Construction method of Parkinson's disease animal model, gRNA specifically targeting Parkin gene, and applications thereof

Abstract

This application belongs to the field of genetic engineering, and specifically relates to a method for constructing a Parkinson's animal model, a gRNA specifically targeting the Parkin gene, and its applications. By injecting a gene editing system specifically targeting the Parkin gene, the Parkin gene of the recipient animal is edited. After two months of viral expression, pathological features similar to those in the brains of PD patients, such as degeneration and death of dopamine neurons, reduced dopamine synthesis in the striatum, and significant aggregation of a-synuclein, can occur. Compared with the PINK1 gene knockout monkey model using the CRISPR / Cas9 technology, the Parkin gene knockout animal model highly coincides with PD patients in terms of gene mutations and selective degeneration and death characteristics of dopamine neurons, providing a more ideal and important animal model for the pathological mechanism of PD and the development of therapeutic targets.

Description

Technical Field

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

[0002] Parkinson's disease (hereinafter referred to as PD) is the second most common neurodegenerative disease after Alzheimer's disease. The main pathological feature is the degenerative lesion of dopaminergic neurons in the substantia nigra striatum and the formation of Lewy bodies by the aggregation of misfolded a-synuclein. The clinical manifestations of PD patients include bradykinesia, muscle rigidity, resting tremor, and gait disorders. At present, the treatment methods 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 curable method.

[0003] Genetic gene mutations can cause Parkinson's disease. The typical pathological feature of Parkinson's disease is the selective death of dopamine neurons and the formation of Lewy bodies by the aggregation of a-synuclein. Among them, the Parkin gene mutation is the leading pathogenic gene for recessive genetic Parkinson's disease, with a relatively high incidence. 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 the loss of gene function, and the premature onset age brings great pain to patients. Point mutations and large fragment deletions of the Parkin gene both exist in PD patients. However, neither the Parkin knockout mouse nor pig models can effectively simulate the important pathological features of progressive degeneration and death of nerve cells and significant aggregation of a-synuclein in the brains of Parkinson's patients, which greatly hinders the clinical translational value of these animal models.

[0004] Non-human primate monkey models are closer to humans in terms of genetics, physiology, and brain structure and 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 found that the PINK1 protein is specifically expressed only in the primate brain and is crucial for maintaining primate brain function (Yang et.al. 2019, Cell Res; Yang et.al. 2021, Protein Cell). Further studies have found that the PINK1 kinase specifically expressed in the primate brain can activate Parkin by phosphorylating Parkin (Chen et al.,2024; Han et al., 2024). Although knockout of the PINK1 gene can lead to the death of primate nerve cells, PINK1 phosphorylates many substrates, and the nerve cell death caused by PINK1 deficiency does not selectively occur in dopamine neurons, and large fragment deletions of the PINK1 gene caused by CRISPR / Cas9 in animal models have not 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 phosphorylated substrates, but it cannot mimic the important pathological feature of selective dopamine neuron death that occurs with aging in PD.

[0006] In summary, existing mouse and pig models with Parkin knockout cannot effectively mimic the important pathological features of progressive degeneration and death of nerve cells and significant aggregation of a-synuclein in the brains of Parkinson's 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 animal model, a gRNA specifically targeting the Parkin gene, and their applications.

[0008] One aspect of the present application provides a method for constructing a Parkinson's disease animal model, including: targeting and knocking out the Parkin gene in the substantia nigra of the target animal's brain to construct a Parkinson's disease animal model.

[0009] In one embodiment, it includes knocking out the Parkin gene by using the CRISPR / Cas9 gene editing method.

[0010] In one embodiment, it includes co-injecting an sgRNA targeting the second exon and the third exon of the Parkin gene of the target animal and a CRISPR nuclease into the substantia nigra region of the target animal's brain;

[0011] The sequences of the sgRNAs are shown in SEQ ID NO.1 to SEQ ID NO.2.

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

[0013] injecting the virus-packaged sgRNA and CRISPR nuclease into the substantia nigra of the test animal to targetedly knockout the Parkin gene in the substantia nigra 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 that the number of injection sites in the unilateral part of the substantia nigra of the test animal is 2 - 3, the injection volume at each site is 5 μL, and the virus titer is 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 an 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, and the gene editing product includes the sgRNA that specifically targets the second exon and the third exon of the Parkin gene as claimed in claim 7 and the CRISPR nuclease.

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

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

[0023] Details of one or more embodiments of the present application are set forth in the following description, and other features, objects and advantages of the present application will become apparent from the specification and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the technical solutions in the embodiments of the present application and to more comprehensively understand the present application and its beneficial effects, the following provides a brief introduction to the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

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

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

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

[0028] Figure 4 It is that the Parkin protein deficiency caused by the Parkin sgRNA recombinant vector virus can cause neurodegenerative death in the substantia nigra of the monkey brain at two different age groups, young and old;

[0029] Figure 5 It is that the Parkin protein deficiency caused by the Parkin sgRNA recombinant vector virus can cause neurodegenerative death in the substantia nigra of the young monkey brain and significantly reduce dopamine synthesis in the striatum;

[0030] Figure 6 It is that the Parkin protein deficiency caused by the Parkin sgRNA recombinant vector virus can cause significant aggregation of endogenous phosphorylated a-synuclein in the substantia nigra of the monkey brain at two different age groups, young and old. Detailed implementation manners

[0031] The following further elaborates on the present application in combination with the implementation manners and embodiments. It should be understood that these implementation manners and embodiments are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and embodiments described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more comprehensive understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by one of ordinary skill in the technical field to which this application pertains.

[0033] The term

[0034] Unless otherwise stated or in case of contradiction, the terms or phrases used herein have the following meanings:

[0035] As used herein, the selection range of the terms “and / or”, “or / and”, “and / or” includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from “and / or”, “or / and”, “and / or” are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by “logical AND” and also undoubtedly includes the technical solution connected by “logical OR”. For example, “A and / or B” includes three parallel solutions: A, B, and A + B. Another example is the technical solution of “A, and / or, B, and / or, C, and / or, D”, which includes any one of A, B, C, D (i.e., the technical solution connected by “logical OR”), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the four-item combination of A, B, C, D (i.e., the technical solution connected by “logical AND”).

[0036] In this application, when it comes to “multiple”, “multiple types”, “multiple times”, “multiple elements”, etc., unless otherwise specified, it means greater than 2 or equal to 2 in quantity. For example, “one or more types” means one type or greater than or equal to two types.

[0037] As used herein, “their combinations”, “any combination thereof”, “any combination mode thereof”, etc. include all suitable combination modes of any two or any two or more of the listed items.

[0038] In this article, the “suitable” in “suitable combination mode”, “suitable mode”, “any suitable mode”, etc. is subject to being able 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 this application, “further”, “even further”, “especially”, etc. are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the protection scope of this application.

[0040] In this application, "optionally", "optional", and "option" mean that something is either present or absent, that is, it refers to either one of two alternative scenarios: "present" or "absent". If the term "optional" appears multiple times in a technical solution, and there is no special explanation, no contradiction, or mutual restraint relationship, then each "optional" is independent of the others.

[0041] In this application, for technical features described in an open-ended manner, it includes both a closed technical solution composed of the listed features and an open technical solution that includes the listed features.

[0042] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints of this numerical range (i.e., the minimum value and the maximum value), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of this numerical range, as well as every integer between the two endpoints. In this document, it is equivalent to directly listing each integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the set of integers 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 in this document should be understood to include any and all sub-ranges subsumed therein.

[0043] For the temperature parameter in this application, unless otherwise specifically defined, it allows for both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0044] In this application, both %(w / w) and wt% represent weight percentages, %(v / v) represents volume percentage, and %(w / v) represents mass-volume percentage.

[0045] All documents mentioned in this application are cited as references in this application, as if each document was cited separately as a reference. Unless it conflicts with the inventive purpose and / or technical solution of this application, the cited documents related to this application are cited in their entirety and for all purposes. When this application involves citing documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited together. When this application involves citing documents, examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be appropriately amended according to the description in this application.

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

[0047] The term "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. An exon is the gene sequence that finally appears in mature RNA, also known as the expressed sequence.

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

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

[0050] Since existing Parkin knockout mouse and pig models cannot effectively simulate the important pathological features of progressive degeneration and death of nerve cells and significant aggregation of a-synuclein in the brains of Parkinson's disease patients, establishing an animal model that can effectively simulate the important neurodegenerative pathology of PD patients using non-human primates is of great significance for exploring the pathogenic mechanism of PD and developing therapeutic targets.

[0051] Based on this, on the one hand, this application provides a method for constructing an animal model of Parkinson's disease, including: targeting and knocking out the Parkin gene in the substantia nigra of the target animal to construct an animal model of Parkinson's disease.

[0052] In a specific example, it includes knocking out the Parkin gene by using the CRISPR / Cas9 gene editing method.

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

[0054] The sequences of the sgRNAs are 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, it includes packaging the sgRNA and the CRISPR nuclease with a virus, and

[0058] injecting the virus-packaged sgRNA and CRISPR nuclease into the substantia nigra of the test animal to targetedly knockout the Parkin gene in the substantia nigra 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 the 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 that the number of injection sites in the unilateral part of the substantia nigra of the test animal is 2 - 3, the injection volume at each site is 5 μL, and the virus titer is 1×10 13 GC / mL;

[0064] On the other hand, the present application provides an 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 a 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 described above.

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

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

[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 efficiently knockdown Parkin protein expression by single administration. In vivo, it can be administered by single injection with fewer administration times, and important pathological features in the brains of Parkinson's disease patients can be simulated shortly after administration.

[0071] In a specific example, the CRISPR nuclease is Cas9. Cas9 is a nuclease that can bind to a designed single-guide RNA (sgRNA) to form a complex. This complex can recognize and bind to the target DNA sequence and then cut the DNA at a specific position. This cleavage can introduce the desired gene alterations through the natural repair mechanisms of the cell (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 the CRISPR nuclease;

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

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

[0075] Since existing mouse and pig models with Parkin knockout cannot simulate the important pathological features of neurodegeneration and death of nerve cells and aggregation of a-synuclein in the brains of PD patients, in this application, sgRNAs are specifically designed for exons 2 and 3 of the monkey Parkin gene. By injecting a gene editing system specific for the Parkin gene into the substantia nigra region of recipient monkeys, the Parkin gene of the recipient monkeys is edited. After two months of viral expression, pathological features similar to those of PD patients, such as degeneration and death of dopamine neurons, reduced dopamine synthesis in the striatum, and significant aggregation of a-synuclein, can occur. Compared with the PINK1 gene knockout monkey model using the CRISPR / Cas9 technology in this application, the Parkin gene knockout monkey model highly matches PD patients in terms of gene mutations and selective degeneration and death characteristics of dopamine neurons, providing a more ideal and important animal model for the pathological mechanism of PD and the development of therapeutic targets.

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

[0077] The implementation scheme of this application will be described in detail below in combination with examples. It should be understood that these examples are only used to illustrate this application and not to limit the scope of this application. For the experimental methods without specific conditions noted in the following examples, priority should be given to the guidance given in this application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0078] In the following specific examples, for the measurement parameters of raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

[0080] Example 1

[0081] I. Modification of the recombinant expression vector

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

[0083] The schematic diagram of the viral vector for constructing the expression target plasmid is as shown in Figure 1 , and 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 enzyme sites on the AAV2-U6sgRNA-EFs-RFP vector, the sequence carrying the MluI and KpnI restriction enzyme sequences and the Parkin sgRNA backbone sequence was ligated into 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 enzyme 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 enzyme sequences was ligated, thereby enabling the expression of SpCas9 in various cells in the brain to obtain AAV-CMV-spCas9.

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

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

[0088] AAV2-U6 sgRNA-EFs-RFP and AAV-CMV-spCas9 were transfected into primary cultured monkey glial cells by electroporation, and the control plasmid was transfected into the control group. Samples were collected after 72 hours for Western blot verification, and the identification results are as shown in Figure 2 .

[0089] The specific steps are as follows:

[0090] (1) Digest and count the P1 generation of monkey glial cells, and distribute them into centrifuge tubes at 1×10 6 cells / sample, and centrifuge 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, carefully aspirate the remaining PBS with a pipette tip, add 100 µL of electroporation solution (82 µL of cell-specific nucleofection solution and 18 µL of additive, freshly prepared and pre-warmed to room temperature; LONZA) to each sample, mix well, then add 1 µg of the plasmid to be transfected and pipette to mix evenly so that it is fully distributed in the electroporation solution and the cells.

[0093] (4)After thoroughly mixing with a pipette tip, aspirate the mixture into the electroporation cuvette and select the electroporation program “A033” for electroporation.

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

[0095] (6)Transfer to a 12-well plate coated with PDL, supplement with complete medium (DMEM + 10% FBS), culture in a cell incubator at 37 °C, change the medium every other day, and harvest the samples for relevant assays 72 h later.

[0096] III. Establishment of animal models and pathological and imaging examinations

[0097] 1. Virus packaging

[0098] Virus packaging was performed on the above-mentioned modified and constructed virus vectors AAV-U6-Parkin sgRNA T1-U6-Parkin sgRNA T2-CMV-RFP (abbreviation: AAV-Parkin sgRNA) and AAV-CMV-Cas9. The specific steps are described in the literature Yang et.al., J Clin Invest, 2017 (doi:10.1172 / JCI92087), and they were injected into the substantia nigra of the monkey brain by stereotactic injection.

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

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

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

[0102] 2. Virus injection

[0103] The specific injection protocol refers to the published paper (Yang et al., Protein Cell 2022), which specifically includes stereotactic localization of the substantia nigra region of the monkey brain through magnetic resonance imaging (MRI) scans, and the parameters are subject to the actual MRI measurements. Mix AAV-Cas9 and AAV-sgRNA-Parkin in a ratio of 4:1, and then inject 15 μL into the substantia nigra of the recipient monkey through stereotactic injection. Among them, the virus concentrations are all pre-adjusted to 10 12 vg / mL; the virus injected into the control side is AAV controlsgRNA-CMV-RFP / AAV Cas9. To avoid individual differences, the brain injection virus protocol in this example is: inject AAV control sgRNA-CMV-RFP / AAV Cas9 and AAV-sgRNA-Parkin / AAV Cas9 viruses into the left and right sides of the same animal respectively.

[0104] 3. Immunofluorescence staining and T7E1 detection

[0105] The experimental steps of T7E1 in this example are as follows: The specific primer sequences are shown in Table 1 below. Amplify exon 2 and exon 3 by PCR. Take 3 μL - 5 μL of the PCR product and first anneal to re-bind and pair the DNA double strands. The PCR program is as follows: 95°C, 10 min; 85°C, 1 min; 75°C, 1 min; 65°C, 1 min; 55°C, 1 min; 45°C, 1 min; 35°C, 1 min; 25°C, 1 min; 4°C, 10 min. Subsequently, add T7E1 enzyme for enzymatic digestion (the specific operation refers to the instruction manual), incubate at 37°C for 45 min, and the result examples are shown in Figure 3 .

[0106] Table 1

[0107]

[0108] According to Figure 3 the results, after anesthetizing, perfusing, euthanizing, and sampling the monkeys injected with the virus two months later for immunofluorescence staining and T7E1 detection, the substantia nigra (SN) can highly express the injected AAV virus, and the Parkin protein can be effectively targeted and knocked down. Figure 3 A in Figure 3In B, the low-magnification microscope image shows that the RFP injected into the substantia nigra region of the monkey reflects successful infection of the substantia nigra region by the virus. TH (tyrosine hydroxylase): tyrosine hydroxylase; Figure 3 In C, immunofluorescence staining shows that Parkin expression is reduced in neurons infected with AAV9-Parkin gRNA-RFP / Cas9 in the substantia nigra region (as indicated by the arrows); Figure 3 In D, T7E1 detection confirmed mutations in the Parkin gene in the brains of monkeys 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 caused obvious degeneration and death of TH-positive neurons, which was effectively verified in both young and old adult monkeys (as Figure 4 shown).

[0110] Figure 4 Images of immunofluorescence staining in A–B show that targeting Parkin in the hippocampus of 6-year-old or 25-year-old monkeys reduced the number of TH-positive neurons compared with the hippocampus injected with AAV Control-gRNA. Magnified images of virus-infected neurons in the Control-gRNA group show the virus infection of neurons. Figure 4 A and B are representative immunofluorescence staining images of multiple technical replicates of at least three biological replicate samples; Figure 4 In C, 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 group 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 the data from specific monkeys were represented in specific colors. Paired two-tailed t-tests were performed using the average count of TH-positive cells in each animal to obtain the P value. Error mean was ±SEM (6–8 years, n = 4; 25–28 years, n = 3); Figure 4 In D, Western blotting was used to analyze neuronal and mitochondrial proteins in the substantia nigra of 6-year-old control monkeys and Parkin-targeted monkeys.

[0111] Figure 5 In A, electron microscopy results showed that targeting Parkin with AAV9 Parkin gRNA / Cas9 caused degeneration of neurons (upper panel) and axons (lower panel) 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 similarly prove that Parkin knockdown can lead to the degeneration and death of nerve cells in the substantia nigra of the brains of young adult monkeys (as shown by D in Figure 4 and A in Figure 5 ).

[0113] 4. PET / CT Detection

[0114] PET-CT scans were performed at the PET / CT-MRI Center of the First Affiliated Hospital of Jinan University. The PET tracer [18F]DOPA was radiolabeled at the Cyclotron and PET Radiopharmaceutical Center (CCPR) of Jinan University. Briefly, the animals were fasted for 12 hours before tracer injection. The average dose of [18F]DOPA injected was 0.5 mCi / kg body weight (18.5 MBq / kg). The animals were placed in the imaging room and anesthetized with a 2.0% isoflurane / oxygen mixture throughout the process. During anesthesia, the body temperature was maintained at 37°C and monitored. The head position was fixed with a stereotaxic frame. First, CT scans were performed, and then static positron emission data collection was carried out for 10 minutes 60 minutes after injection. CT data were acquired in a breath-hold state, modulated using GE AutomA technology (GE Medical System, Milwaukee, USA), at 140 kV, 230 mA, with a noise index of 30, a slice thickness of 3.75 mm, a slice interval of 3.27 mm, a matrix size of 256×256, and a scan FOV of 70 cm. Data analysis was performed using PMOD 4.1 software (Pmod Technologies LLC, Zurich, Switzerland). The uptake of [18F]DOPA in the occipital cortex was used as a reference for SUVr analysis (Xiao Z, Acta Pharm Sin B. 2022). The PET images were registered with individual MR images and then converted to a brain template MR image. The bilateral striatum was analyzed, regions of interest (VOIs) were drawn, and they were resampled to the PET space, and SUVs were extracted by applying Carimas software (Turku, Finland) and PMOD software (Zurich, Switzerland). SUVs were corrected by the initial injection dose and body weight of the monkeys. Standardization of PET imaging analysis was performed by SUVRR / L (SUV of the right striatum / SUV of the left striatum).

[0115] An important pathology in Parkinson's disease patients is that the death of dopamine neurons in the substantia nigra causes a decrease in dopamine synthesis in the striatum and the aggregation of pathological a-synuclein. In this application, the PET / CT method was also used to detect that Parkin knockdown caused a significant decrease in dopamine synthesis in the striatum of brain-injected monkeys, as shown in Figure 5As shown in B of , representative DOPA PET / CT imaging showed that two months after injection of AAVParkin gRNA / Cas9 into the ipsilateral substantia nigra of 8-year-old adult monkeys, dopamine in the striatum decreased.

[0116] Using immunohistochemistry, it was detected that Parkin knockdown could cause obvious pathological aggregation of a-synuclein S129 protein in the substantia nigra of the monkey midbrain (as Figure 6 shown). Among them, Figure 6 The immunostaining image in A of showed that compared with the Control-gRNA / Cas9-injected monkeys, the staining of pS129-α-syn increased in the substantia nigra region of young monkeys (9 years old) and old monkeys (22 years old) injected with AAV9-Parkin gRNA / Cas9; Figure 6 B in is a high-power micrograph showing the accumulation (arrow indicates) and aggregates of pS129-α-syn in the cytoplasm of 22-year-old monkeys; Figure 6 C in is the quantification of the number 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 was statistically analyzed using the images of the injected substantia nigra region (40× or every 0.04 mm 2 ). The number of aggregates was recorded, and specific colors were used to represent that the data came from a certain specific monkey; Figure 6 D in detected pS129-a-syn aggregates using DAB staining of pS129-a-syn and immunofluorescence staining of NeuN. These aggregates were present both in the cell bodies (arrows) and outside the cell bodies or in neuronal processes that could not be recognized by staining. Among them, the representative immunostaining images were from multiple technical replicates of at least two monkey brains.

[0117] Therefore, in this application, a new PD model that can highly simulate the important pathological features of PD patients was established by knocking out the Parkin gene, which is in sharp contrast to the current situation that the previously reported Parkin knockout mouse and pig models cannot simulate the degeneration and death of nerve cells and pathological a-synuclein aggregation in PD patients, providing a new model for studying the pathogenic mechanism of PD and developing potential treatment methods.

[0118] The above-described embodiments merely represent several implementation manners of the present application, facilitating the specific and detailed understanding of the technical solution of the present application. However, it should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art 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 those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solution provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing an animal model of Parkinson's disease, characterized in that, Comprising: Co-injecting the sgRNA targeting the second exon and the third exon of the Parkin gene of the target animal and the CRISPR nuclease into the substantia nigra of the target animal; constructing an animal model of Parkinson's disease; The sequences of the sgRNA are as shown in SEQ ID NO.1~SEQ ID NO.2; The target animal is a non-human primate.

2. The method for constructing an animal model of Parkinson's disease according to claim 1, characterized in that, Comprising: Packaging the sgRNA and the CRISPR nuclease with a virus, and Inject the packaged sgRNA and CRISPR nuclease into the substantia nigra of the test animal, and targetedly knock out the Parkin gene in the substantia nigra of the test animal.

3. The method for constructing an animal model of Parkinson's disease according to claim 1, wherein The molar ratio of the sgRNA to the CRISPR nuclease is 1:(1~4).

4. The method for constructing an animal model of Parkinson's disease according to claim 1, wherein The CRISPR nuclease is Cas9.

5. The method for constructing an animal model of Parkinson's disease according to claim 2, wherein, The virus is an adenovirus.

6. The method for constructing an animal model of Parkinson's disease according to claim 2, characterized in that, The injection conditions include that the number of injection sites in the unilateral part of the substantia nigra of the test animals is 2 to 3, the injection volume at each site is 5 μL, and the virus titer is 1×10 13 GC / mL.

7. Use of the cells, tissues or organs of the Parkinson's animal model constructed by the method for constructing an animal model of Parkinson's disease according to any one of claims 1~6 in screening drugs for treating Parkinson's disease.

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