of iPSCs neurons, 3B is a fluorescence staining image of SOX2 and Tra-1-60, a pluripotency marker for directed differentiation of iPSCs neurons, 3C is a fluorescence staining image of PAX6 and Nestin, a neural progenitor cell marker for directed differentiation of iPSCs neurons, 3D is a schematic diagram of the steps of exosome extraction, 3E is a TEM identification image of exosomes; 3F is an NTA particle size distribution image of exosomes; 3G is a characteristic protein marker of exosomes detected by WB; 3H is the uptake of fluorescently labeled exosomes (green) by retinal cells after intravitreal injection (red is RGCs labeled with RBPMS antibody); 3I is the result of RBPMS fluorescence staining of RGC markers in retinal sections after intravitreal injection of PKH-67-labeled exosomes;

[0043] Figure 4 A is a schematic diagram of the preparation of the optic nerve injury model and the timeline of various tests. 4B is a cross-sectional image and quantitative analysis of the in vivo GCC complex of the COH model experimental group and the control group treated with NPCs-derived exosomes. 4C is a retinal flat mount image of the COH model experimental group and the control group treated with NPCs-derived exosomes in the animal model prepared from Thy1-GFP mice. 4D is the number of surviving RGCs in the COH model experimental group and the control group treated with NPCs-derived exosomes at 7 days after the model was prepared. 4E is the number of surviving RGCs in the COH model experimental group and the control group treated with NPCs-derived exosomes at 14 days after the model was prepared. 4F is the VEP detection and quantitative analysis of the COH model experimental group and the control group treated with NPCs-derived exosomes. 4G is a schematic diagram summarizing the research results.

[0044] Figure 5 A shows the effect of electroporation and incubation conditions on the miR-29 loading amount in the prepared engineered exosomes, 5B shows the miR-29a-3p loading amount in the engineered exosomes constructed by agomir-29a or miR-29a overexpression lentiviral plasmids, 5C shows the schematic diagram of the process of constructing engineered exosomes, 5D shows that engineered exosomes rescue the electrophysiological function of the optic nerve, and 5E shows the visual qMOR analysis of the experimental and control groups of the COH model treated with engineered exosomes;

[0045] Figure 6A is a retinal flat mount of the COH model experimental group and the control group treated with engineered exosomes, 6B is a representative optical coherence tomography (OCT) image of the in vivo GCC complex cross-sectional image of the COH model experimental group and the control group treated with engineered exosomes, 6C is a quantitative analysis of RGCs in the COH model experimental group and the control group treated with engineered exosomes; 6D is a quantitative analysis of the in vivo GCC complex cross-sectional thickness of the representative optical coherence tomography (OCT) image of the COH model experimental group and the control group treated with engineered exosomes, 6E is a schematic diagram of the directed differentiation of stem cells into neurons, 6F is an immunofluorescence staining of the directed differentiated neurons in the COH model experimental group and the control group treated with engineered exosomes, and 6G is a quantitative analysis of the immunofluorescence staining of the directed differentiated neurons in the COH model experimental group and the control group treated with engineered exosomes. DETAILED DESCRIPTION

[0046] As mentioned above, in view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be further disclosed and explained in conjunction with examples below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0047] Example 1. Preparation of a chronic ocular hypertension mouse model

[0048] The mice used in the experiment were provided by Wenzhou Medical University Eye Hospital and approved by the Ethics Committee of Wenzhou Medical University Eye Hospital. We used 2-3 month old mice, and after 1 week of adaptive feeding, we performed intraperitoneal injection anesthesia. After general anesthesia, the eyeball was anesthetized with iodine tincture and disinfected. About 2 μl of silicone oil was pushed from the posterior chamber into the anterior chamber to cause aqueous humor blockage and create a chronic ocular hypertension (COH) model. The model was successfully prepared. Figure 1 As shown in A, after silicone oil was injected into the anterior chamber, the anterior segment OCT showed that the angle was closed. The intraocular pressure changes of the mice were monitored for one week. The standard for successful modeling was an increase in intraocular pressure of more than 5 mmHg. Figure 1 As shown in Figure B, we observed a significant increase in intraocular pressure, with an average increase of about 15 mmHg. As shown in Figure CD, the thickness of the retinal ganglion cell complex GCC was thinner in the chronic high intraocular pressure model compared with the control group.

[0049] Example 2: Protective effect of miR-29b-3p on optic nerve injury in an optic nerve injury model

[0050] (1) Expression of miR-29s in the retina of chronic ocular hypertension model mice

[0051] The mice in the chronic ocular hypertension model and the control group were euthanized, the mouse retinas were isolated, and the retinal RNA was extracted for detection. The expression of miR-29s in the retina of the mice in the chronic ocular hypertension model was significantly downregulated. Figure 1 As shown in E.

[0052] (2) agomiR-29b-3p protects against optic nerve damage in chronic ocular hypertension model mice

[0053] effect

[0054] When the agonist agomiR-29b-3p1ug / eye was injected into the vitreous cavity of chronic ocular hypertension model mice, the expression level in the retina was increased by 10-20 times. Figure 1 As shown in F, agomiR-29b-3p was successfully synthesized. agomiR-29b-3p was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0055] The negative control of agomiR-NC and the agonist agomiR-29b were injected into the vitreous cavity of the COH experimental group and the control group, respectively. The electrophysiological damage of the photoreceptor cells and bipolar cells in the COH experimental group and the control group was detected by ERG. The results are as follows Figure 2 As shown in A and B, the ERG test results showed that the amplitudes of a wave and b wave in the agomiR-29b-3p group were higher than those in the COH experimental group, suggesting that the retinal electrophysiological function could be protected;

[0056] The results are as follows Figure 2 As shown in C and D, the damage degree of the electrophysiological function of the entire optic nerve conduction pathway in the COH experimental group and the control group was detected by VEP. The VEP test results showed that the N1-P1 amplitude in the agomiR-29b-3p group was higher than that in the COH experimental group, indicating that the physiological function of the optic nerve can be protected;

[0057] The results are as follows Figure 2 As shown in E, the visual motor ability and visual function damage degree of animals in the COH experimental group and the control group were analyzed by qOMR. The qMOR analysis results showed that the agomiR-29b-3p group protected the visual function;

[0058] After the experiment, mice in each group were euthanized, and the eyeball samples of the mouse model were obtained. The retina was separated from the eyeball, and the RGCs in the retinal flat mounts of the COH model made by fluorescently labeling the tool mouse Thy1-GFP were stained. 200x images were taken under the LSM880 confocal microscope to quantify the average number of RGCs in each retina. The test results are shown in Figure 2As shown in F and G. The results showed that the control group mice had abundant RGC numbers, the number of surviving RGCs in the COH model was significantly reduced, and the agomiR-29b-3p treatment group could significantly increase the number of surviving RGCs compared with the agomiR-NC negative control treatment group.

[0059] Example 3: Preparation of NPCs-derived exosomes

[0060] Human induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs) differentiated from iPSCs were cultured in vitro using a serum-free culture system. As shown in Figure C, neural progenitor cells were successfully differentiated. The supernatant of iPSCs and NPCs cell culture was collected and exosomes were extracted by differential ultracentrifugation, specifically: at 300g, 4°C, 10min, the supernatant was taken after centrifugation, then at 2000g, 4°C, 10min, the supernatant was taken after centrifugation, then at 10000g, 4°C, 30min, the supernatant was taken after centrifugation, then at 100000g, 4°C, 70min, the supernatant was taken after centrifugation, then at 100000g, 4°C, 70min, the iPSCs-derived exosomes and NPCs-derived exosomes were obtained.

[0061] Transmission electron microscopy analysis, particle size analysis, and marker protein quantitative analysis of iPSCs-derived exosomes and NPCs-derived exosomes were performed. The identification results were consistent with the classic characteristics of exosomes, and it was found that they had a typical cup-shaped structure ( Figure 3 E). NanoSight showed that the main peak particle size of the exosomes from iPSCs and NPCs was 138.7 nm, and the main peak particle size of the exosomes from NPCs was 115.3 nm, which is consistent with the size of plasma exosomes ( Figure 3 F). Immunoblotting experiments verified the exosome-specific positive and negative proteins: Alix, SDCBP, TSG101 and Calnexin. Figure 3 As shown in G, Alix, SDCBP, and TSG101 were significantly enriched in plasma exosomes, while Calnexin was not detected in the exosome samples, proving that the extracted samples were exosomes.

[0062] Furthermore, the collected exosomes were fluorescently labeled (PKH67) and then subjected to in vivo cell uptake experiments. 3I is the intravitreal injection of PKH-67-labeled exosomes, and the retinal sections were fluorescently stained with the RGC marker RBPMS, showing that the green-labeled exosomes co-localized with the red-labeled RGCs, indicating that the exosomes were internalized by RGCs. The results showed that intravitreal injection showed good tissue / cell uptake efficiency without any auxiliary transfection reagents ( Figure 3 HI).

[0063] Example 4: Protective effect of NPCs-derived exosomes on optic nerve injury in an optic nerve injury model

[0064] The optic nerve clamp injury (ONC) model was established using Thy1-GFP tool mice (RGCs fluorescently labeled) Figure 4 A), the preparation method is to take eight-week-old mice, and use a mixed anesthetic of 1% sodium pentobarbital and 0.18% xylazine at a weight of 0.006mL / g for general anesthesia, and at the same time, drop proparacaine hydrochloride on the eye surface for surface anesthesia. Under an ophthalmic surgical microscope, the conjunctiva was cut open with ophthalmic scissors, and the optic nerve was exposed with ophthalmic forceps, and then clamped with Dumont forceps for 15s. The extracted iPSCs and their NPCs exosomes were injected into the vitreous cavity at 500ng / eye. The results showed that both exosomes showed RGC protective effects, and the effect of NPCs exosomes was more significant ( Figure 4 BE). Visual evoked potential (VEP) detection also showed that NPCs-derived exosomes partially restored the electrophysiological function of the optic nerve ( Figure 4 F).

[0065] Example 5: Construction of engineered exosomes ExomiR-29

[0066] The method for constructing engineered exosomes ExomiR-29 can be selected as follows: first, NPCs cells are transfected with agomir-29 or miR-29 overexpression lentiviral plasmids and then exosomes are collected, and exosomes are extracted by differential ultracentrifugation, and the differential ultracentrifugation method is the same as in Example 3; or two schemes are selected: first, exosomes are collected by differential ultracentrifugation and then transfected with agomir-29 or miR-29 overexpression lentiviral plasmids.

[0067] In the specific embodiments provided by the present invention, Figure 5 As shown in A, a control group and an experimental group were set up, and RT-qPCR was used to evaluate the miR-29 loading amount in the engineered exosomes prepared under different conditions. The exosomes of NPCs cells were first collected by differential ultracentrifugation.

[0068] Experimental group: Lipofectamine RNAiMAX (1 μg agomir-29 and 1×10 9 The exosomes were mixed); the BTXGeminiX2 system in the laboratory was used for electroporation. The exosome and agomir-29a mixture was added to the electrode cup for electroporation. The electroporation parameters selected the decay mode, adjusted the perforation electric field strength to 1000V / cm, the electroporation duration to 10ms, the number of drive cycles to 1, and incubated at 37°C for 30min to restore the exosome membrane structure. The engineered exosomes were concentrated by nuclease treatment and ultrafiltration centrifugation to remove free agomir-29a and other impurities.

[0069] Control group 1: Lipofectamine RNAiMAX was used as the transfection reagent to transfect 1 μg agomir-29 with 1×10 9 After the exosomes were mixed, they were incubated at 37°C for 30 minutes to restore the exosome membrane structure. The engineered exosomes were concentrated by nuclease treatment and ultrafiltration centrifugation to remove free agomir-29 and other impurities.

[0070] Control group 2: Transfection reagent using LipofectamineRNAiMAX (1 μg agomir-29 and 1×10 9 The exosomes were mixed); the BTXGeminiX2 system of the laboratory was used for electroporation. The exosome and agomir-29a mixture was added to the electrode cup for electroporation. The attenuation mode was selected as the electroporation parameter. The perforation electric field strength was adjusted to 1000 V / cm, the electroporation duration was 10 ms, and the number of driving cycles was 1. The engineered exosomes were concentrated by nuclease treatment and ultrafiltration centrifugation to remove free agomir-29a and other impurities.

[0071] The results are as follows Figure 5 As shown in A, engineered exosomes efficiently loaded with miR-29a were successfully constructed, and the scheme of electroporation and simultaneous incubation of miR-29a agonist had the highest small nucleic acid drug loading efficiency.

[0072] In addition, RT-qPCR was used to evaluate the miR-29a loading in engineered exosomes prepared by transfection with agomir-29a or miR-29a overexpression lentiviral plasmids, as Figure 5 As shown in B and 5C, NPCs cells were first transfected with agomir-29a or miR-29a overexpression lentiviral plasmids, and then exosomes were extracted for quantitative detection of miR-29a. A control group of invalid sequence and empty plasmid were set up to transfect NPCs cells, and then exosomes were extracted for quantitative detection of miR-29a.

[0073] The miR-29a overexpression lentiviral plasmids were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0074] The results are as follows Figure 5 As shown in B, in the scheme where NPCs cells were transfected with agomir-29a or miR-29a overexpression lentiviral plasmids and then exosomes were collected, the small nucleic acid drug loading efficiency of the agomir-29a group was higher.

[0075] Electrophysiological and visual function analysis of wild-type mice, COH mouse model, COH mouse model with intravitreal injection of invalid NPCs exosomes, and COH mouse model with intravitreal injection of ExomiR-29 showed that engineered exosomes ExomiR-29 showed better optic nerve repair effect ( Figure 5 D, E). Further, the histopathological phenotype analysis after treatment with engineered exosomes ExomiR-29 (shown as sEVs-miR-29+COH in the figure) showed that the number of retinal ganglion cells (RGC) in the engineered exosomes ExomiR-29 treatment group was significantly higher than that in the NPCs exosomes group, and the thickness of the GCC layer in the engineered exosomes ExomiR-29 treatment group was increased compared with that in the NPCs exosomes group, which also verified that engineered exosomes have a better protective effect against optic nerve damage ( Figure 6 AD). In addition, in vitro neuronal differentiation experiments also showed that the delivery of miR-29 gene drugs based on engineered exosomes significantly promoted the efficiency of neuronal differentiation ( Figure 6 EG). In summary, we successfully constructed engineered exosomes ExomiR-29 that effectively loaded small nucleic acid drugs, and confirmed its neuroprotective effect in the optic nerve injury model through in vivo and in vitro experiments, and it can promote the differentiation of stem cells into neurons.

[0076] Finally, it is necessary to explain here that the above embodiments are only used to further illustrate the technical solution of the present invention in detail and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above content of the present invention all fall within the scope of protection of the present invention.

Claims

1. Application of MiR-29 in the preparation of drugs for optic nerve damage.

2. The use of MiR-29 in the preparation of optic nerve injury drugs according to claim 1, characterized in that: The drug includes an agonist agomiR-29 or a miR-29 overexpression lentiviral plasmid, The agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, agomiR-29c-3p, The effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1, The stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.

2. The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.

3. The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5, The effective working sequence of agomiR-29c-3p is shown in SEQ ID NO.6, The stem-loop sequence of agomiR-29c-3p is shown in SEQ ID NO.

7.

3. The use of MiR-29 in the preparation of optic nerve injury drugs according to claim 1 or 2, characterized in that: The optic nerve damage drug is a drug for treating optic nerve damage caused by glaucoma, autoimmunity, inflammation, traumatic, ischemic or hereditary optic neuropathy.

4. The use of exosomes in the preparation of a drug for treating optic nerve damage, characterized in that: The exosomes are derived from neural progenitor cells.

5. Use of the exosomes according to claim 4 in preparing a drug for treating optic nerve damage, characterized in that: The optic nerve damage drug is a drug for treating optic nerve damage caused by glaucoma, autoimmunity, inflammation, traumatic, ischemic or hereditary optic neuropathy.

6. Use of the exosomes according to claim 4 in preparing a drug for treating optic nerve damage, characterized in that: The exosomes also carry drugs that are easily metabolized and degraded by human tissues or cells before exerting their therapeutic effects, including but not limited to drugs and molecules used in gene therapy.

7. Use of exosomes loaded with agomiR-29 in the preparation of a drug for treating optic nerve damage, characterized in that: Exosomes loaded with agomiR-29 are derived from neural progenitor cells; The agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, agomiR-29c-3p, The effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1, The stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.

2. The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.

3. The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5, The effective working sequence of agomiR-29c-3p is shown in SEQ ID NO.6, The stem-loop sequence of agomiR-29c-3p is shown in SEQ ID NO.

7.

8. The use of exosomes loaded with agomiR-29 in the preparation of a drug for treating optic nerve damage according to claim 7, characterized in that: The method for preparing exosomes loaded with agomiR-29 comprises the following steps: (1) extracting carrier exosomes: extracting exosomes from the supernatant of neural progenitor cells using an exosome extraction kit, and resuspending them in PBS to obtain an exosome suspension; (2) Acquisition of agomiR-29: artificially synthesizing agomiR-29, wherein the agomiR-29 includes any one or more of agomiR-29a-3p, agomiR-29b-3p, and agomiR-29c-3p. The effective working sequence of agomiR-29a-3p is shown in SEQ ID NO.1, The stem-loop sequence of agomiR-29a-3p is shown in SEQ ID NO.

2. The effective working sequence of agomiR-29b-3p is shown in SEQ ID NO.

3. The stem-loop sequence of agomiR-29b-3p is shown in SEQ ID NO.4 or SEQ ID NO.5, The effective working sequence of agomiR-29c-3p is shown in SEQ ID NO.6, The stem-loop sequence of agomiR-29c-3p is shown in SEQ ID NO.

7.

9. The use of exosomes loaded with agomiR-29 in the preparation of a drug for treating optic nerve damage according to claim 8, characterized in that: In step (3), by 8 -1×10 9 1 μg of agomir-29 and transfection reagent were added to each exosome to obtain a mixed solution, the mixed solution was added to an electrode cup for electrotransfection, and the exosome membrane structure was restored at 37°C to obtain engineered exosomes. The engineered exosomes were concentrated by nuclease treatment and ultrafiltration centrifugation to remove free agomir-29 and other impurities.

10. The use of exosomes loaded with agomiR-29 in the preparation of a drug for treating optic nerve damage according to claim 9, characterized in that: The parameters of the electroporation include: selecting the attenuation mode, adjusting the perforation electric field strength to 1000 V / cm, the electroporation duration to 10 ms, and the driving cycle number to 1 time.

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