Parkinson's disease animal model, construction method and application

Abstract

The invention discloses a Parkinson's disease animal model, a construction method and application, and relates to the field of animal genetic engineering and disease model construction. According to the model, seven mutation sites related to the human familial Parkinson's disease are introduced behind an initiation codon of a fourth exon of an endogenous SNCA gene, and the mutation sites are A18T, A29S, A30P, E46K, H50Q, G51D and A53T and have genotypes related to the human familial Parkinson's disease. The pig model shows nigra dopaminergic neuron reduction and cortical neuron reduction in the newborn period, and the core pathological process of the human Parkinson's disease can be systematically reproduced. The model can be widely applied to Parkinson's disease pathogenesis research, drug screening and cell therapy effect evaluation, and has important scientific research and preclinical application values.

Description

Technical Field

[0001] This invention relates to the field of animal genetic engineering and disease model construction, and in particular to an animal model of Parkinson's disease, its construction method, and its application. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide after Alzheimer's disease, with its global prevalence increasing significantly with age. Its core pathological features include the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and the deposition of Lewy bodies, primarily formed by the abnormal aggregation of α-synuclein. This leads to typical motor symptoms such as tremor, rigidity, bradykinesia, and postural instability, accompanied by non-motor symptoms such as cognitive impairment, mood disorders, and autonomic dysfunction.

[0003] Numerous genetic and molecular pathological studies have shown that abnormalities in α-synuclein are a key driver of Parkinson's disease (PD). Encoded by the SNCA gene, α-synuclein normally participates in synaptic vesicle circulation and neurotransmitter release; however, when its structure or expression is abnormal, it easily forms oligomers and fibrinous aggregates, triggering mitochondrial dysfunction, oxidative stress, lysosomal / autophagy disorders, and neuroinflammatory responses, ultimately leading to neurodegenerative changes. In 1997, Polymeropoulos et al. first reported a direct association between the SNCA gene A53T mutation and familial PD.2 Subsequently, multiple mutations, including A18T, A29S, A30P, E46K, H50Q, and G51D, have been found to be closely associated with early-onset and malignantly progressive familial PD. These mutations can enhance the tendency for α-synuclein aggregation and accelerate neurotoxicity.3-7

[0004] Traditional rodent models (chemically induced or single-gene transgenic mice / rats) have played a crucial role in PD research. However, due to significant differences in brain size, neural circuit complexity, and immune system compared to humans, they struggle to fully simulate the long-term progression of the disease, neuroinflammation, and multi-system damage. In contrast, pigs, as large animal models, possess highly similar neuroanatomical structures, white matter ratios, brain volume, and immune metabolic characteristics to humans, and have a longer lifespan, facilitating long-term follow-up and preclinical pharmacodynamic and toxicological validation. Therefore, gene-edited miniature pigs are considered a key bridge connecting basic research and clinical translation. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-site mutated SNCA gene-edited pig for constructing an animal model of Parkinson's disease and its preparation method. By introducing mutations at seven sites—A18T, A29S, A30P, E46K, H50Q, G51D, and A53T—after the start codon of exon 4 of the pig SNCA gene, a genotype associated with human familial PD is obtained, thereby establishing an animal model that can systematically reproduce the core pathological process of Parkinson's disease.

[0006] To achieve the above objectives, the present invention proposes the following technical solution: An animal model of Parkinson's disease is proposed, which introduces seven mutation sites related to human familial Parkinson's disease after the start codon of exon 4 of the endogenous SNCA gene. These mutation sites are A18T, A29S, A30P, E46K, H50Q, G51D, and A53T, and the model possesses genotypes associated with human familial Parkinson's disease. This pig model exhibits a reduction in dopaminergic neurons in the substantia nigra and a reduction in cortical neurons during the neonatal period, systematically reproducing the core pathological processes of human Parkinson's disease. This model can be widely used in research on the pathogenesis of Parkinson's disease, drug screening, and evaluation of cell therapy efficacy, possessing significant scientific research and preclinical application value.

[0007] Preferably, the SNCA start codon is ATG, the mutation site is edited at the endogenous site in exon 4, and the mutation in exon 5 is covered.

[0008] The present invention also provides a method for constructing an animal model of Parkinson's disease. The animal model is the Parkinson's disease animal model described above. Using gene editing technology, seven mutation sites related to human familial Parkinson's disease are introduced after the start codon of exon 4 of the endogenous SNCA gene in the animal model. The mutation sites are: A18T, A29S, A30P, E46K, H50Q, G51D, and A53T, thereby obtaining a genotype related to human familial Parkinson's disease.

[0009] Preferably, the relevant mutation sites are introduced via the CRISPR / Cas9 system.

[0010] Preferably, the mutation is introduced by non-homologous end joining (NHEJ).

[0011] Preferably, the nuclease system used in gene editing technology is SpCas9.

[0012] Preferably, it includes the following steps: (1) Design a targeting sgRNA after the start codon of exon 4 of the animal SNCA gene and construct a CRISPR / Cas9 gene editing system; (2) Construct donor plasmids containing target sequences and seven human familial Parkinson's disease-related mutation sites: A18T, A29S, A30P, E46K, H50Q, G51D, A53T and animal SNCA gene; (3) The constructed donor plasmid and Cas9 / gRNA vector were co-transfected into animal fetal fibroblasts. Monoclonal cells were obtained by drug screening and positive cells were obtained by PCR identification. (4) Using the positive clones obtained in step (3) as donor cells, embryos were obtained by somatic cell nuclear transfer (SCNT). (5) The embryos obtained in step (4) are transplanted into recipient sows to obtain an animal model of Parkinson's disease containing seven site mutations.

[0013] Preferably, the sgRNA sequence described in step (1) is as shown in SEQ ID No:1.

[0014] The present invention also provides the uses of the above-mentioned animal models of Parkinson's disease or the animal models of Parkinson's disease obtained by the above methods, for the study of the pathogenesis of Parkinson's disease; for the screening and efficacy evaluation of Parkinson's disease drugs; for the verification of cell / gene therapy effects and the development of companion diagnostic biomarkers; and for the preclinical feasibility assessment of drug delivery routes and devices (DBS, intrathecal administration, brain-targeted delivery).

[0015] The animal described in this invention is a pig.

[0016] Compared with the prior art, the present invention has the following substantial progress: This study, based on CRISPR / Cas9 technology, introduced mutations at seven sites—A18T, A29S, A30P, E46K, H50Q, G51D, and A53T—in exons 4 and 5 of the SNCA gene to construct a miniature pig model of SNCA multi-point mutation gene editing. This model highly mimics human familial PD in terms of genetic background, neuroanatomy, and pathological phenotype. It not only helps to elucidate the molecular pathogenic mechanism of α-synuclein multi-point mutations but can also be used to explore early diagnostic biomarkers and validate novel gene or cell therapy strategies, providing a powerful experimental platform for PD pathogenesis research and clinical translation.

[0017] Specifically as follows: Multi-site in situ editing, one-time modeling: reproduces the pathogenic background of seven sites in a single animal individual, avoiding the genetic background noise and lengthy cycle caused by multiline hybridization.

[0018] Endogenous regulation, highly anthropomorphic: Mutant SNCA is regulated by endogenous promoters and spatiotemporal expression, which is closer to the human disease course than exogenous overexpression or random integration.

[0019] Early detectable pathology: The neonatal period shows significant reduction in dopamine and midbrain neurons, accompanied by amplified inflammation, which is beneficial for early-onset-progression longitudinal studies.

[0020] Clinically translational friendly: The brain volume, white matter ratio, and immune metabolism of large animals are similar to those of humans, making them suitable for integrated validation through imaging, pharmacology, surgery, and behavior. Attached Figure Description

[0021] Figure 1 This invention relates to a gene editing identification strategy; Figure 2 This is the result of positive target cell identification in this invention; Figure 3 The results of genotyping of the gene-edited pigs in this invention; Figure 4 The images show the detection results of dopaminergic neurons in the substantia nigra and cortical neurons in wild-type and gene-edited pigs of this invention; where A is the immunofluorescence staining of dopaminergic neurons (TH-labeled, yellow) in the substantia nigra region of the midbrain of newborn wild-type and SNCA gene-edited pigs; B is the immunofluorescence staining of midbrain neurons (NeUN-labeled, green) in newborn wild-type and SNCA gene-edited pigs; TH: Tyrosine Hydroxylase; Nuc: Nuclei; NeUN: Neuronal Nuclei. Detailed Implementation

[0022] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0023] Example 1 An animal model of Parkinson's disease has seven mutation sites related to human familial Parkinson's disease introduced after the start codon of exon 4 of the endogenous SNCA gene. The mutation sites are: A18T, A29S, A30P, E46K, H50Q, G51D, and A53T, which have genotypes associated with human familial Parkinson's disease.

[0024] Preferably, the SNCA start codon in this embodiment is ATG, the mutation site is edited at the endogenous site in exon 4, and the mutation in exon 5 is covered.

[0025] This pig model exhibits a reduction in dopaminergic neurons in the substantia nigra and a reduction in cortical neurons during the neonatal period, systematically reproducing the core pathological processes of Parkinson's disease in humans. This invention's model can be widely applied to research on the pathogenesis of Parkinson's disease, drug screening, and evaluation of cell therapy efficacy, possessing significant scientific research and preclinical application value.

[0026] Example 2 according to Figure 1 The strategy involves constructing targeting vectors and editing SNCA point mutations.

[0027] A method for constructing an animal model of Parkinson's disease includes the following steps: (1) Design a targeting sgRNA after the start codon of the fourth exon of the porcine SNCA gene and construct a CRISPR / Cas9 gene editing system; the nuclease system preferred in this embodiment is SpCas9, and the alternatives can be high-fidelity Cas9 variants (egSpCas9-HF1, eSpCas9(1.1)), Cas12a / Cpf1, Cas12i, etc.

[0028] The first 200 bp of the ATG genome sequence was selected to design an sgRNA (5'-CCATGGATGTATTCATGAAAGGA-3'). The PX458 plasmid was digested with BBSI restriction enzyme, and the linearized plasmid was purified and recovered. The sgRNA recognition sequence was then denatured and renatured to convert it from single-stranded nucleotides to double-stranded oligonucleotides. The double-stranded DNA was then ligated to the linearized PX458 plasmid using T4 DNA ligase. The plasmid was transformed, plated, and cultured. Positive bacterial cultures were further identified by sequencing, and the plasmid was extracted for later use.

[0029] (2) Construct donor plasmids containing the target sequence and the seven relevant mutation sites of human familial Parkinson's disease: A18T, A29S, A30P, E46K, H50Q, G51D, A53T and porcine SNCA gene; The insertion sequence design requirements are as follows: a porcine SNCA gene coding sequence (CDS) carrying mutations at seven sites: A18T, A29S, A30P, E46K, H50Q, G51D, and A53T; an genomic sequence preceding the SNCA gene ATG at the front of the porcine SNCA gene coding sequence (CDS), which must contain the designed sgRNA; and the porcine SNCA gene 3'-UTR sequence (as shown in SEQ ID No: 2) and the bGH poly(A)signal sequence (as shown in SEQ ID No: 4) at the rear of the porcine SNCA gene coding sequence (CDS). After the sequence design is completed, it will be submitted to the company for DNA synthesis and construction into a standard T vector as the Donor vector required for gene editing.

[0030] (3) The constructed donor plasmid and Cas9 / gRNA vector were co-transfected into porcine fetal fibroblasts. Monoclonal cells were obtained by drug screening and positive cells were obtained by PCR identification. Porcine fetal fibroblasts cultured to 90% confluence were digested into single cells using trypsin. The cells were resuspended in 100 μL of electroporation (nuclear or liposome transfection can also be used in practice) solution. Donor vector (1 μg), PX458 plasmid (1 μg), and puromycin-resistant vector (20 ng) were added to the cell suspension. After transfection using an electroporator, culture medium was added and the cells were incubated overnight. The next day, after cell attachment, puromycin (2 μg / mL) was administered for drug screening. Cells were aliquoted into 10 cm culture dishes at a density gradient of 200-500 cells per dish. After 10 days of culture, single clones were picked and cultured. Genotyping of the single-cell clones at the target site was performed (PCR and sequencing). Two gene-edited cell clones were obtained, one of which showed good growth. Genotyping of this clone revealed recombination with multiple point mutations in the SNCA gene (see results). Figure 2 ).

[0031] (4) Using the positive clone cells as donor cells, embryos were obtained by somatic cell nuclear transfer (SCNT); The establishment of reconstructed porcine embryos in this embodiment was performed using a two-step method: First, enucleation was performed. In this embodiment, enucleation of the porcine embryo was done blindly. A manipulation solution with a final concentration of 7.5 μg / mL CB was placed on the stage of an inverted microscope in a micromanipulator. Then, the cytoplasm surrounding the first polar body was aspirated using a micromanipulation needle to remove the nucleus from the oocyte. The enucleated oocytes were preserved in the manipulation solution and placed in a 38°C incubator with maintained humidity. After all enucleation was completed, dead or poorly shaped oocytes were discarded. For cell injection, porcine fetal fibroblasts with regular shape, uniform size, homogeneous cytoplasm, and high viability were selected and injected into the perivitelline space of the enucleated oocytes. The injected porcine oocytes were then placed in a drop of PZM3 embryo culture medium to recover for approximately 0.5 hours. The restored enucleated and enucleated oocytes were transferred in batches to the fusion medium and washed and equilibrated for 2-3 minutes. Then, they were transferred to a fusion tank filled with fusion medium. The position of the reconstructed embryos was adjusted so that the contact surface between the donor cell and the recipient oocyte was perpendicular to the direction of the electric field. Subsequently, two repeated DC pulses of 1.2 kV / cm, 30 μs, with an interval of 1 s were applied using an electrofusion instrument to induce fusion and activate the embryos simultaneously. The electrofused embryos were transferred to porcine embryo culture medium PZM3 for 30 minutes, and the fusion status was determined.

[0032] (5) The embryo is transplanted into a recipient sow to obtain a gene-edited pig containing the seven mutation sites.

[0033] In this embodiment, allylprogesterone was used to synchronize estrus in recipient sows. Each sow was fed 5 mL of allylprogesterone oil solution every afternoon for 18 days, with the first day of feeding designated as D1. On D19, PGF2α was injected in the morning and afternoon, 6 hours apart. On D20, PMSG was injected in the morning. On D23, PMSG was injected, and 80 hours later, hCG was injected to induce ovulation. Gene-edited cell nuclear transfer was performed on the same day. On the morning of D24, the gene-edited cell nuclear transfer embryos prepared on D23 were transferred into the gestating sows. Each recipient sow received 100-400 embryos.

[0034] Each recipient sow was injected with 0.5 mL of domperidone and 10 vials of 50% acetaminophen solution to induce muscle relaxation and anesthesia. The skin was incised along the midline of the abdomen to locate the linea alba and open the abdominal cavity. The oviducts and ovaries were manually removed. Embryos were loaded using a 3-Fr cat catheter and inserted through the oviduct at the fimbriae opening into the ampulla, where the embryos were injected. The abdominal cavity was checked under a stereomicroscope for any missed embryos. After confirming no missed embryos, the cavity was closed. The abdominal cavity was sutured with CR032 absorbable sutures, forming three layers: the linea alba, subcutaneous fat, and skin, using nodular sutures. The sows were injected with amoxicillin-clavulanate potassium solution for three consecutive days after embryo transfer. Appropriate antibiotics and microorganisms were added to the feed to improve their condition.

[0035] Pregnancy checks were performed on day 30 after embryo transfer. Subsequently, ultrasound examinations were conducted on the recipient sows every three weeks. If a recipient sow showed signs of returning to estrus, this was confirmed by ultrasound. Sows with continued pregnancy entered their expected delivery date at day 110 and were transferred to the farrowing crate to await delivery.

[0036] Example 3 Newborn piglet sample collection and genotyping Oral epithelial cells were collected from freshly born piglets using sterile oral swabs. The swabs were placed in physiological saline, stirred, and centrifuged to obtain the oral epithelial cells. 1 μL of the oral epithelial cell suspension was added to 1 μL of Proteinase K solution and treated at 56℃ for 10 min and 95℃ for 5 min. The treated liquid could be directly used as a template for PCR detection. Genotyping analysis (PCR and sequencing) was performed on the obtained piglet samples. A total of 16 gene-edited miniature pigs were obtained in this study. Genotyping results showed that they were SNCA gene-edited miniature pigs with multiple point mutations. Figure 3 ).

[0037] Example 4: Pathological phenotype detection in pigs with multi-site mutation SNCA gene editing model.

[0038] Histopathological analysis of the midbrain of pigs with SNCA point mutation gene editing model: Histopathological analysis was performed on the midbrain of pigs with SNCA multi-point mutation gene editing.

[0039] Conclusion: Significant reductions were found in cortical neurons and dopaminergic neurons in gene-edited pigs, with smaller cell bodies, reduced cell-to-cell body contact, fewer or absent axons, and distal synaptic breaks. Figure 4 ).

[0040] Pathology: TH / NeuN immunohistochemistry showed a reduction in neonatal substantia nigra dopamine neurons and midbrain neurons, with shrinking cell bodies and axonal / synaptic breaks; Example 5 This embodiment also provides the uses of the above-mentioned animal models of Parkinson's disease or the animal models of Parkinson's disease obtained by the above methods, for the study of the pathogenesis of Parkinson's disease (such as α-syn aggregation-mitochondrial stress-lysosomal / autophagy disorder-neuroinflammatory coupling); for the screening and efficacy evaluation of Parkinson's disease drugs (such as L-DOPA, MAO-B inhibitors, α-syn aggregation inhibitors, autophagy / lysosomal modulators, microglial immunomodulators, etc.); for the verification of cell / gene therapy efficacy and the development of companion diagnostic biomarkers; and for the preclinical feasibility assessment of drug delivery routes and devices (DBS, intrathecal administration, brain-targeted delivery).

Claims

1. An animal model of Parkinson's disease, characterized in that, The model introduces the following seven mutation sites related to human familial Parkinson's disease after the start codon of exon 4 of the endogenous SNCA gene. The mutation sites are: A18T, A29S, A30P, E46K, H50Q, G51D, and A53T, which have genotypes associated with human familial Parkinson's disease.

2. The animal model of Parkinson's disease according to claim 1, characterized in that, The SNCA start codon is ATG. The mutation site is edited at the endogenous site in exon 4 and covers the mutation in exon 5.

3. A method for constructing an animal model of Parkinson's disease, characterized in that, The Parkinson's disease animal model described in claim 1 uses gene editing technology to introduce seven mutation sites related to human familial Parkinson's disease after the start codon of exon 4 of the endogenous SNCA gene in the animal model. The mutation sites are: A18T, A29S, A30P, E46K, H50Q, G51D, and A53T, thereby obtaining a genotype related to human familial Parkinson's disease.

4. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, The relevant mutation sites were introduced using the CRISPR / Cas9 system.

5. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, The mutation is introduced by non-homologous end joining.

6. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, The nuclease system used in gene editing technology is SpCas9.

7. The method for constructing an animal model of Parkinson's disease according to claim 3, characterized in that, Includes the following steps: (1) Design a targeting sgRNA after the start codon of exon 4 of the animal SNCA gene and construct a CRISPR / Cas9 gene editing system; (2) Construct donor plasmids containing target sequences and seven human familial Parkinson's disease-related mutation sites: A18T, A29S, A30P, E46K, H50Q, G51D, A53T and animal SNCA gene; (3) The constructed donor plasmid and Cas9 / gRNA vector were co-transfected into animal fetal fibroblasts. Monoclonal cells were obtained by drug screening and positive cells were obtained by PCR identification. (4) Using the positive clone cells obtained in step (3) as donor cells, embryos are obtained by somatic cell nuclear transfer. (5) The embryos obtained in step (4) are transplanted into recipient sows to obtain an animal model of Parkinson's disease containing seven site mutations.

8. The method for constructing an animal model of Parkinson's disease according to claim 7, characterized in that, The sgRNA sequence described in step (1) is shown in SEQ ID No:

1.

9. The use of an animal model of Parkinson's disease as described in any one of claims 1-2 or an animal model of Parkinson's disease obtained by the method described in any one of claims 3-8, characterized in that, Used for research on the pathogenesis of Parkinson's disease; for drug screening and efficacy evaluation for Parkinson's disease; for validation of the effects of cell therapy and gene therapy; and for preclinical feasibility assessment of drug delivery routes and devices.

10. An animal model of Parkinson's disease as described in any one of claims 1-2, or the method as described in any one of claims 3-8, or the use as described in claim 9, characterized in that, The animal in question is a pig.

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