Construction method of non-human primate parkinson's disease animal model and application thereof

Abstract

The application belongs to the field of animal model construction, and particularly relates to a construction method of a non-human primate Parkinson's disease animal model and application thereof. The application combines PINK1 knockdown and A53T alpha-synuclein overexpression of two pathogenic factors, can simultaneously induce dopamine neuron death and Lewy body formation in the brain of a young adult monkey in a short time of 4 weeks to 3 months, and significantly shortens the model construction period.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction, specifically relating to a method for constructing a non-human primate Parkinson's disease animal model and its application. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by the progressive death of dopaminergic neurons in the substantia nigra of the midbrain and Lewy bodies formed by the aggregation of pathological α-synuclein. Currently, there is no cure for PD, and research into its pathogenesis and the development of new drugs are severely limited by the lack of animal models that can simultaneously mimic these two core pathological features.

[0003] The existing technology has the following drawbacks:

[0004] Neurotoxin models (such as MPTP and 6-OHDA): Although they can rapidly cause damage to dopamine neurons, they cannot form pathological α-synuclein aggregates and Lewy bodies, which is inconsistent with the pathological characteristics of the vast majority of PD patients.

[0005] Genetically modified rodent models (mice, rats): Due to species differences, most mouse models with knockout of PD-related genes (such as PINK1, Parkin) cannot reproduce the typical dopamine neuron death and Lewy body pathology in PD patients.

[0006] Single-gene modified non-human primate models: The applicant's previous research has successfully established monkey models with PINK1 knockdown or α-synuclein (A53T) overexpression. PINK1 knockdown leads to dopamine neuron death, but Lewy body formation is slow (senescence-dependent); while α-synuclein (A53T) overexpression leads to the accumulation of pathological proteins, but the degree of neuronal death is relatively mild. Currently, no model can rapidly and stably simulate both neuronal death and Lewy body formation—the two core pathologies.

[0007] Therefore, there is an urgent need in this field to develop a novel animal model that can rapidly and stably simulate key pathological features of PD patients (dopamine neuron death and α-synuclein aggregation) for pathogenesis research and therapeutic drug evaluation. Summary of the Invention

[0008] To address the above problems, this invention provides a method for constructing an animal model, comprising the following steps:

[0009] (1) Provide non-human primates;

[0010] (2) Construction and packaging of recombinant viral vectors:

[0011] a. Construct the first recombinant viral vector that targets and knocks down the PINK1 gene;

[0012] b. Construct a second recombinant viral vector overexpressing mutant α-synuclein;

[0013] (3) Stereoscopic injection: The mixture of the first recombinant viral vector and the second recombinant viral vector is injected into the substantia nigra region of the midbrain on one side of the non-human primate using stereoscopic injection technology.

[0014] (4) Feeding and identification: After feeding the animals for 2-6 months, the animal models that successfully simulate the core pathology of PD were screened and confirmed through behavioral assessment, imaging examination and pathological examination after sampling.

[0015] Furthermore, the non-human primate is a cynomolgus monkey or rhesus monkey, an adult monkey aged 5-8 years.

[0016] Furthermore, the first recombinant viral vector that targets and knocks down the PINK1 gene is an AAV virus containing PINK1 sgRNA and Cas9.

[0017] Furthermore, the mutant α-synuclein is a human A53T mutant.

[0018] The present invention also provides an animal model prepared by the above method.

[0019] The present invention also provides the application of the above-mentioned animal model as an animal model for simulating key pathological features of PD patients.

[0020] The present invention has the following beneficial effects:

[0021] Rapid modeling: By combining PINK1 knockdown with A53T α-synuclein overexpression, two pathogenic factors, dopamine neuron death and Lewy body formation can be induced simultaneously in the brains of young adult monkeys in a relatively short period of 4 weeks to 3 months, significantly shortening the model construction cycle.

[0022] More comprehensive pathological simulation: The model of this invention can simultaneously reproduce the two core pathological features of PD patients—degenerative death of dopaminergic neurons and Lewy bodies formed by the aggregation of pathological α-synuclein, overcoming the deficiency of existing models with only one pathological feature.

[0023] Closer to clinical phenotype: The animal model exhibits progressive motor disorders (such as gait abnormalities and limb rigidity) and imaging features (PET / CT showing reduced dopamine synthesis) that are highly consistent with clinical PD patients, providing a more reliable indicator for drug efficacy evaluation.

[0024] Species-specific advantage: By constructing models using non-human primates that are more closely related to human genetics and physiology, the specific functions of PD pathogenic genes in the primate brain can be accurately reflected, and the research results obtained have higher clinical translational value.

[0025] These advantages primarily stem from the key technical aspect of this invention—combining PINK1 loss of function with the toxicity of A53T-mutated α-synuclein, and acting on the substantia nigra of the midbrain in non-human primates. PINK1 loss initially impairs mitochondrial function and the autophagy pathway, weakening the neurons' ability to clear pathological proteins; while A53T overexpression exacerbates the accumulation tendency of α-synuclein. The synergistic effect of both accelerates and aggravates the pathological process, thereby rapidly reproducing the complete pathological spectrum of PD. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Viral vector was injected into the substantia nigra of the brain of adult monkeys.

[0028] Figure 2 Monkeys 1 and 2 underwent AAV virus injection to knock down PINK1 or add A53T α-synuclein overexpression into the left substantia nigra, while the right substantia nigra served as a control. Four weeks after injection, FDOPA PET / CT showed that A53T overexpression and PINK1 knockdown significantly reduced dopamine synthesis in the ipsilateral striatum.

[0029] Figure 3 Monkey 2 remained active 2 weeks after viral injection, but developed motor impairment 4 weeks later, especially difficulty feeding with its right hand. This suggests that the neuronal damage caused by the overexpression of A53T and knockdown of PINK1 in the substantia nigra of the left brain resulted in motor impairment of the right limb innervated by the substantia nigra.

[0030] Figure 4Immunofluorescence staining analysis of monkey brain substantia nigra tissue showed that both AAV virus knockdown of PINK1 and A53T overexpression could reduce TH positive cells, and PINK1 KD combined with A53T overexpression caused more severe TH neuronal death.

[0031] Figure 5 Immunohistochemical staining analysis of monkey brain substantia nigra tissue showed that both AAV virus knockdown of PINK1 group and A53T overexpression group increased α-synuclein and phosphorylated α-synuclein S129 aggregates, and the PINK1KD plus A53T overexpression group produced significantly more α-synuclein S129 aggregates. Detailed Implementation

[0032] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1 (Construction of a PINK1 KD / A53T OE double-gene mutant PD monkey model)

[0038] Animals: Six adult crab-eating macaques aged 5-8 years were selected, half male and half female.

[0039] Virus construction:

[0040] AAV9-PINK1 gRNA / Cas9 virus was constructed to knock down PINK1.

[0041] AAV-α-synuclein (A53T) virus was constructed for overexpression of mutant α-synuclein.

[0042] Injection method:

[0043] Stereoscopic localization of the monkey brain was performed using MRI.

[0044] To reduce individual variability, a self-control design was used: AAV9-PINK1 gRNA / Cas9 + AAV-GFP was injected into one side of the substantia nigra (control group); AAV9-PINK1 gRNA / Cas9 + AAV-α-syn A53T was injected into the contralateral substantia nigra (experimental group) (see [link to experimental design]). Figure 1 ).

[0045] Effectiveness evaluation:

[0046] Behavioral studies: Four weeks after injection, the experimental group experienced motor dysfunction and difficulty feeding in the contralateral limbs (e.g., the right hand) (see [link to relevant documentation]). Figure 3 ).

[0047] Imaging: 4 weeks post-injection,¹ 8 F-FDOPA PET / CT showed that, compared with the control group, the experimental group showed a significant reduction in dopamine synthesis signals in the striatum (see [link to PET / CT]). Figure 2 ).

[0048] Pathology: Immunohistochemistry of samples taken 3-6 months after injection showed that the number of TH⁺ dopaminergic neurons in the substantia nigra region of the experimental group was significantly reduced compared with that of the control group (see...) Figure 4 ), and the typical Lewy body structure of p-S129-α-synuclein⁺ appears (see Figure 5 ).

[0049] Comparative Example 1 (Single-gene model vs. the two-gene model of this invention):

[0050] Model A: AAV-PINK1 KD virus was injected only into the substantia nigra of the monkey brain. Although this model could lead to the death of dopamine neurons, Lewy bodies formed slowly in young adult monkeys, usually taking months or even depending on aging.

[0051] Model B: AAV-α-syn A53T virus was injected only into the substantia nigra of the monkey brain. This model can form pathological α-synuclein accumulation, but its toxicity to dopamine neurons is relatively weak.

[0052] Conclusion: Compared with single-gene models A or B, the PINK1 KD / A53T OE dual-gene model constructed in this invention achieves significant dopamine neuron death and typical Lewy body formation in a shorter time (4 weeks to 3 months), with a phenotype that is closer to the end-stage pathological state of human PD patients and better stability.

[0053] Comparative Example 2 (Rodent Model in the Prior Art):

[0054] In mouse models with PINK1 or Parkin gene knockout, the characteristic dopamine neuron death and Lewy body pathology of PD patients could not be observed.

[0055] Conclusion: The non-human primate model constructed in this invention overcomes the problem of pathological simulation failure caused by species differences in rodent models, and has irreplaceable value.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing an animal model, characterized in that, Includes the following steps: (1) Provide non-human primates; (2) Construction and packaging of recombinant viral vectors: a. Construct the first recombinant viral vector that targets and knocks down the PINK1 gene; b. Construct a second recombinant viral vector overexpressing mutant α-synuclein; (3) Stereoscopic injection: The mixture of the first recombinant viral vector and the second recombinant viral vector is injected into the substantia nigra region of the midbrain on one side of the non-human primate using stereoscopic injection technology. (4) Feeding and identification: After feeding the animals for 2-6 months, the animal models that successfully simulate the core pathology of PD were screened and confirmed through behavioral assessment, imaging examination and pathological examination after sampling.

2. The method according to claim 1, characterized in that, The non-human primates mentioned are cynomolgus monkeys or rhesus monkeys, adult monkeys aged 5-8 years.

3. The method according to claim 1, characterized in that, The first recombinant viral vector that targets and knocks down the PINK1 gene is an AAV virus containing PINK1 sgRNA and Cas9.

4. The method according to claim 1, characterized in that, The mutant α-synuclein is a human A53T mutant.

5. An animal model, characterized in that, It is prepared by the method described in claim 1.

6. The application of the animal model as described in claim 5 as an animal model for simulating key pathological features of PD patients.

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