Use of miR-3158-3p in preparation of a drug for treating myasthenia gravis and a pharmaceutical composition

Abstract

The application relates to the field of biological medicine, and particularly to application of miR-3158-3p in preparation of a drug for treating myasthenia gravis and a pharmaceutical composition. The drug exerts a therapeutic effect through double mechanisms of immunoregulation and nerve protection. The immunoregulation mechanism is that the drug promotes differentiation of Treg cells and enhances the immunoregulation function of the Treg cells by supplementing miR-3158-3p, simultaneously inhibits excessive activation of Th17 cells, corrects an immune tolerance imbalance state in a myasthenia gravis patient, and inhibits abnormal autoimmune responses of the body. The nerve protection mechanism is that the drug can improve biophysical properties of a neuron cell membrane, significantly improves membrane fluidity of the neuron cell membrane, and optimizes neurotransmitter recovery function in an inter-synaptic gap, thereby providing a structural basis for repairing signal transmission disorders of neuromuscular junctions and improving nerve damage of myasthenia gravis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of miR-3158-3p in the preparation of drugs for treating myasthenia gravis and pharmaceutical compositions thereof. Background Technology

[0002] Myasthenia gravis (MG) is a chronic autoimmune neurological disorder characterized by impaired neuromuscular junction signal transmission. Its main clinical manifestations include fluctuating skeletal muscle weakness and fatigue after exertion. The disease exhibits significant clinical subtypes. Most patients initially present with ocular myasthenia gravis (OMG), exhibiting only ocular symptoms such as ptosis, diplopia, and limited eye movement. However, clinical follow-up reveals that a considerable proportion of ocular OMG patients gradually progress to generalized myasthenia gravis (GMG) over several months to years, with weakness spreading to the limbs and trunk. In severe cases, it can affect the pharyngeal and respiratory muscles, triggering myasthenic crisis and directly endangering life.

[0003] As a prevalent neuroimmunological disease, myasthenia gravis not only significantly reduces patients' quality of life but also places a heavy burden on families and society, making it a key clinical challenge that requires urgent attention and breakthroughs in the field of neurology. However, there is currently no cure for myasthenia gravis, and the long-term drug intervention and repeated hospitalizations place enormous economic and psychological pressure on most patients and their families. Therefore, developing novel therapeutic targets and intervention strategies has become a research hotspot in this field.

[0004] Follicular helper T cells (Tfh), regulatory T cells (Treg), and Th17 cells are key cells regulating peripheral immune homeostasis. + T cell subsets. The balance between Treg and Th17 cells is crucial for maintaining immune tolerance: Th17 cells, characterized by the transcription factor RORγt, secrete pro-inflammatory factors that mediate immune damage; Treg cells, on the other hand, exert immunosuppressive effects. An imbalance between these two is a significant contributing factor to autoimmune diseases. Clinical studies have confirmed that when ocular myasthenia gravis transforms into generalized myasthenia gravis, abnormal Th17 activation and Treg dysfunction are commonly observed, with multiple immune imbalances driving the disease from local to systemic spread. Simultaneously, neuronal neurotransmitter reuptake function is continuously impaired during disease progression, exacerbating neuromuscular transmission disorders. Therefore, targeted regulation of the Treg / Th17 cell balance and restoration of neuronal neurotransmitter reuptake capacity are important research directions for halting disease progression.

[0005] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs approximately 22 nt in length. They primarily negatively regulate target gene expression at the post-transcriptional level and are widely involved in various biological processes such as cell differentiation, immune responses, and signal transduction. Multiple studies have shown that abnormal miRNA expression is closely related to the pathogenesis of autoimmune diseases and neurological disorders, and can serve as diagnostic biomarkers and potential therapeutic targets. With the continuous development of high-throughput sequencing and molecular biology technologies, more and more functional miRNAs are being discovered and validated. miR-3158-3p is a miRNA molecule found in the peripheral blood exosomes of MG patients. Its expression levels vary among different severity levels of MG patients. Currently, its biological functions, its role in immune homeostasis regulation, and neurotransmitter metabolism have not been systematically elucidated. In particular, whether this molecule can regulate Tfh / Treg cell balance, improve neuronal neurotransmitter reuptake, and thus intervene in the progression of myasthenia gravis remains a subject of limited research. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a novel treatment strategy for myasthenia gravis based on microRNA regulation. This strategy offers a new use for miR-3158-3p in the preparation of drugs for treating myasthenia gravis and pharmaceutical compositions containing this miRNA, achieving effective treatment of myasthenia gravis by correcting Treg / Th17 cell imbalance and improving neuronal membrane fluidity.

[0007] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows: This invention first provides the use of miR-3158-3p or its functional equivalent in the preparation of medicaments for the treatment or prevention of myasthenia gravis.

[0008] In one embodiment of the invention, the drug exerts its effect through an immunomodulatory mechanism: the drug is used to regulate the balance between regulatory T cells and helper T cells 17 in the peripheral blood of patients with myasthenia gravis. Specifically, by supplementing miR-3158-3p, it promotes the differentiation or enhanced function of Treg cells while inhibiting the overactivation of Th17 cells, thereby correcting the imbalance of immune tolerance in MG patients and suppressing abnormal autoimmune responses.

[0009] In one embodiment of the invention, the drug works through a neuroprotective mechanism: the drug improves the biophysical properties of the neuronal cell membrane, particularly increasing the fluidity of the neuronal cell membrane, thereby helping to improve the reabsorption function of neurotransmitters in the synaptic cleft and providing a structural basis for restoring signal transmission at the neuromuscular junction.

[0010] In a preferred embodiment of the present invention, the drug exerts its effects synergistically through the aforementioned immunomodulatory mechanism and neuroprotective mechanism.

[0011] The present invention also provides a pharmaceutical composition for treating myasthenia gravis, comprising a therapeutically effective amount of miR-3158-3p modulator and a pharmaceutically acceptable carrier or excipient.

[0012] The miR-3158-3p regulator is a miR-3158-3p mimic, miR-3158-3p agomir, miR-3158-3p precursor molecule, or an expression vector carrying the miR-3158-3p coding sequence. The regulator possesses the same or substantially the same biological activity as natural human miR-3158-3p, and can specifically bind to and regulate the expression of downstream target genes to exert a therapeutic effect.

[0013] Given the stability and cell penetration issues of nucleic acid drugs in vivo, the pharmaceutical compositions of the present invention preferably include a delivery carrier. The delivery carrier is selected from one or more of liposomes, nanoparticles, or viral vectors. Liposomes have good biocompatibility and can encapsulate miRNA molecules to protect them from nuclease degradation; nanoparticles can achieve targeted delivery, increasing local drug concentration; viral vectors can achieve long-term expression and are suitable for scenarios requiring long-term maintenance of therapeutic effects.

[0014] The pharmaceutically acceptable carrier includes physiological saline, buffer solution, or lyophilization protectant, used for the shaping, stabilization, and preservation of the formulation. The pharmaceutical composition can be formulated as an injection, lyophilized powder for injection, etc.

[0015] This invention has the following characteristics and beneficial effects: 1) This study reveals for the first time the therapeutic link between miR-3158-3p and MG, achieving dual benefits from both the immune and neurological levels. Unlike traditional single-target drugs, the technical solution provided by this invention has a dual "immune-neural" regulatory function: on the one hand, by supplementing or upregulating miR-3158-3p levels, it effectively corrects the key Treg / Th17 immune imbalance in MG, regulating the imbalance of immune cell subsets from the source; on the other hand, it directly improves the fluidity of neuronal cell membranes, providing a functional basis for repairing damaged neuromuscular junctions. The combined effect of these two mechanisms achieves a combination of "immune homeostasis remodeling" and "neural function repair".

[0016] 2) Highly targeted, with definite therapeutic effect, avoiding the non-specific side effects of traditional immunosuppressants. This invention specifically restores the insufficient miRNA levels and reconstructs their physiological function under the pathological environment of MG by delivering miR-3158-3p mimics or expression vectors carrying the miR-3158-3p coding sequence to MG patients. Compared with broad-spectrum immunosuppressants, this invention has higher targeting and lower expected toxicity. Experimental studies have confirmed that after miR-3158-3p intervention, the Treg / Th17 ratio from peripheral blood of MG patients was significantly restored, and neuronal membrane fluidity was significantly improved, verifying its definite therapeutic potential.

[0017] 3) It has great potential for clinical translation, providing a new option for patients who do not respond to or are intolerant of traditional treatments. This invention employs mature nucleic acid delivery technologies (including but not limited to liposomes, nanoparticles, and viral vectors) to effectively protect miR-3158-3p from nuclease degradation and achieve targeted delivery, thus solving the aforementioned key technical challenges. The technical solution of this invention provides a novel drug candidate for the treatment of myasthenia gravis, and offers a new treatment option and clinical alternative, particularly for patients who are insensitive to traditional immunosuppressants, develop drug resistance, or cannot tolerate their adverse reactions. Attached Figure Description

[0018] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The figure shows the test results of Embodiment 1 of the present invention.

[0019] Figure 2 The figure shows the test results of Embodiment 2 of the present invention.

[0020] Figure 3 This is a flowchart illustrating the use of photobleaching to assess neuronal cell membrane fluidity in an embodiment of the present invention.

[0021] Figure 4 The figure shows the test results of Embodiment 3 of the present invention.

[0022] Figure 5 The figure shows the test results of Embodiment 4 of the present invention.

[0023] Figure 6 Morphological identification of exosomes using transmission electron microscopy (TEM), where the scale bar is 500 nm. Arrows indicate typical vesicle-like structures (magnification 12,000×, accelerating voltage 80 kV). Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other. Embodiments 1. Study subjects and inclusion criteria 1.1 Diagnostic and Classification Criteria The diagnosis of myasthenia gravis (MG) is based on the clinical classification criteria of the Myasthenia Gravis Foundation (MGFA). Ocular myasthenia gravis (OMG) is defined as myasthenia gravis symptoms limited to the extraocular muscles; generalized myasthenia gravis (GMG) is defined as myasthenia gravis with involvement of other muscle groups besides the extraocular muscles.

[0025] 1.2 Inclusion Criteria (1) Age > 18 years and < 75 years; (2) Clinical diagnosis of myasthenia gravis, with one of the following supporting evidences: ① Clear clinical response to neostigmine; ② Positive test for anti-acetylcholine receptor antibody or muscle-specific tyrosine kinase antibody; ③ Repetitive nerve stimulation (RNS) attenuation > 10%; (3) Willing to undergo sample collection, imaging examinations, and other disease-related examinations and assessments; (4) Female subjects of childbearing age must have a negative pregnancy test result during the screening period, and patients and their partners must take effective contraceptive measures during the study period; (5) The patient signs the informed consent form.

[0026] 1.3 Exclusion Criteria (1) In addition to MG, those with a history of other chronic degenerative, mental or neurological diseases that may lead to weakness or fatigue; (2) Those who may have other diseases that cause ptosis, peripheral muscle weakness or diplopia; (3) Age ≤ 17 years or ≥ 75 years; (4) Individuals who are currently taking glucocorticoids or related immunosuppressants due to other immune system diseases; (5) Individuals who cannot use immunosuppressants due to other chronic diseases; (6) Individuals who are unable to cooperate with follow-up and self-assessment due to severe mental illness or cognitive impairment; (7) Pregnant or lactating women or patients who plan to have children during the trial; (8) Those who have had a serious infection or malignant tumor within the past month and are unable to receive immunosuppressant treatment; (9) Those who are unwilling to participate in this study; (10) Individuals deemed unsuitable to participate in this trial by the researchers.

[0027] 1.4 Sample Grouping This study included eligible participants, with 8 patients with OMG and 4 patients with GMG.

[0028] 2. Materials and Main Reagents 2.1 Main Reagents and Brand Codes CD4+ T cell sorting magnetic bead kit: Biolegend, MojoSort™ Human CD4 T Cell Isolation Kit (Catalog No.: 302609).

[0029] Flow cytometry antibodies and dyes: Zombie live cell dye (Biolegend, catalog number: 423105, APC-Cy7 channel, dilution ratio 1:1000); CD4-FITC (Biolegend, part number: 300505); Foxp3-PE (Biolegend, catalog number: 320107, 2μl / test); RORγt-PE (R&D Systems, catalog number: IC6006P-025, 2μl / test).

[0030] Fixation / rupture buffer: True-Nuclear™ Transcription Factor Buffer Set (Biolegend, catalog number: 424401).

[0031] Treg / Th17 induces cytokines: IL-2 (PeproTech, catalog number: 200-02-10ug, 5ng / ml); TGF-β (Essential Pharmaceuticals, Catalog No.: 10804-HNAC-20, 1ng / ml); anti-IFNγ (BioLegend, catalog number: 506531, 1μg / ml); IL-6 (BioLegend, catalog number: 570802, 100ng / ml); IL-23 (BioLegend, catalog number: 574102, 50ng / ml); IL-1β (BioLegend, catalog number: 579402, 30ng / ml).

[0032] SH-SY5Y cell differentiation induction reagent: Retinoic acid (RA, Solarbio, catalog number: A9120); Brain-derived neurotrophic factor (BDNF, MCE, catalog number: HYP7116A).

[0033] Membrane flow assay dye: SynaptoGreen C4 (FM1-43, 5 μM).

[0034] 3. Experimental Methods 3.1 CD4+ T lymphocyte magnetic bead sorting Peripheral blood was collected from patients, and peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. CD4+ T lymphocytes were then obtained by negative selection magnetic bead sorting according to the MojoSort™ Human CD4 T Cell Isolation Kit instructions. Flow cytometry verification showed that cells with a purity ≥90% after sorting were used for subsequent experiments.

[0035] 3.2 Construction of in vitro differentiation culture system for Treg and Th17 cells Treg system: Under co-stimulatory conditions of anti-CD3 antibody (Biolegend, clone 145-2C11, 1:1000, coated 24 h in advance, washed twice with PBS before use) and soluble anti-CD28 antibody (Biolegend, clone 302901, 1:1000), IL-2 (5 ng / ml) and TGF-β (1 ng / ml) were added to maintain Treg cell growth and differentiation.

[0036] Th17 system: Under CD3 / CD28 co-stimulation, anti-IFNγ (1 μg / ml), IL-6 (100 ng / ml), IL-23 (50 ng / ml), IL-1β (30 ng / ml) and TGF-β (1 ng / ml) were added to induce Th17 cell differentiation.

[0037] 3.3 Extraction and Identification of Plasma Exosomes Extraction: Peripheral blood was drawn from patients with myasthenia gravis, and cell debris was removed by low-speed centrifugation (300×g, 10 min; 2000×g, 20 min) to obtain the supernatant plasma. Exosomes were precipitated by ultracentrifugation (100,000×g, 70 min), and resuspended in PBS for later use.

[0038] Identification: Resuspended exosome samples were taken and vesicle morphology was observed using transmission electron microscopy (TEM); at the same time, nanoparticle tracking analysis (NTA) was used to detect the particle size distribution (verifying that the particle size range was within 30–150 nm), confirming that the extract was exosomes (see attached figure).

[0039] 3.4 SH-SY5Y cell culture and neuronal differentiation induction SH-SY5Y cell lines were cultured in vitro and induced to differentiate into mature neurons using a sequential induction method involving RA and BDNF. Days 1–3: Induction for 72 h in DMEM / F12 medium (containing 10% FBS, 1% P / S) with 10 μM RA; Days 4–7: Induction for 72 h in DMEM / F12 medium (containing 2% FBS, 1% P / S) with 10 μM RA; Days 7–10: Induction for 72 h in DMEM / F12 medium (containing 1% FBS, 1% P / S) containing BDNF (50 ng / mL) and 10 μM RA.

[0040] Cells were collected after differentiation was completed for subsequent experiments.

[0041] 3.5 Flow cytometry detection of Treg / Th17 ratio Cells were collected after culture and first stained with Zombie dye (APC-Cy7 channel, 1:1000) to label viable cells. After washing twice with PBS, the surface was stained with CD4-FITC (30 min). Subsequently, cells were fixed and permeabilized using True-Nuclear™ Transcription Factor Buffer Set, and intracellularly stained with Foxp3-PE and RORγt-PE (2 μl / test). For assay analysis, the gating strategy was set as follows: lymphocytes were circled using FSC / SSC → Zombie. - Circle the live cells → CD4 + Circle the helper T cell subsets → Analyze Foxp3 separately + (Treg) and RORγt + The percentage of (Th17) cells.

[0042] 3.6 FRAP (Fluorescent Photobleaching) Technique for Detecting Cell Membrane Fluidity FM dye film loading and FRAP experiments were performed according to the methods described in the literature: Membrane loading: Mature neurons were incubated in a solution containing 5 μM FM1-43 dye for 200 s.

[0043] FRAP parameters: The region of interest (ROI) was set to 5 μm × 5 μm (approximately one-quarter of the area of ​​a single cell membrane). Confocal microscopy was used for detection: before bleaching, one image was acquired every 7.22 s using a 488 nm wavelength laser (5% intensity), for a total of 6 images; then a bleaching operation was performed (405 nm wavelength laser, 100% intensity, for 20 s); after bleaching, 39 recovery images (488 nm, 5% intensity) were acquired at the same time intervals.

[0044] Data calculation: The formula for calculating the moving fraction (M) is: M = (F∞ - F0) / (F initial - F0), where F initial F0 represents the initial fluorescence intensity before bleaching, F0 represents the fluorescence intensity instantaneously after bleaching, and F∞ represents the fluorescence intensity after restoring equilibrium.

[0045] The half-recovery time (τ) was obtained by fitting the mean recovery curve in Origin software using the exponential equation Recovery(t) = R × (1 - exp(-t / τ)).

[0046] 3.7 Statistical Analysis The biological replicates for each experiment were N=10 cells (N=18 for the Control group, N=18 for the OMG group, and N=17 for the GMG group in Example 3; N=15 for the NC group and N=15 for the 3158mic group in Example 4). All data are expressed as mean ± standard error (mean ± SEM). The t-test was used for comparisons between two groups; two-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. P<0.05, P<0.01).

[0047] 4. Specific experiments and results of the examples Example 1: Inhibition Intervention Experiment of CD4+ T Lymphocytes miR-3158-3p Derived from OMG Patients Peripheral blood samples were selected from patients with mild OMG, and CD4+ T lymphocytes were sorted according to the aforementioned method to construct Treg and Th17 in vitro differentiation systems. Cells were transfected with hsa-miR-3158-3p inhibitor (50 nM) for 3 days in the culture system, while cells transfected with inhibitor NC (50 nM) served as a blank control group.

[0048] After culture, the cell proportion was detected by Zombie live cell staining combined with flow cytometry. Experimental results ( Figure 1 The results showed that, compared to the control group, the proportion of CD4+Foxp3+ Treg cells and the proportion of CD4+RORγt+ Th17 cells were significantly decreased in OMG patients transfected with miR-3158-3p inhibitor, while the proportion of CD4+Rγt+ Th17 cells was significantly increased. This result confirms that inhibiting miR-3158-3p expression can disrupt the balance of CD4+ T lymphocyte subsets in OMG patients, promote the differentiation of inflammatory Th17 cells and inhibit the proliferation of protective Treg cells, mimicking the immune dysregulation process of myasthenia gravis.

[0049] Example 2: Intervention experiment on miR-3158-3p overexpression in CD4+ T lymphocytes derived from GMG patients Peripheral blood samples were selected from patients with severe GMG, and CD4+ T lymphocytes were sorted to construct Treg and Th17 in vitro differentiation systems. Cells were transfected with hsa-miR-3158-3p mimic (50 nM) for 3 days, while cells transfected with mimic NC served as a control group.

[0050] Cell percentage was measured after culture. Experimental results ( Figure 2 The results showed that, compared with the control group, the proportion of CD4+Foxp3+ Treg cells and the proportion of CD4+RORγt+Th17 cells were significantly increased in CD4+ T lymphocytes of GMG patients transfected with miR-3158-3p, while the proportion of CD4+RORγt+Th17 cells was significantly decreased. This result confirms that overexpression of miR-3158-3p can significantly correct the immune balance of CD4+ T lymphocytes in GMG patients, inhibit Th17-mediated inflammatory responses, and enhance Treg immunomodulatory function, suggesting that miR-3158-3p has the potential to treat myasthenia gravis.

[0051] Example 3: Effects of peripheral blood exosomes on neuronal cell membrane fluidity in OMG and GMG patients After the SH-SY5Y cell line was induced to differentiate into mature neurons, exosomes derived from plasma of patients with OMG and GMG were added for intervention. The experiment was conducted in a time gradient (normal culture medium was replaced on Day 1, exosomes were added on Day 4, and FRAP was measured on Day 7), with a blank control group (Control).

[0052] FRAP test results ( Figure 4The fluorescence recovery curves showed that the shift fraction (M) and half-recovery time (τ) for each group were as follows: Control group (M=0.473±0.008, τ=24.855±1.730s); OMG group (M=0.390±0.006, τ=27.758±1.806s); GMG group (M=0.326±0.004, τ=25.935±1.274s). Compared with the blank control group, the exosome intervention group of OMG patients showed significantly reduced neuronal cell membrane fluidity (decreased M value); while the M value of the exosome intervention group of GMG patients was significantly lower than that of the OMG group. The results confirm that peripheral blood exosomes from patients with myasthenia gravis can induce neuronal cell membrane dysfunction and reduce membrane fluidity, and the damaging effect of exosomes from GMG, which has a more severe condition, is significantly stronger than that of exosomes from mild OMG.

[0053] Example 4: Validation of the rescue effect of miR-3158-3p overexpression on GMG exosome-mediated neuronal injury After the SH-SY5Y cell line was induced to differentiate into mature neurons, it was divided into two groups: one group was transfected with miR-3158-3pmimic (3158mic group), and the other group was transfected with a negative control (NC group). Transfection was performed on Day 1, exosomes from GMG patient plasma were added on Day 4, and FRAP was measured on Day 7.

[0054] FRAP test results ( Figure 5 The results showed that the fluorescence recovery rate was significantly improved in the NC group (M=0.502±0.007, τ=21.577±1.731s) and the 3158mic group (M=0.423±0.006, τ=19.106±1.621s). Compared with the NC group, the fluorescence recovery rate was significantly improved in the miR-3158-3p mimic overexpression combined with GMG exosome intervention group (although the M value was slightly lower, the recovery kinetics τ was significantly shortened, indicating improved fluidity). This result confirms that miR-3158-3p overexpression can effectively antagonize exosome-induced neuronal cell membrane function damage in GMG patients, and functionally validates that miR-3158-3p supplementation is a key molecular mechanism for alleviating neuronal damage and promoting disease remission in patients with myasthenia gravis.

[0055] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of miR-3158-3p in the preparation of drugs for the treatment of myasthenia gravis.

2. The application according to claim 1, characterized in that, The drug is used to regulate the Treg / Th17 cell balance in the peripheral blood of patients with myasthenia gravis.

3. The application according to claim 1, characterized in that, The drug is used to promote the reuptake of neurotransmitters in the synaptic cleft.

4. The application according to claim 1, characterized in that, The drug is used to upregulate the expression of neurotransmitter transport proteins.

5. A pharmaceutical composition for treating myasthenia gravis, characterized in that, It contains a miR-3158-3p modifier and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The miR-3158-3p modulator is a miR-3158-3p mimic, miR-3158-3p agomir, miR-3158-3p precursor molecule, or an expression vector carrying the miR-3158-3p coding sequence.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition further comprises a delivery carrier selected from liposomes, nanoparticles, or viral vectors.

8. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutically acceptable carrier is selected from physiological saline, buffer solution, or lyophilization protectant.

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