Construction method of Parkinson's animal model, gRNA of specific targeting Parkin gene and application of gRNA

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a construction method of a Parkinson's animal model, gRNA of a specific targeting Parkin gene and application of the gRNA. According to the application, the Parkin gene of a receptor animal is edited by injecting a gene editing system specifically aiming at the Parkin gene, and after virus expression is performed for two months, pathological characteristics, similar to important pathological characteristics in the brain of a PD patient, such as dopamine nerve cell degeneration death, dopamine synthesis reduction at a striatum part and a-synuclein significant aggregation can appear. Compared with a PINK1 gene knockout monkey model, the CRISPR / Cas9 technology has the advantages that the Parkin gene knockout animal model is highly matched with a PD patient in the aspects of gene mutation and selective dopamine nerve cell degeneration and death characteristics, and a more ideal important animal model is provided for a PD pathological mechanism and development of treatment targets.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering, and specifically relates to a method for constructing a Parkinson's disease animal model, a gRNA specifically targeting the Parkin gene, and its application. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Its main pathological features are degenerative lesions of dopaminergic neurons in the substantia nigra and striatum and Lewy bodies formed by the aggregation of misfolded a-synuclein. The clinical manifestations of PD patients are bradykinesia, muscle rigidity, resting tremor and gait disorders. The current treatments for Parkinson's disease can alleviate the condition to a certain extent, but cannot prevent the progressive degeneration and death of brain nerve cells, and there is still no cure.

[0003] Genetic gene mutations can cause Parkinson's disease. The typical pathological characteristics of Parkinson's disease are Lewy bodies formed by selective dopamine neuron death and a-synuclein aggregation. Among them, Parkin gene mutation is the number one pathogenic gene for recessive Parkinson's disease, with a high incidence rate. Parkin protein is a phosphorylation substrate of PINK1 kinase and functions as an E3 ubiquitin-protein ligase. Its mutation will cause juvenile Parkinson's disease due to gene function loss, and the premature onset age brings great pain to patients. Point mutations and large fragment deletions of the Parkin gene are present in PD patients. However, Parkin knockout mouse and pig models cannot effectively simulate the important pathological characteristics of progressive degeneration and death of neurons and significant aggregation of a-synuclein in the brains of Parkinson's patients, which greatly hinders the clinical translation value of these animal models.

[0004] Non-human primate models are closer to humans in terms of genetics, physiology, and brain function. Although in vitro studies have reported that PINK1 can phosphorylate and activate Parkin, in vivo experiments in small animals have not confirmed PINK1-mediated phosphorylation of Parkin. The inventors previously discovered that PINK1 protein is specifically expressed only in the primate brain and is essential for maintaining primate brain function (Yang et al. 2019, Cell Res; Yang et al. 2021, Protein Cell). It was further discovered that PINK1 kinase specifically expressed in the primate brain can activate Parkin by phosphorylating Parkin (Chen et al., 2024; Han et al., 2024). Although PINK1 gene knockout can cause primate neuronal cell death, PINK1 phosphorylates many substrates, and neuronal cell death caused by PINK1 deficiency does not occur selectively in dopamine neurons, and large fragment deletions of the PINK1 gene caused by CRISPR / Cas9 in animal models have not yet been reported in PD patients (Yang et al., 2019; 2022).

[0005] Therefore, the PINK1 gene knockout monkey model can be used to study the important physiological functions of the PINK1 gene and how it affects the functions of Parkin and other phosphorylation substrates, but it cannot simulate the important pathological characteristics of selective dopamine neuron death that occurs in PD with aging.

[0006] In summary, the existing Parkin knockout mouse and pig models are unable to effectively simulate the important pathological characteristics of Parkinson's disease, such as the progressive degeneration and death of neurons and the significant aggregation of a-synuclein in the brains of patients, which greatly limits the search for drugs and methods that can effectively treat PD. Summary of the invention

[0007] Based on this, an embodiment of the present application provides a method for constructing a Parkinson's disease animal model, a gRNA specifically targeting the Parkin gene, and its application.

[0008] On the one hand, the present application provides a method for constructing an animal model of Parkinson's disease, comprising: targeted knockout of the Parkin gene in the substantia nigra of a target animal to construct an animal model of Parkinson's disease.

[0009] In one embodiment, the Parkin gene is knocked out using CRISPR / Cas9 gene editing method.

[0010] In one embodiment, the method comprises co-injecting sgRNA targeting the second exon and the third exon of the Parkin gene of the target animal and CRISPR nuclease into the substantia nigra of the brain of the target animal;

[0011] The sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.2.

[0012] In one embodiment, the method comprises: packaging the sgRNA and CRISPR nuclease with a virus, and

[0013] The virus-packaged sgRNA and CRISPR nuclease are injected into the substantia nigra of the midbrain of the test animal to target and knock out the Parkin gene in the substantia nigra of the midbrain of the test animal.

[0014] In one embodiment, the molar ratio of the sgRNA to the CRISPR nuclease is 1:(1-4).

[0015] In one embodiment, the CRISPR nuclease is Cas9;

[0016] In one embodiment, the virus is an adenovirus;

[0017] In one embodiment, the injection conditions include 2 to 3 injection sites in the unilateral part of the substantia nigra of the subject brain, 5 μL injection volume at each site, and a virus titer of 1×10 13 GC / mL;

[0018] In one embodiment, the target animal is a non-human primate.

[0019] On the other hand, the present application provides a sgRNA that specifically targets the second exon and the third exon of the Parkin gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.2.

[0020] On the other hand, the present application provides a gene editing product, which includes the sgRNA specifically targeting the second exon and the third exon of the Parkin gene as described in claim 7 and the CRISPR nuclease.

[0021] On the other hand, the present application also provides the cells, tissues or organs of the Parkinson's disease animal model constructed by the method for constructing the Parkinson's disease animal model.

[0022] On the other hand, the present application also provides the use of the cells, tissues or organs in screening drugs for treating Parkinson's disease.

[0023] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0025] Figure 1 This is the AAV Parkin sgRNA plasmid map;

[0026] Figure 2 This is the RNA verification diagram of the in vitro synthesized cas9 protein and Parkin targeting sgRNA site;

[0027] Figure 3 The Parkin sgRNA recombinant vector virus can be efficiently expressed in the substantia nigra of the monkey brain, and the Parkin protein is effectively knocked down;

[0028] Figure 4 Parkin protein deficiency caused by Parkin sgRNA recombinant vector virus can cause neurodegeneration and death in the substantia nigra of monkeys of two different age groups, young and old;

[0029] Figure 5 Parkin protein deficiency caused by Parkin sgRNA recombinant vector virus can cause neurodegeneration and death in the substantia nigra of young monkeys and a significant reduction in dopamine synthesis in the striatum;

[0030] Figure 6 The loss of Parkin protein caused by the Parkin sgRNA recombinant vector virus can cause significant aggregation of endogenous phosphorylated a-synuclein in the substantia nigra of monkeys of two different age groups, young and old. DETAILED DESCRIPTION

[0031] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] the term

[0034] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0035] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0036] In the present application, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0037] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0038] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0039] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0040] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0041] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0042] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0043] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0044] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0045] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0046] The term "sgRNA" (small guide RNA) is a guide RNA (gRNA) that guides the insertion or deletion of uridine residues into the kinetoplastid during the RNA editing process. It is a small non-coding RNA that can pair with pre-mRNA.

[0047] The term "exon" Exon (expressed region) is a part of a eukaryotic gene. It is preserved after splicing and can be expressed as a protein during protein biosynthesis. Exon is the gene sequence that appears last in mature RNA, also known as the expressed sequence.

[0048] A nucleotide sequence that is present in both the initial transcript and the mature RNA molecule. The term exon also refers to the region of DNA that encodes the corresponding RNA exon. All exons together make up the genetic information that is manifested in proteins. Although exons only account for 1% to 2% of the human genome, 85% of known disease-causing mutations are contained in exons.

[0049] Through research, this application found that Parkin deficiency can cause degeneration and death of dopamine neurons in the substantia nigra of non-human primates, reduced dopamine synthesis in the striatum, and significant aggregation of endogenous a-synuclein to form Lewy bodies. It can well simulate the important pathological characteristics of Parkinson's patients in non-human primates and can be used for large-scale drug screening for the treatment of Parkinson's disease.

[0050] Since the existing Parkin knockout mouse and pig models cannot effectively simulate the important pathological characteristics of Parkinson's disease, such as the progressive degeneration and death of neurons and the significant aggregation of a-synuclein in the brains of patients, the use of non-human primates to establish an animal model that effectively simulates the important neurodegenerative pathologies of PD patients is of great significance for exploring the pathogenic mechanism of PD and developing therapeutic targets.

[0051] Based on this, the present application provides, on the one hand, a method for constructing an animal model of Parkinson's disease, comprising: targeted knockout of the Parkin gene in the substantia nigra of the target animal brain to construct an animal model of Parkinson's disease.

[0052] In a specific example, the Parkin gene is knocked out using a CRISPR / Cas9 gene editing method.

[0053] In a specific example, sgRNA targeting the second exon and the third exon of the Parkin gene of the target animal is co-injected with CRISPR nuclease into the substantia nigra of the target animal's brain;

[0054] The sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.2.

[0055] PARK2 T1: 5'-CTCCAGCCATGGTTTCCCAG-3' (SEQ ID NO.1)

[0056] PARK2 T2: 5'-CAAGAAATGAATGCAACTGG-3' (SEQ ID NO.2)

[0057] Specifically including: using viruses to package the sgRNA and CRISPR nuclease, and,

[0058] The virus-packaged sgRNA and CRISPR nuclease are injected into the substantia nigra of the midbrain of the test animal to knock out the Parkin gene in the substantia nigra of the midbrain of the test animal; the molar ratio of the sgRNA to the CRISPR nuclease is 1:(1-4).

[0059] For example, the molar ratio of the sgRNA or its recombinant expression vector to the CRISPR nuclease or its recombinant expression vector is 1:1, 1:2, 1:3, or 1:4.

[0060] Optionally, the CRISPR nuclease is Cas9.

[0061] Further optionally, the virus is an adenovirus, including an expression vector containing an adenovirus, and the expression vector of the adenovirus contains a fluorescent protein tag.

[0062] Optionally, the protein tag includes one or both of RFP and zsGreen.

[0063] In a specific example, the injection conditions include 2 to 3 injection sites in the unilateral part of the substantia nigra of the subject's brain, 5 μL injection volume at each site, and a virus titer of 1×10 13 GC / mL;

[0064] On the other hand, the present application provides a sgRNA that specifically targets the second exon and the third exon of the Parkin gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.2.

[0065] In one specific example, the target animal is a non-human primate, such as a monkey.

[0066] On the other hand, the present application provides a gene editing product, which includes the sgRNA specifically targeting the second exon and the third exon of the Parkin gene and the CRISPR nuclease.

[0067] It will be appreciated that other reagents related to CRISPR gene editing are also included.

[0068] The present application also provides the cells, tissues or organs of the Parkinson's disease animal model constructed by the method for constructing the Parkinson's disease animal model.

[0069] On the other hand, the present application provides the use of the cells, tissues or organs in screening drugs for treating Parkinson's disease.

[0070] The present application can effectively knock down Parkin protein expression through a single administration, can be injected in vivo with a single dose, requires fewer administrations, and can simulate important pathological features in the brain of Parkinson's patients within a short period of time after administration.

[0071] In a specific example, the CRISPR nuclease is Cas9. Cas9 is a nuclease that can be combined with a designed single guide RNA (sgRNA) to form a complex that can recognize and bind to the target DNA sequence and then cut the DNA at a specific location. This cutting can introduce the desired genetic changes through the cell's natural repair mechanisms (such as non-homologous end joining NHEJ or homology-directed repair HDR).

[0072] In a specific example, the CRISPR nuclease expression vector is an adenovirus vector carrying CRISPR nuclease;

[0073] In a specific example, the virus-packaged sgRNA or recombinant expression vector and CRISPR nucleic acid or its recombinant expression vector are simultaneously injected into the substantia nigra of the brain of the test animal.

[0074] On the other hand, the present application provides the method for constructing a Parkinson's disease animal model and the use of the Parkinson's disease animal model constructed by the method in screening drugs for treating Parkinson's disease.

[0075] In view of the fact that the existing Parkin knockout mouse and pig models cannot simulate the important pathological characteristics of neuronal cell degeneration and death and a-synuclein aggregation in the brain of PD patients, this application specifically designs sgRNA for exons 2 and 3 of the monkey Parkin gene, and edits the Parkin gene of the recipient monkey by injecting a gene editing system specifically targeting the Parkin gene into the substantia nigra region of the recipient monkey. After two months of viral expression, pathological characteristics similar to the important pathological characteristics in the brain of PD patients, such as dopamine neuronal cell degeneration and death, reduced dopamine synthesis in the striatum, and significant aggregation of a-synuclein, can appear. Compared with the PINK1 gene knockout monkey model using CRISPR / Cas9 technology, the Parkin gene knockout monkey model of this application is highly consistent with PD patients in terms of gene mutation and selective dopamine neuronal cell degeneration and death characteristics, providing a more ideal and important animal model for PD pathological mechanism and development of therapeutic targets.

[0076] Furthermore, the convenient, rapid, single-dose model construction method provided in the present application can provide an important model for Parkinson's disease drug screening, stem cell therapy, gene defect repair therapy, and pathogenic mechanism research of Parkin gene deletion, etc., and has great economic value and preclinical research significance.

[0077] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0078] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0079] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0080] Example 1

[0081] 1. Transformation of recombinant expression vector

[0082] The AAV2-U6 sgRNA backbone vector used in this example was purchased from Guangzhou Paizhen Biotechnology Co., Ltd. The vector carries an RFP fluorescent protein tag driven by the EFs promoter, and restriction endonucleases MluI and KpnI were purchased from NEB, USA.

[0083] Schematic diagram of constructing a viral vector for expressing the target plasmid Figure 1 , the specific process is as follows:

[0084] 1. Specific sgRNAs were designed for exons 2 and 3 of the monkey Parkin gene. Using the MluI and KpnI restriction sites on the AAV2-U6sgRNA-EFs-RFP vector, the vector carrying the MluI and KpnI restriction sequences and the ParkinsgRNA backbone sequence was connected to the vector to obtain the AAV-U6-Parkin sgRNA T1-U6-Parkin sgRNA T2-EFs-RFP vector.

[0085] 2. The Mecp2 promoter sequence was deleted using the Xba1 and AgeI restriction sites on the pAAV-pMecp2-SpCas9-spA (purchased from Addgene px551 plasmid # 60957) backbone plasmid, and the CMV promoter sequence carrying the Xba1 and AgeI restriction sequences was connected to achieve the expression of SpCas9 in various cells in the brain to obtain AAV-CMV-spCas9.

[0086] 3. The successful expression of the modified AAV plasmid vector was verified by sequencing.

[0087] 2. Verification of the effectiveness of the constructed plasmid and viral packaging in primary monkey glial cells

[0088] AAV2-U6 sgRNA-EFs-RFP and AAV-CMV-spCas9 were electroporated into primary cultured monkey glial cells, and the control group was transfected with a control plasmid. After 72 hours, samples were collected for Western blot verification, and the identification results were as follows Figure 2 shown.

[0089] The specific steps are as follows:

[0090] (1) Digest and count the P1 monkey glial cells according to 1×10 6 The cells / sample were distributed into centrifuge tubes and centrifuged at 300 × g for 5 minutes.

[0091] (2) Discard the supernatant, add 5 mL of sterile PBS solution to resuspend the cells, and centrifuge at 300 × g for 5 minutes.

[0092] (3) Discard the supernatant and carefully aspirate the remaining PBS with a pipette tip. Add 100 µL of electroporation solution (82 µL of cell-specific nuclear transfection solution and 18 µL of additives, prepared before use and brought to room temperature in advance; LONZA) to each sample and mix thoroughly. Then add 1 µg of the plasmid to be transfected and pipette evenly to distribute it in the electroporation solution and cells.

[0093] (4) Mix thoroughly with a pipette tip and suck the mixture into the electroporation cup. Select the electroporation program "A033" for electroporation.

[0094] (5) After electroporation, add 200 µL of serum-free culture medium preheated at 37°C to the electroporation cup and let it stand for 2 minutes.

[0095] (6) Transfer the cells to a PDL-coated 12-well plate and add complete culture medium (DMEM + 10% FBS). Culture the cells in a 37°C cell culture incubator and change the medium every other day. Collect the samples after 72 hours for relevant tests.

[0096] 3. Animal model construction and pathological and imaging tests

[0097] 1. Virus packaging

[0098] The modified and constructed viral vectors AAV-U6-Parkin sgRNA T1-U6-Parkin sgRNAT2-CMV-RFP (abbreviated as: AAV-Parkin sgRNA) and AAV-CMV-Cas9 were packaged. The specific steps are shown in the literature Yang et al., J Clin Invest, 2017 (doi: 10.1172 / JCI92087), and injected into the substantia nigra of the monkey brain by stereotaxic injection.

[0099] The AAV vector contains the tandem U6-Parkin sgRNA-T1- U6-Parkin sgRNA-T2- sequences:

[0100] PARK2 T1: 5'-CTCCAGCCATGGTTTCCCAG-3'

[0101] PARK2 T2: 5'-CAAGAAATGAATGCAACTGG-3'

[0102] 2. Virus injection

[0103] The specific injection scheme refers to the published paper (Yang et al., Protein Cell 2022), which specifically includes stereolocalization of the substantia nigra of the monkey brain through magnetic resonance imaging (MRI), and the parameters are based on the actual MRI measurement. AAV-Cas9: AAV-sgRNA-Parkin injection ratio of 4:1 is mixed, and then injected into the substantia nigra of the recipient monkey through positioning injection, a total of 15μL, of which the virus concentration is pre-adjusted to 10 12 vg / mL; the virus injected on the control side was AAV controlsgRNA-CMV-RFP / AAV Cas9. In order to avoid individual differences, the brain injection virus scheme in this example was: AAV control sgRNA-CMV-RFP / AAV Cas9 and AAV-sgRNA-Parkin / AAV Cas9 viruses were injected into the left and right sides of the same animal, respectively.

[0104] 3. Immunofluorescence staining and T7E1 detection

[0105] The T7E1 experimental steps in this example are as follows: The specific sequences of the primers are designed as shown in Table 1 below, exon 2 and exon 3 are amplified by PCR, 3 μL~5 μL of the PCR product is first annealed and the DNA double strands are re-paired, and the PCR program is as follows: 95℃, 10min; 85℃, 1min; 75℃, 1min; 65℃, 1min; 55℃, 1min; 45℃, 1min; 35℃, 1min; 25℃, 1min; 4℃, 10min. Then add T7E1 enzyme for enzyme digestion (refer to the instructions for specific operations), incubate at 37℃ for 45min, and the results are shown in the example. Figure 3 .

[0106] Table 1

[0107]

[0108] according to Figure 3 The results showed that monkeys injected with the virus for two months were anesthetized, perfused and euthanized to obtain samples for immunofluorescence staining and T7E1 detection. The substantia nigra (SN) could efficiently express the injected AAV virus, and the Parkin protein could be effectively targeted and knocked down. Figure 3 A in the figure is the use of stereotaxic technology to inject AAV9-Parkin gRNA-RFP / Cas9 to target the Parkin gene (PARK2) in the monkey brain area. The control group was injected with AAV-Cas9 and control gRNA-RFP at the same time; Figure 3B is a low-power microscope image showing that the RFP in the substantia nigra of the injected monkey reflects the successful infection of the substantia nigra by the virus. TH (tyrosinehydroxylase): tyrosine hydroxylase; Figure 3 C in the figure shows that Parkin expression is reduced in neurons infected with AAV9-Parkin gRNA-RFP / Cas9 in the substantia nigra (indicated by arrows) by immunofluorescence staining. Figure 3 D in the figure shows that T7E1 detection confirmed the mutation of the Parkin gene in the brain of the monkey injected with AAV9-Parkin gRNA / cas9.

[0109] Further staining analysis of the substantia nigra dopamine neuron marker TH showed that knocking out Parkin in the substantia nigra of the monkey brain can cause obvious degeneration and death of TH-positive neurons, which has been effectively verified in both young and old adult monkeys (e.g. Figure 4 as shown).

[0110] Figure 4 The immunofluorescence staining images in A~B show that the number of TH-positive neurons was reduced when Parkin was targeted in the hippocampus of monkeys aged 6 or 25 years old compared with the hippocampus injected with AAV Control-gRNA. The magnified image of the virus-infected nerve cells in the Control-gRNA group shows the virus infection of the nerve cells. Figure 4 A and B in the figure are representative immunofluorescence staining images of multiple technical replicates of at least three biological replicate samples; Figure 4 C in the figure is a statistical analysis of the number of TH-positive neurons in the substantia nigra region of monkeys injected with AAV-Cas9 / Control-gRNA and AAV-Cas9 / Parkin-gRNA. The experimental groups included 4 young monkeys aged 6-8 years and 3 old monkeys aged 25-28 years. The number of TH-positive cells in each image (20×) was recorded, and specific colors were used to indicate that the data came from specific monkeys. The average count of TH-positive cells in each animal was used for paired two-tailed t-test to obtain the P value. The error mean is ±SEM (6-8 years old, n=4; 25-28 years old, n=3); Figure 4 D in the figure shows the analysis of neuronal and mitochondrial proteins in the substantia nigra of 6-year-old control monkeys and Parkin-treated monkeys using Western blotting.

[0111] Figure 5 The electron microscopy results in A show that AAV9Parkin gRNA / Cas9 targeting Parkin caused degeneration of neurons (upper figure) and axons (lower figure) in the substantia nigra compared with AAV Control-gRNA injected into the substantia nigra of 6-year-old monkeys.

[0112] In addition, this application also uses immunoblotting experiments and electron microscopy experiments to prove that Parkin knockdown can lead to the degeneration and death of neurons in the substantia nigra of young adult monkeys (such as Figure 4 D and Figure 5 ).

[0113] 4. PET / CT

[0114] PET-CT scanning was performed at the PET / CT-MRI Center of the First Affiliated Hospital of Jinan University. The PET tracer [18F]DOPA was radiolabeled at the Center for Cyclometry and PET Radiopharmaceuticals (CCPR) of Jinan University. Briefly, animals were fasted for 12 h before tracer injection. The average dose of [18F]DOPA injected was 0.5 mCi / kg body weight (18.5 MBq / kg). Animals were placed in the imaging room and anesthetized with a 2.0% isoflurane / oxygen mixture throughout. During anesthesia, body temperature was maintained at 37°C and monitored. The head position was fixed with a stereotaxic frame. CT scanning was performed first, followed by 10 min of static positron emission data collection 60 min after injection. CT data were acquired in a breath-hold state using GE AutomA technology (GE Medical System, Milwaukee, USA) modulated at 140 kV, 230 mA, noise index of 30, slice thickness of 3.75 mm, inter-slice spacing of 3.27 mm, matrix size of 256 × 256, and scanning FOV of 70 cm. Data were analyzed using PMOD4.1 software (Pmod Technologies LLC, Zurich, Switzerland). [18F]DOPA uptake in the occipital cortex was used as a reference for SUVr analysis (Xiao Z, Acta Pharm Sin B. 2022). PET images were coregistered with individual MR images and then converted to brain template MR images. Bilateral striatum was analyzed, and regions of interest (VOIs) were drawn and resampled to PET space, and SUVs were extracted by applying Carimas software (Turku, Finland) and PMOD software (Zürich, Switzerland). SUVs were corrected for the monkeys’ initial injection dose and body weight. Normalization of PET imaging analysis was performed by SUVRR / L (SUV of right striatum / SUV of left striatum).

[0115] The important pathology of Parkinson's disease patients is the death of dopamine neurons in the substantia nigra, which will lead to reduced dopamine synthesis in the striatum and pathological a-synuclein aggregation. In this application, the PET / CT method was also used to detect that Parkin knockdown caused a significant reduction in dopamine synthesis in the striatum of brain-injected monkeys. Figure 5Figure 2 (B) shows representative DOPA PET / CT imaging showing dopamine reduction in the striatum two months after injection of AAVParkin gRNA / Cas9 into the ipsilateral substantia nigra of an 8-year-old adult monkey.

[0116] Immunohistochemistry detected that Parkin knockdown could cause obvious pathological aggregation of a-synuclein S129 protein in the substantia nigra of the monkey midbrain (such as Figure 6 ). Among them, Figure 6 The immunostaining images in A show increased pS129-α-syn staining in the substantia nigra region of young monkeys (9 years old) and old monkeys (22 years old) injected with AAV9-Parkin gRNA / Cas9 compared to Control-gRNA / Cas9 injected monkeys; Figure 6 B is a high-power micrograph showing pS129-α-syn accumulation (indicated by arrows) and aggregates in the cytoplasm of a 22-year-old monkey; Figure 6 Figure C shows the quantification of pS129-α-syn aggregates in the substantia nigra of young monkeys (7 and 9 years old) and old monkeys (22 and 25 years old). The average number of pS129-α-syn aggregates (40× or per 0.04 mm) was calculated using images of the injected substantia nigra area. 2 ). The number of clusters was recorded, and a specific color was used to indicate that the data came from a specific monkey; Figure 6 D in the figure shows the detection of pS129-a-syn aggregates by DAB staining of pS129-a-syn and immunofluorescence staining of NeuN. These aggregates are present both in the cell body (arrows) and outside the cell body or in neuronal processes that cannot be identified by staining. Representative immunostaining images are from multiple technical replicates of at least two monkey brains.

[0117] Therefore, the present application established a new PD model that can highly simulate the important pathological characteristics of PD patients by knocking out the Parkin gene. This is in sharp contrast to the previous reported Parkin knockout mouse and pig models that cannot simulate the neuronal degeneration and death and pathological a-synuclein aggregation of PD patients, and provides a new model for studying the pathogenic mechanism of PD and developing potential treatments.

[0118] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for constructing an animal model of Parkinson's disease, characterized in that: include: Targeted knockout of the Parkin gene in the substantia nigra of the target animal's brain was used to construct an animal model of Parkinson's disease.

2. The method for constructing an animal model of Parkinson's disease according to claim 1, characterized in that: The method comprises using CRISPR / Cas9 gene editing method to knock out the Parkin gene.

3. The method for constructing an animal model of Parkinson's disease according to claim 1, characterized in that: Including, co-injecting sgRNA targeting the second exon and the third exon of the Parkin gene of the target animal and CRISPR nuclease into the substantia nigra of the brain of the target animal; The sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.

2.

4. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that: include: using viruses to package the sgRNA and CRISPR nuclease, and The virus-packaged sgRNA and CRISPR nuclease are injected into the substantia nigra of the midbrain of the test animal to target and knock out the Parkin gene in the substantia nigra of the midbrain of the test animal.

5. The method for constructing an animal model of Parkinson's disease according to claim 4, characterized in that: The molar ratio of the sgRNA to the CRISPR nuclease is 1:(1-4).

6. The method for constructing an animal model of Parkinson's disease according to claim 5, characterized in that: The method includes one or more of the following features: (1) The CRISPR nuclease is Cas9; (2) The virus is an adenovirus; (3) Injection conditions include 2 to 3 injection sites in the unilateral substantia nigra of the test animal, 5 μL injection volume at each site, and a virus titer of 1×10 13 GC / mL; (4) The target animal is a non-human primate.

7. An sgRNA specifically targeting the second exon and the third exon of the Parkin gene, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.

2.

8. A gene editing product, characterized in that: The gene editing product includes the sgRNA specifically targeting the second exon and the third exon of the Parkin gene as described in claim 7 and the CRISPR nuclease.

9. The cells, tissues or organs of the Parkinson's disease animal model constructed by the method for constructing a Parkinson's disease animal model according to any one of claims 1 to 6.

10. Use of the cells, tissues or organs of the Parkinson's disease animal model according to claim 9 in screening drugs for treating Parkinson's disease.

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