A tick-borne encephalitis virus-derived circular RNA and application thereof in prevention and treatment of Parkinson's disease

Abstract

The present application relates to a tick-borne encephalitis virus-derived circular RNA and its application in preventing and treating Parkinson's disease, and belongs to the technical field of biological medicine. The present application first finds that the virus circular RNA vcNS2B derived from the tick-borne encephalitis virus can specifically bind to P4HB protein, thereby inhibiting S-nitrosylation modification of P4HB, maintaining the oxidoreductase activity and molecular chaperone function of P4HB, and breaking the cascade reaction caused by endoplasmic reticulum stress and downstream alpha-synuclein aggregation at the root. Further, the present application adopts a gene therapy method of AAV9 to deliver vcNS2B, under the pathological condition of PD, the expression of vcNS2B mediated by AAV9 continuously protects P4HB from S-nitrosylation, effectively reduces the unfolded protein response, and further reduces the excessive aggregation of alpha-synuclein. Therefore, vcNS2B has wide application value in the prevention and treatment of PD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a circular RNA derived from tick-borne encephalitis virus and its application in the prevention and treatment of Parkinson's disease. Background Technology

[0002] The clinical manifestations of Parkinson's disease (PD) include motor symptoms such as resting tremor, bradykinesia, rigidity, and postural instability, as well as non-motor symptoms such as decreased sense of smell, sleep disturbances, and cognitive impairment. The main pathological feature of PD is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNPc) and striatum of the midbrain, and a weakened ability of tyrosine hydroxylase (TH) to synthesize dopamine.

[0003] Mounting evidence suggests that protein misfolding and aggregation are common pathological bases for neurodegenerative diseases such as Parkinson's disease (PD). A hallmark of PD is Lewy bodies, whose main component is aggregated α-synuclein. In PD, persistently activated microglia and damaged neurons induce the production of large amounts of nitric oxide (NO), leading to nitrosative stress. Excess NO can combine with superoxide anions to form peroxynitrite (ONOO). - This process triggers mitochondrial damage, DNA damage, and α-synuclein aggregation. It can also transfer NO groups to the sulfhydryl group on the cysteine ​​residue at the active site, forming SNO groups, which S-nitrosylate and modify various key proteins. S-nitrosylated Parkin protein (SNO-Parkin) inhibits its E3 ubiquitin ligase activity, leading to impaired clearance of abnormal proteins and damaged mitochondria; S-nitrosylated PTEN induces putative kinase 1 (SNO-PINK1) to inhibit mitophagy; and S-nitrosylated Parkinson's disease-associated gene 7 encoded protein (SNO-DJ-1) reduces cellular antioxidant capacity, further exacerbating oxidative stress. These processes collectively lead to protein homeostasis imbalance, mitochondrial dysfunction, amplified neuroinflammation, and the death of dopaminergic neurons in the substantia nigra, ultimately driving the occurrence and progression of Parkinson's disease.

[0004] In addition, the proline 4-hydroxylase β subunit (P4HB, belonging to the protein disulfide isomerase (PDI) family) is a key oxidoreductase in the endoplasmic reticulum responsible for catalyzing the correct formation and rearrangement of protein disulfide bonds. It is also an important molecular chaperone, playing a crucial role in maintaining protein homeostasis. Numerous studies have shown that S-nitrosylation of PDI is significantly increased in Parkinson's disease patients. This post-translational modification inhibits PDI enzymatic activity, leading to the accumulation of misfolded proteins, promoting the unfolded protein response (UPR), and simultaneously inhibiting the ubiquitination and degradation of α-synuclein and Lewy bodies, ultimately triggering neuronal cell damage and death.

[0005] Currently, the main drugs used clinically to treat Parkinson's disease (PD) fall into six categories: levodopa, dopamine receptor agonists, monoamine oxidase-B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, anticholinergic drugs, and glutamate receptor antagonists. These drugs primarily work by supplementing dopamine or regulating dopaminergic signaling pathways to improve motor symptoms. However, two recent monoclonal antibody drugs targeting α-synuclein were discontinued after Phase III clinical trials showed no significant difference compared to placebo.

[0006] Currently, there are no approved drugs for the treatment of Parkinson's disease (PD) specifically targeting S-nitrosylation. Existing research mainly focuses on neuroprotective effects by reducing pathological S-nitrosylation levels, promoting denitrosylation, or blocking S-nitrosylation of specific target proteins. For example, aminoguanidine is primarily an inhibitor of nitric oxide synthase and diamine oxidase; 1400W is a highly selective, irreversible inducible nitric oxide synthase inhibitor. Inhibition of S-nitrosylation modification through broad-spectrum inhibition of NO production is also possible, but this method lacks specificity and interferes with the normal physiological function of NO. Other studies have found that uric acid can clear ONOO. - It inhibits protein nitrosylation modification, but the clinical trial using inosine nucleoside to increase uric acid levels was terminated because it did not significantly delay the progression of PD. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a specific treatment drug for Parkinson's disease that targets the S-nitrosylation of protein disulfide isomerases.

[0008] To address the aforementioned technical problems, this invention provides a circular RNA derived from tick-borne encephalitis virus and its application in the prevention and treatment of Parkinson's disease. This invention is the first to discover that the viral circular RNA (vcNS2B) derived from tick-borne encephalitis virus can specifically recognize and bind to the P4HB protein, protecting the C4HB domain through a steric hindrance effect generated by physical binding. 53 GHC 56 The site catalytic motif is protected from S-nitrosylation modification (i.e., blocking the formation of SNO-P4HB), maintaining the oxidoreductase activity and molecular chaperone function of P4HB, and ensuring endoplasmic reticulum protein folding homeostasis. This fundamentally blocks the cascade reaction caused by endoplasmic reticulum stress and downstream α-synuclein aggregation. Furthermore, this invention utilizes AAV9 to deliver vcNS2B for gene therapy. It was found that under PD pathological conditions, AAV9-mediated high expression of vcNS2B effectively reduced UPR by continuously protecting P4HB from S-nitrosylation modification, further alleviating excessive α-synuclein aggregation, protecting neuronal function in the striatum and substantia nigra pars compacta of the midbrain, ultimately resulting in significant improvements in motor ability and cognitive function at the whole animal level.

[0009] The first objective of this invention is to provide a viral circular RNA derived from tick-borne encephalitis virus, the nucleotide sequence of which is shown in SEQ ID NO.1, or a sequence having at least 95% homology with SEQ ID NO.1.

[0010] Furthermore, SEQ ID NO.1: TGGAGACGTTCCTTTAATGAGCCTATGACTGTCATAGGAGTGATGCTGACTTTGGCCAGTGGAATGATGCGCCACACGTCTCAGGAGGCGGTGTGTGCCATGGCGCTGGCGGCTTTCCTTCTACT CATGCTCACGCTTGGAACTAGGAAAATGCAGTTGTTGGCTGAGTGGAGTGGGAACATTGAGTGGAATCCAGAACTGACCAGTGAGGGCGGTGAAGTGAGCCTGCGCGTCAGACAAGATGCCCTTGG AAACTTGCACCTGACTGAACTGGAGAAGGAGGAACGCATGATGGCTTTTTGGCTGGTGGTAGGCTTGATTGCCTCGGCCTTTCACTGGTCGGGGATCCTTATCGTCATGGGACTGTGGACCATTT CGGAGATGCTTGGCTCACCCAGGAGAACGGATCTTGTGTTTTCAGGATGCTCCGAAGGGCGAAGTGACAGCCGTCCGCTCGATGTAAAAAATGGAGTGTACCGCATATATACTCCAGGCCTGCTC.

[0011] A second objective of this invention is to provide a recombinant vector containing the aforementioned viral circular RNA.

[0012] A third objective of this invention is to provide the application of the above-mentioned viral circular RNA or the above-mentioned recombinant vector in the preparation of Parkinson's disease treatment products.

[0013] Furthermore, the Parkinson's disease treatment product is used to increase the expression level of tyrosine hydroxylase and reduce the phosphorylation level of α-synuclein.

[0014] Furthermore, the viral circular RNA is delivered via an adeno-associated virus vector, a lentiviral expression vector, a nanoliposome vector, or a mammalian cell expression plasmid.

[0015] Further, the adeno-associated virus vector includes any one of adeno-associated virus vector 9, adeno-associated virus vector rh10, adeno-associated virus PHP.B / PHP.eB, adeno-associated virus CAP-B10, and adeno-associated virus HSC15 / 17. Preferably, the serotype of adeno-associated virus vector 9 achieves long-term and stable expression of vcNS2B in the central nervous system through systemic administration.

[0016] Furthermore, the mammalian cell expression plasmid includes elements that promote the circularization of viral circular RNA.

[0017] Furthermore, the mammalian cell expression plasmid is selected from any one of puc57, pZW1, pcDNA3.1, pRc / CMV, and pUC19.

[0018] A fourth object of the present invention is to provide a pharmaceutical composition for the treatment of Parkinson's disease, the pharmaceutical composition comprising a viral circular RNA with a nucleotide sequence as shown in SEQ ID NO.1, or a sequence having at least 95% homology with SEQ ID NO.1.

[0019] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0020] Furthermore, the pharmaceutical composition is administered via intravenous injection, intracerebral injection, or intrathecal injection.

[0021] Compared with the prior art, the above-described technical solution of the present invention has the following advantages: This invention utilizes AAV9 to deliver vcNS2B. Under PD pathological conditions, AAV9-mediated high expression of vcNS2B effectively reduces UPR by continuously protecting P4HB from S-nitrosylation and further alleviates the excessive aggregation of α-synuclein, protecting neuronal function in the striatum and substantia nigra pars compacta of the midbrain, significantly improving the phenotype of Parkinson's disease, and providing a new therapeutic target for the treatment of Parkinson's disease. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0023] Figure 1 It is in vcNS2B wKI -α-synuclein A53T vcNS2B treatment in mice reduced α-synuclein A53T The results of SNO-P4HB in mouse brain tissue are shown in Figure A, where A is the experimental flowchart and B is the Western blot image of the protein. Figure 2 It is in vcNS2B wKI -α-synuclein A53T VCNS2B treatment in mice improves α-synuclein A53T The results of the mice's cognitive abilities are shown in the diagrams. A and B represent the results of open field movement, C represents the results of wheel movement, and D represents the results of new object recognition. express p <0.05, express p <0.01, express p <0.001, the same applies below; Figure 3 It is in vcNS2B wKI -α-synuclein A53T VCNS2B treatment in mice improves α-synuclein at the molecular level. A53T The results of the Parkinson's disease phenotype in mice are shown in the figure. In the figure, A is the expression level of tyrosine hydroxylase in the striatum and B is the expression level of S-nitrosylated P4HB in the substantia nigra. Figure 4 AAV9-vcNS2B treatment reduces α-synuclein. A53T The results of SNO-P4HB in mouse brain tissue are shown in Figure A, where A is the experimental flowchart and B is the Western blot image of the protein. Figure 5 AAV9-vcNS2B treatment improves α-synuclein A53T The results of the cognitive abilities of mice are shown in the figure. A and B are the results of open field movement, C is the results of running wheel movement, and D is the results of new object recognition. ns indicates no significant difference. Figure 6 AAV9-vcNS2B therapy improves α-synuclein at the molecular level. A53T The results of the Parkinson's disease phenotype in mice are shown in the figure. In the figure, A is the expression level of tyrosine hydroxylase in the striatum, B is the expression level of tyrosine hydroxylase in the substantia nigra, and C is the expression level of phosphorylated α-synuclein in the substantia nigra. Figure 7 The figure shows the experimental results of AAV9-vcNS2B treatment reducing SNO-P4HB in the brain tissue of mice with MPTP-induced Parkinson's disease. In the figure, A is the experimental flowchart and B is the protein immunoblot image. Figure 8 The results of AAV9-vcNS2B treatment improved the motor and cognitive abilities of MPTP-induced Parkinson's disease mice. A and B are the results of open field movement, C is the results of running wheel movement, and D is the results of new object recognition. Figure 9 The AAV9-vcNS2B treatment improves MPTP-induced Parkinson's disease symptoms in mice at the molecular level. Here, A is the expression level of tyrosine hydroxylase in the striatum, B is the expression level of tyrosine hydroxylase in the substantia nigra, and C is the expression level of phosphorylated α-synuclein in the substantia nigra. Figure 10 It is P4HB protein C53 GHC 56 Mass spectrum of site S-nitrosylation modification; Figure 11 The diagram shows the results of the verification of the mechanism of action of vcNS2B. In the diagram, A shows the result of vcNS2B inhibiting LGTV-induced S-nitrosylation modification of P4HB protein, B shows the result of vcNS2B inhibiting glutamate-induced LGTV-induced S-nitrosylation modification of P4HB protein, and C shows the result of vcNS2B failing to inhibit P4HB S-nitrosylation after P4HB double mutation. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] The animal models involved in the following examples are as follows: vcNS2B wKI Mice: This mouse is a vcNS2B gene knock-in mouse, where vcNS2B was conditionally knocked into intron 1 at the ROSA26 site (NCBI reference sequence: NR_027008.1) of C57BL / 6JCya mice via genome editing mediated by clustered regularly spaced short palindromic repeats and related proteins (CRISPR / Cas). vcNS2B was obtained by co-injecting gRNA, donor DNA, and Cas9 protein into fertilized eggs of C57BL / 6JCya mice. fl / + Mice. Subsequently with E2a-Cre Tool mice (from Cyagen (Suzhou) Biotechnology Co., Ltd., product number: I001024) were hybridized to breed vcNS2B with high systemic expression. wKI Mice.

[0026] The gRNA sequence used for genome editing is shown in SEQ ID NO.2 (5'-CTCCAGTCTTTCTAGAAGATGGG-3'). The donor DNA sequence is shown in SEQ ID NO.3.

[0027] SEQ ID NO.3: GAGTTCTAAAATTAAACTATGTGGAGTCATGTCCAACCGCACAATGCATCTTTATGTGAAACTTGCTAGAGTTTTTGTTTTCCTTCTATGTAAAAGTCCAGTTGGGAAGCTTTATTTCTGATAGATTAAATGGTATAGGTCTTTCAGTTTTCTCTTCATTTCTGACAACTGAACTGCTCTCGCCTTGAACCTGTTTTGGCACTAAAATAAAATCTGTTCAATTAACGAATTCTGAAAATATGCTATCTTACAGTGGAGACGTTCCTTTAATGAGCCTATGACTGTCATAGGAGTGATGCTGACTTTGGCCAGTGGAATGATGCGCCACACGTCTCAGGAGGCGGTGTGTGCCATGGCGCTGGCGGCTTTCCTTCTACTCATGCTCACGCTTGGAACTAGGAAAATGCAGTTGTTGGCTGAGTGGAGTGGGAACATTGAGTGGAATCCAGAACTGACCAGTGAGGGCGGTGAAGTGAGCCTGCGCGTCAGACAAGATGCCC TTGGAAACTTGCACCTGACTGAACTGGAGAAGGAGGAACGCATGATGGCTTTTTGGCTGGTGGTAGGCTTGATTGCCTCGGCCTTTCACTGGTCGGGATCCTTATCGTCATGGGACTGTGGAC CATTTCGAGATGCTTGGCTCACCCAGGAGAACGGATCTTGTGTTTTCAGGATGCTCCGAAGGGCGAAGTGACAGCCGTCCGCTCGATGTAAAAAATGGAGTGTACCGCATATATACTCCAGGCC TGCTCGTGAATATATTTTTTCTTGAGGATCCACTAATTTGGGATGATAACGCCAAAACAGGTTCAAGGCGAGAGCAGTCAGTTGTCAGAAATGAAGAGAAAACTGAAAGACCTATACCATTTAATCTATCAGAAATAAAGCTTCCCAACTGGACTTTTACATAGAAGGAAAACAAAAACTCTAGCAAGTTTCACATAAAGATGCATTGTGCGGTTGGACATGACTCCACATAGTTTAATTTTAGAACTC α-synuclein A53T Mice: These mice are PD transgenic mice from the Jackson laboratory, with the strain name B6;C3-Tg(Prnp-SNCA). A53T)83Vle / J, item number 004479.

[0028] vcNS2B wKI -α-synuclein A53T Mice: vcNS2B wKI Mouse and α-synuclein A53T Offspring bred from mouse hybrids.

[0029] The detection methods involved are: I. Stereotactic Injection of the Brain For 6-month-old α-synuclein A53T Mice were anesthetized and fixed on a stereotaxic apparatus. A hole was made in the mouse's brain using a hand-held skull drill. The needle was then slowly inserted into the midbrain, and after 5 minutes, 10 mg of [unspecified substance] was injected. 10 The mice were injected with AAV9-vcNS2B (the control group received AAV9-Vector). After injection, the needle was removed after 5 minutes. The wound was sutured and penicillin was applied to prevent infection. One month after the stereotactic injection, open field tests, wheel tests, and new / old object recognition tests were performed to assess the mice's motor and cognitive abilities.

[0030] II. Open Field Experiment The open field test is a common method for assessing exploratory behavior and motor abilities in mice. We conducted the experiment in a dimly lit and soundproof room. Mice were gently placed with their backs to the experimenter into a white plastic open field test chamber (40 cm × 40 cm × 40 cm). A camera positioned directly above the chamber tracked and recorded the movement of each mouse over 5 minutes. After each mouse's experiment, the chamber was cleaned with 75% ethanol to maintain a clean environment and avoid potential interference between different subjects.

[0031] III. Running Wheel Experiment The running wheel experiment was used to assess the motor abilities of mice. The running wheel had a diameter of 21 cm. The distance the mice traveled on the running wheel within 5 minutes was recorded. The data was transmitted to an integrator via an electromagnetic sensor, so that software programs could collect, store, and analyze the data on the mice's movement speed.

[0032] IV. Experiments on the Recognition of Old and New Things In the Novel Object Recognition (NOR) test, mice underwent short-term recognition memory assessment 24 hours after the open field experiment. First, the mice were gently placed with their backs to the experimenter into a white plastic open field test box (40 cm × 40 cm × 40 cm). Two identical objects (object 1 and object 2) were placed in the box, and the mice were allowed to freely familiarize themselves with the objects for 10 minutes. Following this, a 30-minute memory retention period was allowed. After 30 minutes, the mice were returned to the same test box for a new object recognition test, but one of the familiar objects was replaced with a new object of a different color and shape (e.g., object 2 was replaced with object 3, which has a different color and shape). The apparatus was cleaned with 75% ethanol solution before each test. The Novel Object Recognition Index (NOR Index) was calculated as: (New object exploration time - Familiar object exploration time) / (New object exploration time + Familiar object exploration time). An index greater than 0 indicates normal cognitive ability, while an index less than 0 indicates cognitive impairment.

[0033] V. Immunofluorescence staining of phosphorylated α-synuclein in mouse brain tissue sections Mice were anesthetized and their hearts were perfused twice with 25 mL of 1×phosphate-buffered saline (PBS). Brain tissue was then removed and fixed in 4% paraformaldehyde at room temperature for 48 h. Subsequently, dehydration and embedding were performed. The tissue was then incubated in soft paraffin for 90 min after each of the following incubation processes: 3 h in 75% ethanol, 30 min in 85% ethanol, 30 min in 95% ethanol, 30 min in 100% ethanol solution 1, 30 min in 100% ethanol solution 2, 30 min in 100% xylene solution 1, 30 min in 100% xylene solution 2, and 90 min in soft paraffin. The prepared samples were cut into 5 μm thick sections using a HistoCore Autocut microtome, with two coronal sections placed on each slide. Before immunofluorescence staining, the slides were incubated at 65 °C for 2 h, followed by dewaxing and hydration. Xylene solution 1, 10 min; Xylene solution 2, 10 min; 100% ethanol solution 1, 5 min; 100% ethanol solution 2, 5 min; 95% ethanol solution, 5 min; 85% ethanol solution, 5 min; 75% ethanol solution, 5 min; distilled water, 5 min. Then wash three times with 1× phosphate-buffered saline (PBS), 5 min each time. After washing, antigen retrieval is performed by immersing the sections in boiling antigen retrieval buffer and incubating on low heat for 10 min, then removing the sections and allowing them to cool naturally to room temperature. After cooling, wash three times with 1× phosphate-buffered saline (PBS), 5 min each time. After washing, incubate the tissue sections at room temperature with endogenous peroxidase blocking buffer for 25 min. After blocking endogenous peroxidase, wash three times with 1× phosphate-buffered saline (PBS), 5 min each time. Then block the sections in blocking solution containing 5% normal goat serum (Solarbio, catalog number SL038) for 1 h. Then wash three times with 1× phosphate-buffered saline (PBS), 5 min each time. The primary antibody (phosphorylated α-synuclein monoclonal rabbit antibody (CST, catalog number 23706S)) was diluted with blocking buffer and incubated overnight at 4 °C. The next day, the sample was washed three times with 1× phosphate-buffered saline (PBS) for 5 min each time. The secondary antibody (CST, catalog number #4414) was diluted with blocking buffer containing 5% normal goat serum and incubated at room temperature for 1 h. The sample was washed three times with 1× phosphate-buffered saline (PBS) for 5 min each time, stained with 4',6-diamino-2-phenylindole (DAPI), and finally mounted.

[0034] VI. Immunohistochemical staining of mouse brain tissue sections with tyrosine hydroxylase (TH) Paraffin sections were prepared according to the above procedure, followed by baking, dewaxing, and hydration. The sections were then washed three times with 1×PBS for 5 min each time. After washing, antigen retrieval was performed by immersing the sections in boiling antigen retrieval buffer and incubating on low heat for 10 min, then removing the sections and allowing them to cool naturally to room temperature. After cooling, the sections were washed three times with 1×PBS for 5 min each time. After washing, the tissue sections were incubated at room temperature with endogenous peroxidase blocking buffer for 25 min. After blocking endogenous peroxidase, the sections were washed three times with 1×PBS for 5 min each time. The sections were then blocked for 1 h in blocking buffer containing 5% normal goat serum (Solarbio, catalog number SL038). The sections were then washed three times with 1×PBS for 5 min each time. The primary antibody (anti-tyrosine hydroxylase polyclonal rabbit antibody (proteintech, catalog number 25859-1-AP)) was diluted with blocking buffer and incubated overnight at 4 °C. The next day, the sections were washed three times with 1×PBS for 5 min each time. Rabbit secondary antibody (Zhongshan Jinqiao, catalog number PV-9001) was incubated at room temperature for 1 h. The slides were washed three times with 1×PBS for 5 min each time, and then developed using a 3,3'-diaminobenzidine (DAB) kit (Biosharp, catalog number BL732A). After color development, the slides were rinsed with tap water to stop the process. Hematoxylin was counterstained for 2 min, differentiated for 30 s, and rinsed with running water for 10 min to achieve blue reversion. The slides were then rinsed with 75%, 85%, 95%, and 100% ethanol for 1 min each, followed by xylene solution for 1.5 min and xylene solution for 2.5 min. Finally, the slides were mounted and air-dried.

[0035] VII. SNO-P4HB Detection The S-nitrosylated biotin replacement kit (Cayman Chemical, catalog number 10006518) was used, and the assay was performed according to the manufacturer's instructions. Mouse brain tissue homogenate supernatant was lysed in blocking buffer containing 1× protease inhibitor and phosphatase inhibitor, and incubated at 4 °C on ice for 30 min, followed by centrifugation at 12000 g for 15 min. The supernatant was transferred and 2 mL of pre-chilled acetone was added, and the mixture was precipitated at -20 °C for 1 h, followed by centrifugation at 3000 g for 10 min. After centrifugation, the protein precipitate was resuspended in reducing and labelling solution under dark conditions and incubated at room temperature for 1 h. Then, 2 mL of pre-chilled acetone was added, and the biotinylated protein was precipitated at -20 °C, followed by assay using BeyoMag. TMStreptavidin magnetic beads (brand: Beyotime, catalog number P2151) were enriched overnight at 4°C. After washing three times with washing buffer, Western blot was performed to detect S-nitrosylation (SNO-P4HB) of P4HB.

[0036] VIII. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model MPTP (brand: MCE, catalog number: HY-W114750) dissolved in sterile saline was administered intraperitoneally (ip) to AAv9-Vector or AAv9-vcNS2B mice at a dose of 30 mg / kg once daily for 5 consecutive days. Control mice received the same volume of saline at the same time intervals. Nine days after the last injection, the mice's behavior was assessed using open field, wheel running, and novel object recognition tests. The mice were then sacrificed, and brain tissue was harvested. Immunohistochemistry was used to detect tyrosine hydroxylase expression to assess the loss of dopaminergic neurons in the substantia nigra pars compacta and striatum, combined with Western blotting to detect TH and α-synuclein, to comprehensively assess Parkinson's disease symptoms.

[0037] IX. Western Blot (1) Cell collection: After discarding the culture medium, wash the remaining culture medium with PBS and aspirate it. Add trypsin to digest the cells. After the cells are completely digested, add culture medium to stop the digestion. Mix well by pipetting and resuspend the cells in a centrifuge tube. Centrifuge at 3000 rpm for 3 min. Discard the supernatant and add PBS to resuspend the cells. Centrifuge at 3000 rpm for 3 min. Discard the supernatant and keep the precipitate.

[0038] (2) Cell lysis and total protein extraction: Place the cell pellet on ice, add an appropriate amount of prepared cell lysis buffer, mix by pipetting, and lyse on ice for 30 min. Then place it in a centrifuge at 4℃ and centrifuge at 16000 rpm for 10 min. After centrifugation, aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube. Take 2 μL of the supernatant and determine the protein concentration using a quinolinic acid (BCA) kit. Then add a certain amount of loading buffer according to the protein concentration, mix well, and heat denature in a metal bath at 100℃ for 10 min. After centrifugation, place it on ice to wait for gel running or freeze it in a freezer at -80℃.

[0039] (3) Polyacrylamide gel electrophoresis: After loading equal amounts of protein samples, run the gel. First, set the electrophoresis apparatus to a constant voltage of 80V. After electrophoresis for about 30 minutes, wait for the protein samples to enter the separating gel. Then, set the electrophoresis apparatus to a constant voltage of 120V and continue electrophoresis for about 60 minutes. Stop electrophoresis when the protein samples are fully separated in the separating gel.

[0040] (4) Transfer: First, the polyvinylidene fluoride membrane (PVDF membrane) is activated by soaking in methanol. After electrophoresis, the membrane is transferred by a semi-dry method. The voltage of the transfer instrument is set to 18 V and the transfer time is 1 h 40 min.

[0041] (5) Blocking: After the transfer is completed, take out the PVDF membrane, put it into 5% skim milk powder blocking solution, place it on a shaker and shake it gently, and block it at room temperature for 1 hour or at 4°C overnight.

[0042] (6) Primary antibody incubation: After blocking, the PVDF membrane is washed with phosphate buffer (PBST) containing Tween-20 and then cut. The primary antibody of the protein to be detected is diluted in the primary antibody dilution solution at a certain ratio. The PVDF membrane is placed in the solution and incubated on a shaker at room temperature for 2 h or at 4°C overnight.

[0043] (7) Secondary antibody incubation: recover the primary antibody, wash the membrane with PBST 3 times, each time for 5-10 min, prepare the corresponding secondary antibody dilution solution with PBST, put the membrane in it after washing, and incubate on a shaker at room temperature for 1-2 h.

[0044] (8) After the secondary antibody incubation is completed, wash the membrane with PBST 3 times, each time for 5-10 min. Then prepare the enhanced chemiluminescence solution (ECL colorimetric solution), add it evenly to the membrane, and use a chemiluminescence imaging instrument for detection.

[0045] Example 1: vcNS2B in α-synuclein A53T Therapeutic effects in transgenic mice 1. vcNS2B hybrid mice (vcNS2B) wKI -α-synuclein A53T (in mice) Reduced α-synuclein A53T SNO-P4HB in mouse brain tissue Mice with systemic hyperexpression of vcNS2B were compared with α-synuclein. A53T Transgenic mice were hybridized to breed vcNS2B. wKI -α-synuclein A53T Mice, and 9-month-old α-synuclein A53T Mice were used as a negative control. Total protein was extracted from mouse brain tissue homogenates, and biotinylated-P4HB (Biotin-P4HB) was prepared from SNO-P4HB using a biotin exchange assay. The protein was then enriched using streptavidin magnetic beads, and SNO-P4HB was detected by Western blotting. The results showed that, compared to α-synuclein... A53T Mice, vcNS2B wKI -α-synuclein A53TSNO-P4HB levels in mouse brain tissue were significantly reduced ( Figure 1 ).

[0046] 2. VCNS2B hybrid therapy for the prevention of α-synuclein A53T Parkinson's disease symptoms in transgenic mice vcNS2B wKI -α-synuclein A53T Mice were used as the experimental group, and 9-month-old α-synuclein... A53T Mice served as negative controls. When the mice reached 9 months of age, they underwent open field tests, wheel running tests, and new / old object recognition tests to measure the distance the mice traveled in the open field. Figure 2 A), trajectory ( Figure 2 B); distance and speed on the running wheel ( Figure 2 (C); The cognitive abilities of two types of mice were tested in the new and old object cognition experiment (C). Figure 2 (D). The results show that vcNS2B wKI -α-synuclein A53T Mice showed significant recovery of α-synuclein A53T The study of motor and cognitive abilities in mice indicates that vcNS2B hybridization therapy can prevent α-synuclein... A53T Parkinson's disease phenotype in mice.

[0047] 3. VCNS2B hybrid therapy improves α-synuclein at the molecular level. A53T Mouse Parkinson's disease phenotype Mouse brain tissue was collected and subjected to immunohistochemical and immunofluorescence staining experiments. Immunohistochemistry was used to detect the expression level of tyrosine hydroxylase (TH) in the striatum of mouse brain tissue, and immunofluorescence was used to detect the expression level of phosphorylated α-synuclein in the substantia nigra of mice. Figure 3 The results showed that vcNS2B hybridization therapy protected α-synuclein. A53T The expression level of TH in the striatum of mice ( Figure 3 A); reduced the level of S-nitrosolated P4HB in substantia nigra ( Figure 3 (B). Effectively improves α-synuclein at the molecular level. A53T Symptoms of Parkinson's disease in mice.

[0048] Example 2: AAV9-vcNS2B in α-synuclein A53T Therapeutic effects in transgenic mice 1. AAV9-vcNS2B treatment reduces α-synuclein. A53T SNO-P4HB in mouse brain tissue Construction of AAV9-vcNS2B: The adeno-associated virus was prepared by Vivegen Biotech. Using a recombinant adeno-associated virus (AAV) vector, preferably AAV9 serotype, vcNS2B as shown in SEQ ID NO.1 was inserted into the AAV9-Vector backbone. Construction of AAV9-Vector: AAV9-Vector was purchased from Weizhen Biotechnology, product number AV200208-AV9.

[0049] Take α-synuclein from 6-month-old infants A53T Mice were randomly divided into a model group (injected with 10...) 10 vg's AAV9-Vector) and the treatment group (injection 10) 10 Mice were treated with AAV9-vcNS2B (vg) once, followed by feeding for another month. When mice reached 7 months of age, brain tissue homogenates were collected to extract total protein. Biotinylated-P4HB (SNO-P4HB) was prepared using a biotinylation assay, enriched with streptavidin magnetic beads, and detected by Western blotting. The results showed that AAV9-vcNS2B treatment increased α-synuclein... A53T SNO-P4HB was significantly reduced in mice ( Figure 4 ).

[0050] 2. AAV9-vcNS2B treatment improves α-synuclein A53T Parkinson's disease symptoms in transgenic mice Take α-synuclein from 6-month-old infants A53T Mice were randomly divided into a model group (injected with 10...) 10 vg's AAV9-Vector) and the treatment group (injection 10) 10 The mice were treated with AAV9-vcNS2B (vg), and then fed for another month. When the mice reached 7 months of age, they underwent open field tests, wheel tests, and new / old object recognition tests to measure the distance the mice traveled in the open field. Figure 5 A), trajectory ( Figure 5 B); distance and speed on the running wheel ( Figure 5 (C); α-synuclein was detected in experiments involving the cognition of new and old things. A53T Cognitive abilities in mice Figure 5 (D). The results showed that AAV9-vcNS2B treatment significantly restored α-synuclein. A53T The motor and cognitive abilities of mice indicated that α-synuclein protein received by AAV9-vcNS2B treatment... A53T The Parkinson's disease phenotype in mice was improved.

[0051] 3. AAV9-vcNS2B treatment improves α-synuclein at the molecular level. A53T Mouse Parkinson's disease phenotype Mouse brain tissue was collected and subjected to immunohistochemical and immunofluorescence staining experiments. Immunohistochemistry was used to detect the expression level of tyrosine hydroxylase (TH) in the striatum and substantia nigra of mouse brain tissue, and immunofluorescence was used to detect the expression level of phosphorylated α-synuclein in the substantia nigra. Figure 6 The results showed that AAV9-vcNS2B treatment protected α-synuclein. A53T Expression levels of TH in mouse striatum and substantia nigra ( Figure 6 AB); reduced the expression level of phosphorylated α-synuclein in the substantia nigra (AB); Figure 6 (C). Effectively improves α-synuclein at the molecular level. A53T Symptoms of Parkinson's disease in mice.

[0052] Example 3: vcNS2B wKI Therapeutic effects in MPTP-induced PD mouse model 1. vcNS2B wKI Reduced SNO-P4HB in the brain tissue of MPTP-induced Parkinson's disease mice Eight-week-old wild-type mice and vcNS2B were used wKI Mice were randomly divided into a control group and a model group. The model group was intraperitoneally injected with 20 mg / kg MPTP for 5 consecutive days, while the control group was injected with an equal volume of physiological saline. Figure 7 Nine days after the last injection, mouse brain tissue homogenates were collected to extract total protein. SNO-P4HB was prepared into Biotin-P4HB using a biotin exchange assay, and enriched using streptavidin magnetic beads. Western blotting was used to detect SNO-P4HB. The results showed that AAV9-vcNS2B treatment significantly reduced SNO-P4HB in MPTP-induced Parkinson's disease mice. Figure 7 B).

[0053] 2. VCNS2B treatment improves motor and cognitive abilities in MPTP-induced Parkinson's disease mice. Eight-week-old wild-type mice were randomly divided into a control group and a model group. The model group received intraperitoneal injections of MPTP at 20 mg / kg for 5 consecutive days, while the control group received an equal dose of saline. Nine days after the last injection, the mice underwent open field tests, wheel tests, and new / old object recognition tests to assess their motor and cognitive abilities. Figure 8 ). Treatment of α-synuclein with AAV9-vcNS2B A53TConsistent results were observed in mice; VCNS2B treatment significantly improved motor function in MPTP-induced Parkinson's disease mice in both the open field and wheel running tests. Figure 8 AC), and improved the mice's cognitive ability to distinguish between new and old things ( Figure 8 The results (D) indicate that VCNS2B treatment can effectively prevent the disease phenotype of Parkinson's disease in mice.

[0054] 3. VCNS2B treatment improves MPTP-induced Parkinson's disease symptoms in mice at the molecular level. Mouse brain tissue was collected and subjected to immunohistochemical and immunofluorescence staining experiments. Immunohistochemistry was used to detect the expression level of TH in the striatum of mouse brain tissue, and immunofluorescence was used to detect the expression level of phosphorylated α-synuclein in the substantia nigra of mice. Figure 9 The results showed that the expression level of TH in the striatum of Parkinson's disease mice treated with vcNS2B was significantly improved. Figure 9 A); and reduced the expression level of phosphorylated α-synuclein in the substantia nigra (A ...). Figure 9 (B). This indicates that vcNS2B effectively improves Parkinson's disease symptoms in mice at the molecular level.

[0055] Comparative Example: Verification of the Mechanism of Action of vcNS2B This comparative example demonstrates that the effect of vcNS2B depends on its specific binding to P4HB. The binding sites of vcNS2B to P4HB are K401 and K409, which are also the sites where vcNS2B affects C. 53 GHC 56 The basis of site S-nitrosylation is that if vcNS2B and P4HB are not bound, they cannot affect C. 53 GHC 56 S-nitrosylation at the site. This comparative example demonstrates that vcNS2B inhibits C by binding to P4HB K401 and K409 sites by examining the binding of vcNS2B to P4HB and the P4HB double mutant. 53 GHC 56 S-nitrosylation.

[0056] 1. Plasmid construction pcDNA3.1 plasmid: empty vector plasmid; P4HB-FL-Flag plasmid: a pcDNA3.1 plasmid containing a Flag tag and P4HB; P4HB-aTRX mut- Flag plasmid: A pcDNA3.1 plasmid containing a Flag tag and the P4HB mutant, wherein the P4HB mutant is a double-site mutation (aTRX) where C at position 53 is changed to S (C53S) and C at position 56 is changed to S (C56S). mut The amino acid sequence is shown in SEQ ID NO.4: P4HB-K401A-Flag plasmid: a pcDNA3.1 plasmid containing a Flag tag and a P4HB single mutant (amino acid sequence as shown in SEQ ID NO.5) with a K mutation at position 401 to A (K401A); P4HB-K409A-Flag plasmid: a pcDNA3.1 plasmid containing a Flag tag and a P4HB single mutant (amino acid sequence as shown in SEQ ID NO.6) with a K mutation at position 409 to A (K409A); P4HB-K401A / K409A-Flag plasmid: a pcDNA3.1 plasmid containing a Flag tag and a P4HB double mutant (amino acid sequence as shown in SEQ ID NO.7) with K mutation at position 401 being A and K mutation at position 409 being A (K409A); pLCDH plasmid: empty vector plasmid; vcNS2B plasmid: pLCDH plasmid containing vcNS2B.

[0057] 2. P4HB protein C 53 GHC 56 S-nitrosylation modification of the site We infected HT22 cells (a mouse hippocampal neuronal cell line highly sensitive to glutamate and viral infection-induced oxidative toxicity, and a classic in vitro model for studying oxidative stress injury and screening neuroprotective drugs) with LGTV to induce nitrification stress. Protein mass spectrometry analysis was performed 24 hours after viral infection. Results are as follows: Figure 10 As shown, the proteomic results indicate that P4HB protein C 53 GHC 56 The site undergoes S-nitrosylation modification ( Figure 10 ).

[0058] 3. vcNS2B inhibits S-nitrosylation modification of P4HB protein. circGFP plasmid: pLCDH plasmid containing green fluorescent protein (GFP).

[0059] circGFP and vcNS2B plasmids were transfected into HT22 cells, followed by LGTV infection (without LGTV virus as a negative control). After 24 hours of treatment, Western blotting was performed to detect S-nitrosylation of P4HB. Results are as follows: Figure 11 As shown in A, vcNS2B can inhibit the S-nitrosylation modification of P4HB protein caused by viral infection.

[0060] pLCDH or vcNS2B plasmids were transfected into HT22 cells, followed by stimulation with 5 mM glutamate (PBS was used as a control). After 24 hours of treatment, Western blotting was performed to detect S-nitrosylation of P4HB. The results are as follows: Figure 11 As shown in B, vcNS2B can inhibit glutamate-stimulated S-nitrosylation modification of P4HB protein.

[0061] The pcDNA3.1 plasmid, P4HB-FL-Flag plasmid, and P4HB-aTRX were used. mut The P4HB-K401A-Flag plasmid, P4HB-K401A / K409A-Flag plasmid, and P4HB-K401A / K409A-Flag plasmid were combined with either the pLCDH plasmid or the vcNS2B plasmid and transfected into HT22 cells. After stimulation with 5 mM glutamate and incubation for 24 hours, Western blotting was performed to detect S-nitrosylation of P4HB. Results are as follows: Figure 11 As shown in C, in P4HB-FL-Flag plasmid or P4HB-aTRX mut In the presence of the Flag plasmid, the content of S-nitrosylated P4HB decreased when the vcNS2B plasmid was included; however, after the K mutation at positions 401 and 409 of the P4HB protein, vcNS2B lost its function of inhibiting P4HB S-nitrosylation. Figure 11 (C).

[0062] P4HB-aTRX mut -Flag amino acid sequence (SEQ ID NO.4): MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWSGHSKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDELDYKDDDDK .

[0063] Amino acid sequence of P4HB-K401A-Flag (SEQ ID NO.5): MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCAQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDELDYKDDDDK .

[0064] Amino acid sequence of P4HB-K409A-Flag (SEQ ID NO. 6): MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDALGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDELDYKDDDDK .

[0065] Amino acid sequence of P4HB-K401A / K409A-Flag (SEQ ID NO.7): MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWL KKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHIL LFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKN VFVEFYAPWCGHCAQLAPIWDALGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDELDYKDDDDK .

[0066] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A viral circular RNA derived from tick-borne encephalitis virus, characterized in that, The nucleotide sequence of the viral circular RNA is shown in SEQ ID NO.1, or a sequence with at least 95% homology to SEQ ID NO.

1.

2. A recombinant vector containing the viral circular RNA of claim 1.

3. The use of the viral circular RNA of claim 1 or the recombinant vector of claim 2 in the preparation of Parkinson's disease treatment products.

4. The application according to claim 3, characterized in that, The Parkinson's disease treatment product is used to increase the expression level of tyrosine hydroxylase and reduce the phosphorylation level of α-synuclein.

5. The application according to claim 3, characterized in that, The viral circular RNA is delivered via adeno-associated virus vector, lentiviral expression vector, nanoliposome vector, or mammalian cell expression plasmid.

6. The application according to claim 5, characterized in that, The adeno-associated virus vector is selected from any one of adeno-associated virus vector 9, adeno-associated virus vector rh10, adeno-associated virus PHP.B / PHP.eB, adeno-associated virus CAP-B10, and adeno-associated virus HSC15 / 17.

7. The application according to claim 5, characterized in that, The mammalian cell expression plasmid includes elements that promote the circularization of viral circular RNA.

8. The application according to claim 7, characterized in that, The mammalian cell expression plasmid is selected from any one of puc57, pZW1, pcDNA3.1, pRc / CMV, and pUC19.

9. A pharmaceutical composition for the treatment of Parkinson's disease, characterized in that, The pharmaceutical composition comprises viral circular RNA with a nucleotide sequence as shown in SEQ ID NO.1, or a sequence with at least 95% homology to SEQ ID NO.

1.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition may also include a pharmaceutically acceptable carrier or excipient.

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