Construction method and application of a glutamine acyl cyclase transgenic mouse model

Abstract

The application discloses a construction method and application of a glutamine acylase transgenic mouse model, and belongs to the technical field of Alzheimer's disease (AD) animal models. The model construction method comprises the following steps: taking AAV-Syn-MCS-EGFP as a skeleton vector, inserting a human QC gene to obtain a recombinant transfer plasmid AAV-Syn-QPCT-EGFP; co-transfecting HEK293T cells by a three-plasmid co-transfection method to obtain AAV-PHP.eB-Syn-QPCT-EGFP virus; injecting the AAV-PHP.eB-Syn-QPCT-EGFP virus into a mouse tail vein to obtain the QC transgenic mouse model. The mouse presents cognitive impairment and AD-like pathological phenotypes such as senile plaques in the brain after being injected for 4-8 weeks. The model can be used for AD pathological mechanism research, QC inhibitor in-vivo biological function research and action mechanism analysis, anti-AD lead drug discovery, anti-AD drug action mechanism, anti-AD drug pharmacokinetics and pharmacodynamics related research. In addition, the tail vein injection operation is simple and does not need complex equipment such as a stereotaxic instrument, so that large quantities of animals can be modeled, and experimental cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of animal models of Alzheimer's disease, and in particular to a method for constructing and applying a QC transgenic mouse model. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and the main form of dementia in the elderly. There are currently no effective treatments. Research into animal models is crucial for the discovery of anti-AD drugs.

[0003] Existing common AD animal models and their main limitations include: ① Traditional transgenic models (such as APP / PS1 and 5×FAD mice) rely on multi-gene overexpression, have long breeding cycles (6-12 months), and exhibit a single pathological phenotype, making it difficult to simulate the abnormal activation of glutamine acyl cyclase (QC) in sporadic AD; ② Although stereotactic injection-mediated AAV models can achieve targeted expression in the brain, they are highly invasive, easily damaging brain tissue, and have limited transfection range, failing to simulate the pathological spread of AD throughout the brain; ③ While tail vein injection of AAV has the advantages of simple operation and systemic delivery, traditional serotypes (such as AAV9) have low efficiency in crossing the blood-brain barrier (BBB), requiring high-dose injections and easily causing peripheral tissue side effects. Therefore, developing an efficient, convenient animal model that more closely resembles the clinicopathological characteristics of AD is of great significance for research on the pathogenesis of AD and the screening and research of related drugs.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a highly efficient, convenient, and clinically pathologically relevant QC transgenic mouse model, aiming to solve at least one of the above-mentioned technical problems of existing AD animal models.

[0006] The technical solution of the present invention is as follows: A first aspect of the present invention provides a method for constructing a transgenic mouse model of glutamine acyl cyclase (QC), comprising the steps of: Using AAV-Syn-MCS-EGFP as a vector, the mouse QC gene was inserted to obtain the AAV-Syn-QPCT-EGFP recombinant transfer plasmid; The AAV-Syn-QPCT-EGFP recombinant transfer plasmid, the AAV-PHP.eB serotype packaging plasmid, and the adenovirus helper plasmid were combined to form a three-plasmid packaging system. HEK293T cells were co-transfected with the three-plasmid co-transfection method to obtain the AAV-PHP.eB-Syn-QPCT-EGFP virus. The AAV-PHP.eB-Syn-QPCT-EGFP virus was injected into the tail vein of mice, and the QC transgenic mouse model was obtained 4-8 weeks after the injection.

[0007] Optionally, in the method for constructing the QC transgenic mouse model, the AAV-PHP.eB-Syn-QPCT-EGFP virus is non-covalently coupled with Thr BBB shuttle peptide.

[0008] Optionally, in the method for constructing the QC transgenic mouse model, the AAV-PHP.eB-Syn-QPCT-EGFP (titer: 1×10⁻⁶) is used. 11 -1×10 13 The injection dose of the virus (vg) is 100-200 µL per mouse.

[0009] Optionally, in the method for constructing the QC transgenic mouse model, the mouse is a C57BL / 6J, 6-8 weeks old, male, and weighs 20-22 g.

[0010] In a second aspect, the present invention provides an application of a QC transgenic mouse model obtained by the construction method described herein in the study of AD pathological mechanisms.

[0011] A third aspect of the present invention provides an application of the QC transgenic mouse model obtained by the construction method described in the present invention in the evaluation of the biological function and the study of the mechanism of action of QC inhibitors in vivo.

[0012] In a fourth aspect, the present invention provides an application of a QC transgenic mouse model obtained by the construction method described herein in the discovery of anti-AD lead drugs.

[0013] In a fifth aspect, the present invention provides an application of a QC transgenic mouse model obtained by the construction method described herein in the study of the mechanism of action of anti-AD drugs.

[0014] In a sixth aspect, the present invention provides an application of a QC transgenic mouse model obtained by the construction method described herein in pharmacokinetic studies of anti-AD drugs.

[0015] Optionally, in the aforementioned application, the active ingredient of the AD treatment drug is a QC inhibitor.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The core pathological features of Alzheimer's disease (AD) include senile plaques formed by the deposition of β-amyloid (Aβ), neurofibrillary tangles, and neuronal loss, ultimately leading to progressive cognitive decline. Recent studies have confirmed that glutamine acyl cyclase (QC) plays a crucial role in the pathogenesis of AD: QC can specifically catalyze the cyclization of the N-terminal glutamate residue of the Aβ peptide, generating PEAβ, which has strong neurotoxicity. This cyclization modification not only enhances the aggregation and cytotoxicity of Aβ but also inhibits its degradation and clearance, accelerates the formation of senile plaques, etc. Furthermore, the expression level of QC in the brains of AD patients is significantly higher than that in normal individuals, directly associated with disease progression. Therefore, QC transgenic mice are expected to better reflect the pathological process of AD and become a more scientific animal model of AD.

[0017] 2. Highly Efficient Blood-Brain Barrier-Free Delivery System: A QC overexpression vector was constructed using the AAV-PHP.eB serotype. This serotype targets transport receptors on the surface of brain vascular endothelial cells, achieving a 10-100 times higher BBB-crossing efficiency compared to traditional AAV9. It allows for widespread transfection of the entire brain (cortex, hippocampus, amygdala, and other AD-susceptible brain regions) after tail vein injection, enhancing the intrabrain penetration ability of viral particles, reducing the effective injection dose, and minimizing off-target expression in peripheral tissues. A tandem reporter gene (EGFP) is included in the vector for easy and direct tracking of viral transfection efficiency and QC expression localization.

[0018] 3. Optimized model construction efficiency: Compared with the 6-12 month pathological latency period of traditional transgenic models, this method can form a stable AD-like pathological phenotype 4-8 weeks after injection through AAV-mediated transient overexpression, which greatly shortens the experimental cycle; the tail vein injection operation is simple and does not require complex equipment such as stereotaxic instruments, which can realize large-scale animal modeling and reduce experimental costs.

[0019] 4. Comprehensive pathological phenotype: PEAβ generation is directly triggered by QC high expression, and Aβ aggregation, cognitive impairment and other multi-dimensional AD pathological features are simulated simultaneously. Compared with the single Aβ overexpression model, it is closer to the pathogenesis of clinical sporadic AD. Attached Figure Description

[0020] Figure 1 The image shows the qPCR results of QC mRNA expression levels in the hippocampus and cortex of the model group (QC-OE), the empty vector group (QC-NC), and the control group (Control).

[0021] Figure 2 The image shows the immunofluorescence results of QC expression and localization in the hippocampus and cortex in the QC-OE, QC-NC, and Control groups.

[0022] Figure 3The graph shows the quantitative detection results of Aβ and PEAβ in mouse brain tissue in the QC-OE group, QC-NC group and Control group.

[0023] Figure 4 The image shows the results of senile plaque deposition in the brains of mice in the QC-OE, QC-NC, and Control groups.

[0024] Figure 5 , Figure 6 The graph shows the results of the Morris water maze assessment of the learning and memory abilities of mice in the QC-OE group, QC-NC group and Control group.

[0025] Figure 7 The results of the novel object recognition experiment in the QC-OE group, QC-NC group and Control group are shown in the figure. The results evaluate the recognition and memory ability of mice.

[0026] in, refer to p <0.05, the difference is statistically significant; refer to p <0.01, the difference is highly significant; refer to p <0.001, the difference is highly significant; ns refers to p >0.05, the difference was not statistically significant. Detailed Implementation

[0027] This invention provides a method for constructing and applying a transgenic mouse model of glutamine acyl cyclase (QC). To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] Due to its high safety, low immunogenicity, and long-term stable expression, AAV vectors have become the preferred tool for gene delivery in the central nervous system (CNS). In recent years, the development of AAV serotypes with enhanced BBB efficiency (such as AAV-PHP.eB) and BBB shuttle peptide modification technology has provided technical support for achieving efficient whole-brain transfection via tail vein injection, making it possible to establish QC transgenic mouse models through systemic delivery.

[0029] Based on this, embodiments of the present invention provide a method for constructing a QC transgenic mouse model, comprising the following steps: Using AAV-Syn-MCS-EGFP as a backbone vector, the mouse QC gene was inserted to obtain the AAV-Syn-QPCT-EGFP recombinant transfer plasmid; The AAV-Syn-QPCT-EGFP recombinant transfer plasmid, the AAV-PHP.eB serotype packaging plasmid, and the adenovirus helper plasmid were combined to form a three-plasmid packaging system, which was then co-transfected into HEK293T cells using the three-plasmid co-transfection method to obtain the AAV-PHP.eB-Syn-QPCT-EGFP virus. The AAV-PHP.eB-Syn-QPCT-EGFP virus was injected into the tail vein of mice, and the QC transgenic mouse model was obtained 4-8 weeks after the injection.

[0030] Compared with the prior art, this embodiment has the following beneficial effects: 1. The core pathological features of Alzheimer's disease (AD) include senile plaques formed by the deposition of β-amyloid (Aβ), neurofibrillary tangles, and neuronal loss, ultimately leading to progressive cognitive decline. Recent studies have confirmed that glutamine acyl cyclase (QC) plays a crucial role in the pathogenesis of AD: QC can specifically catalyze the cyclization of the N-terminal glutamate residue of the Aβ peptide, generating PEAβ, which has strong neurotoxicity. This cyclization modification not only enhances the aggregation and cytotoxicity of Aβ but also inhibits its degradation and clearance, accelerates the formation of senile plaques, etc. Furthermore, the expression level of QC in the brains of AD patients is significantly higher than that in normal individuals, directly associated with disease progression. Therefore, QC transgenic mice are expected to better reflect the pathological process of AD and become a more scientific animal model of AD.

[0031] 2. Highly Efficient Blood-Brain Barrier-Free Delivery System: A QC overexpression vector was constructed using the AAV-PHP.eB serotype. This serotype targets transport receptors on the surface of brain vascular endothelial cells, achieving a 10-100 times higher BBB-crossing efficiency compared to traditional AAV9. It allows for widespread transfection throughout the entire brain (cortex, hippocampus, amygdala, and other AD-susceptible brain regions) after tail vein injection, enhancing the viral particle's intrabrain penetration ability, reducing the effective injection dose, and minimizing off-target expression in peripheral tissues. A reporter gene is tandemly incorporated into the vector for easy and direct tracking of viral transfection efficiency and QC expression localization.

[0032] 3. Optimized model construction efficiency: Compared with the 6-12 month pathological latency period of transgenic models, this method can form a stable AD-like pathological phenotype 4-8 weeks after injection through AAV-mediated transient overexpression, which greatly shortens the experimental cycle; the tail vein injection operation is simple and does not require complex equipment such as stereotaxic instruments, which can realize large-scale animal modeling and reduce experimental costs.

[0033] 4. Comprehensive pathological phenotype: PEAβ generation is directly triggered by QC high expression, and Aβ aggregation, cognitive impairment and other multi-dimensional AD pathological features are simulated simultaneously. Compared with the single Aβ overexpression model, it is closer to the pathogenesis of clinical sporadic AD.

[0034] In this embodiment, AAV-Syn-MCS-EGFP was used as a vector to insert the mouse QC gene, resulting in the AAV-Syn-QPCT-EGFP recombinant transfer plasmid.

[0035] In this embodiment, the expression of the QC gene is driven by the neuron-specific promoter Syn (Synapsin I promoter), avoiding non-specific inflammatory responses caused by abnormal expression in non-neuronal cells (such as glial cells), which is closer to the characteristics of neuronal Aβ abnormalities in AD pathology.

[0036] In one embodiment, the AAV-PHP.eB-Syn-QPCT-EGFP virus is non-covalently coupled with a ThrBBB shuttle peptide. Non-covalent coupling of the ThrBBB shuttle peptide to the virus can further enhance the brain penetration ability of viral particles, reduce the effective injection dose, and decrease off-target expression in peripheral tissues.

[0037] In one embodiment, the injection volume of the AAV-PHP.eB-Syn-QPCT-EGFP virus is 100-200 µL per mouse, i.e., a titer of 1 × 10⁻⁶. 11 -1×10 13 vg / mouse, preferably 1×10 12 vg / mouse.

[0038] In one embodiment, the mouse is a C57BL / 6J, 6-8 weeks old, male, and weighs 20-22g.

[0039] In one embodiment, the method for constructing the QC transgenic mouse model includes the following steps: Construction of recombinant transfer plasmid: Using AAV-Syn-MCS-EGFP as the backbone vector, a full-length human QC coding sequence (GenBank accession number: NM_012413.4) was inserted to construct the recombinant transfer plasmid AAV-Syn-QPCT-EGFP; the sequence correctness was verified by enzyme digestion and sequencing. Virus preparation and quality control: AAV-PHP.eB virus was packaged using a three-plasmid co-transfection method in HEK293T cells. After purification by gradient centrifugation with iodixanol, the viral genome titer (target ITR sequence) was detected by ddPCR, and the empty shell / intact particle ratio was detected by AUC method to ensure that the viral titer was ≥1×10⁻⁶. 13vg / mL, empty shell rate ≤30%, host cell DNA residue <10ng / 10 12 vg; Tail vein injection: The AAV-PHP.eB-Syn-QPCT-EGFP virus was injected into the tail vein of mice. The QC transgenic mouse model was obtained 4-8 weeks after the injection (4-8 weeks of normal feeding).

[0040] Tail vein injection is a simple and minimally invasive procedure for administering medication to mice, making it more suitable for long-term model observation. After injection, mice are routinely fed, and their mental state, diet, and activity are observed daily. Samples can be collected after 8 weeks for subsequent testing. This mouse model directly triggers PEAβ production through high QC expression, simultaneously simulating multi-dimensional Alzheimer's disease pathological characteristics such as Aβ aggregation and cognitive impairment. It more closely resembles the clinical pathogenesis of sporadic AD, providing a more realistic animal model for research on AD pathogenesis, drug screening, and efficacy evaluation.

[0041] This invention provides an application of a QC transgenic mouse model obtained by the construction method described above in the study of AD pathological mechanisms.

[0042] This invention provides an application of a QC transgenic mouse model obtained by the construction method described above in the in vivo biological function evaluation and mechanism of action study of QC inhibitors.

[0043] This invention provides an application of a QC transgenic mouse model obtained by the construction method described above in the discovery of anti-AD lead drugs.

[0044] This invention provides an application of the QC transgenic mouse model obtained by the construction method described above in the study of the mechanism of action of anti-AD drugs.

[0045] This invention provides an application of a QC transgenic mouse model obtained by the construction method described above in pharmacokinetic studies of anti-AD drugs.

[0046] The active ingredient in the AD treatment drug is a QC inhibitor.

[0047] The present invention will now be described in detail through specific embodiments.

[0048] I. Experimental Procedure (a) Laboratory animals and grouping (1) Animal selection: 21 male C57BL / 6J mice aged 6-8 weeks, weighing 20-22 g, were housed in an SPF-grade environment (temperature 22±2℃, humidity 50±5%, 12 h light-dark cycle).

[0049] (2) Group design: The mice were randomly divided into a control group of 7 mice, an empty vector group of 7 mice, and a model group of 7 mice. The control group was not treated. The empty vector group was injected with AAV-empty vector (AAV-PHP.eB-Syn-EGFP, which was prepared in the same way as AAV-PHP.eB-Syn-QPCT-EGFP, except that the human QC gene was not inserted). The model group was injected with AAV-PHP.eB-Syn-QPCT-EGFP virus (i.e., AAV-PHP.eB-Syn-QPCT-EGFP virus prepared by the following method).

[0050] (II) Vector construction and virus preparation (1) Vector construction: The AAV-Syn-MCS-EGFP plasmid was used as the vector backbone. The Syn promoter was used to drive the expression of the exogenous gene, and EGFP was used to trace the expression of the target gene. Primers were designed based on the full-length coding sequence of human glutaminylcyclase (QPCT). The full-length coding sequence of human QC was obtained from the NCBI GenBank database, accession number NM_012413.4. The full-length CDS fragment of QC was obtained by PCR amplification using the synthetic gene fragment containing the full-length coding sequence of human QC as a template.

[0051] After purification by agarose gel electrophoresis, the PCR product was ligated into a linearized AAV-Syn-MCS-EGFP backbone to construct the recombinant transfer plasmid AAV-Syn-QPCT-EGFP. The ligation product was transformed into competent *E. coli*, and single clones were selected for amplification after antibiotic selection. After plasmid extraction, restriction endonuclease digestion was used to identify the size and orientation of the insert fragment. Sanger sequencing was then performed to confirm that the QC insert sequence, reading frame, and linker region were correct and mutation-free. The sequenced plasmid was used for subsequent AAV virus packaging.

[0052] The AAV-Syn-MCS-EGFP vector backbone was derived from Addgene; the human QC coding sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; and the restriction endonuclease, ligase, high-fidelity DNA polymerase, plasmid extraction kit, and gel recovery kit were all purchased from Thermo Scientific™.

[0053] (2) Virus preparation and quality control: The virus was prepared using a three-plasmid co-transfection method on HEK293T cells. A three-plasmid packaging system consisting of AAV-Syn-QPCT-EGFP, AAV-PHP.eB serotype packaging plasmid, and adenovirus helper plasmid was co-transfected into HEK293T cells; after mixing, it was co-transfected into HEK293T cells. The HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing fetal bovine serum and antibiotics. Lipofectamine 3000 (purchased from Thermo Fisher Scientific) was used as the transfection reagent. After transfection, the cells were cultured for 60 hours, and the cell and culture supernatant were collected. Viral particles were released through repeated freeze-thaw cycles or chemical lysis. After removing free host cell DNA and plasmid DNA by nuclease treatment, the virus was purified by iodixanol density gradient ultracentrifugation. The target gradient layer containing AAV virus was collected, concentrated by ultrafiltration, and buffer replaced to obtain AAV-PHP.eB-Syn-QPCT-EGFP virus. The virus is preserved in sterile PBS or a virus preservation solution containing stabilizers at -80°C, avoiding repeated freeze-thaw cycles.

[0054] Viral quality control includes viral genome titer, empty shell / intact particle ratio, and residual impurity detection. Viral genome titer was detected using digital titer PCR (ddPCR), with the AAV ITR sequence as the detection target; results are expressed as vg / mL. The empty shell / intact particle ratio was detected using analytical ultracentrifugation (AUC). Residual host cell DNA was detected using qPCR or ddPCR. The viral preparation must meet the following quality control standard: viral genome titer ≥ 1 × 10⁻⁶. 13 vg / mL, empty shell rate ≤30%, host cell DNA residue less than 10 ng / 10 12 Only virus batches that meet the above standards can be used for subsequent animal experiments or in vivo drug administration studies.

[0055] The AAV-PHP.eB serotype packaging plasmid and adenovirus helper plasmid were both obtained from Addgene; iodixanol was purchased from Sigma-Aldrich; nuclease and ddPCR reagent were purchased from Thermo Fisher Scientific; and AUC detection service and host cell DNA residue detection were provided by the Jianyi Bio Detection Platform.

[0056] (iii) Tail vein injection (1) Injection dose: Both the empty group and the model group were injected with 1×10 12 The dose is administered as vg / animal, in a volume of 200 μL.

[0057] (2) Operation steps: After the mice were anesthetized by isoflurane inhalation, they were fixed on a constant temperature operating table, the tail vein was exposed, and the virus solution was slowly injected along the distal end of the tail vein using a 30G insulin syringe. After injection, hemostasis was achieved by pressing for 30 seconds. After waking up, the mice were put back into the breeding cage.

[0058] (iv) Feeding and Sample Collection (1) Feeding period: After injection, the mice were fed routinely for 8 weeks, during which their mental state, diet and activity were observed daily.

[0059] (2) Sample collection: After the behavioral experiment, three mice in each group were deeply anesthetized with 3-bromoethanol and then perfused with PBS through the heart until the liver turned white. The mouse head was quickly cut off and the brain tissue was dissected. The left and right hemispheres of the mouse were separated. After separating the hippocampus and cortex of one hemisphere, they were placed in labeled 1.5mL EP tubes, flash-frozen in liquid nitrogen and then stored in a -80℃ freezer. Proteins were extracted later for ELISA detection and Western blot analysis. The other hemisphere was directly placed in a blood analysis bottle containing 4% paraformaldehyde for fixation and stored at 4℃ in the dark for immunofluorescence detection and immunohistochemical analysis.

[0060] (V) Detection Indicators and Methods (1) QC expression verification: ① qPCR detection of the relative expression level of QC mRNA in cortical / hippocampal tissue (internal reference gene: GAPDH); ② Immunofluorescence staining to observe the distribution and expression intensity of QC-EGFP fusion protein in the brain.

[0061] (2) AD pathological examination: ① ELISA method to detect PEAβ (3-42) and total Aβ (1-42) levels in cortical / hippocampal tissue (kit: Thermo Fisher Scientific); ② Immunohistochemical staining (primary antibody: 4G8 antibody, dilution ratio 1:500) to observe senile plaque deposition.

[0062] (3) Cognitive function test: Eight weeks after injection, the Morris water maze (5 days of localization and navigation test and 1 day of spatial exploration test) and new object recognition test were performed to evaluate the spatial learning and memory ability and recognition and memory ability of mice.

[0063] II. Experimental Results (a) AAV-mediated high-efficiency expression of QC in the brain (1) Figure 1 qPCR results showed that 3 weeks after injection, the expression levels of QC mRNA in the cortex and hippocampus of the model group (QC-OE) were significantly higher than those in the control group (cortex: p <0.001; Hippocampus: p <0.01), there was no statistically significant difference between the empty group (QC-NC group) and the control group (ns, p>0.05).

[0064] (2) Figure 2 Immunofluorescence staining results showed that extensive EGFP fluorescence signals were observed in the cortex and hippocampus of the model group, confirming that QC is specifically highly expressed in neurons, while no EGFP signal was observed in the control group.

[0065] (II) Formation of AD-like pathological phenotype (1) Figure 3 Quantitative detection results of PEAβ and PEAβ showed that at 8 weeks, the levels of PEAβ (3-42) in the cortex and hippocampus of the model group were significantly higher than those in the control group. p <0.05), the total Aβ (1-42) level was significantly higher than that in the control group (cortical: p <0.001; Hippocampus: p <0.01).

[0066] (2) Figure 4 The results of senile plaque deposition showed that at 8 weeks in the model group, scattered 4G8-positive senile plaques were visible in the cortex and hippocampus, i.e. Figure 4 In the area indicated by the middle arrow, the plaques are mainly located around neurons, while no obvious plaque formation was observed in the control group.

[0067] (iii) Significant decline in cognitive function in mice (1) Figures 5-6 The Morris water maze results showed that in the positioning and navigation experiment, the escape latency of the model group was significantly longer than that of the control group. p <0.05); In the space exploration experiment, the proportion of time spent in the target quadrant by the model group was significantly lower than that by the control group ( p <0.001), the number of platform crossings decreased ( p <0.05).

[0068] (2) Figure 7 The results of the new object recognition experiment showed that the discrimination index of the model group was significantly lower than that of the control group, indicating that the recognition memory ability was impaired. p <0.001).

[0069] (iv) Safety assessment During the experiment, all mice remained healthy, exhibiting normal mental status, appetite, and weight gain, with no obvious abnormal behavior. Peripheral tissue analysis (liver and kidneys) showed that QC expression levels in the model group were two orders of magnitude lower than in the brain, and no significant tissue damage was observed, confirming the vector's good brain targeting and safety.

[0070] It is known that the present invention successfully constructed a QC transgenic mouse model (a brain-specific C57BL / 6J mouse model that highly expresses QC) by tail vein injection of AAV-PHP.eB. This model can form stable AD-like pathological phenotypes such as Aβ aggregation and cognitive impairment 8 weeks after injection, providing an efficient and reliable animal model for the study of AD pathogenesis and the screening of QC inhibitors.

[0071] In summary, the present invention provides a method for constructing a QC transgenic mouse model and its application. The method includes the following steps: using AAV-Syn-MCS-EGFP as a vector, inserting a human QC gene to obtain an AAV-Syn-QPCT-EGFP recombinant transfer plasmid. The AAV-Syn-QPCT-EGFP recombinant transfer plasmid, the AAV-PHP.eB serotype packaging plasmid, and the adenovirus helper plasmid were combined to form a three-plasmid packaging system, which was then co-transfected into HEK293T cells using the three-plasmid co-transfection method to obtain the AAV-PHP.eB-Syn-QPCT-EGFP virus. The AAV-PHP.eB-Syn-QPCT-EGFP virus was injected into the tail vein of mice, and the QC transgenic mouse model was obtained 4-8 weeks after the injection.

[0072] Compared with the prior art, the present invention has the following beneficial effects: 1. The core pathological features of Alzheimer's disease (AD) include senile plaques formed by the deposition of β-amyloid (Aβ), neurofibrillary tangles, and neuronal loss, ultimately leading to progressive cognitive decline. Recent studies have confirmed that glutamine acyl cyclase (QC) plays a crucial role in the pathogenesis of AD: QC can specifically catalyze the cyclization of the N-terminal glutamate residue of the Aβ peptide, generating PEAβ, which has strong neurotoxicity. This cyclization modification not only enhances the aggregation and cytotoxicity of Aβ but also inhibits its degradation and clearance, accelerates the formation of senile plaques, etc. Furthermore, the expression level of QC in the brains of AD patients is significantly higher than that in normal individuals, directly associated with disease progression. Therefore, QC transgenic mice are expected to better reflect the pathological process of AD and become a more scientific animal model of AD.

[0073] 2. Highly efficient blood-brain barrier delivery system: The QC overexpression vector is constructed using the AAV-PHP.eB serotype. This serotype targets the transport receptor on the surface of brain vascular endothelial cells, and its cross-BBB efficiency is 10-100 times higher than that of traditional AAV9. It can achieve widespread transfection of the whole brain (cortex, hippocampus, amygdala and other AD-susceptible brain regions) after tail vein injection. At the same time, Thr BBB shuttle peptide is non-covalently coupled to the vector to further enhance the brain penetration ability of viral particles, reduce the effective injection dose, and reduce off-target expression in peripheral tissues.

[0074] 3. Optimized model construction efficiency: Compared with the 6-12 month pathological latency period of traditional transgenic models, this method can form a stable AD-like pathological phenotype 4-8 weeks after injection through AAV-mediated transient overexpression, which greatly shortens the experimental cycle; the tail vein injection operation is simple and does not require complex equipment such as stereotaxic instruments, which can realize large-scale animal modeling and reduce experimental costs.

[0075] 4. Comprehensive pathological phenotype: PEAβ generation is directly triggered by QC high expression, and the phenotype characteristics of AD in multiple dimensions such as cognitive impairment are simulated simultaneously. Compared with the single Aβ overexpression model, it is closer to the pathogenesis of sporadic AD in clinical practice.

[0076] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for constructing a QC transgenic mouse model, characterized in that, Including the following steps: Using AAV-Syn-MCS-EGFP as a vector, a human QC gene was inserted to obtain the AAV-Syn-QPCT-EGFP recombinant transfer plasmid; The AAV-Syn-QPCT-EGFP recombinant transfer plasmid, the AAV-PHP.eB serotype packaging plasmid, and the adenovirus helper plasmid were combined to form a three-plasmid packaging system, which was then co-transfected into HEK293T cells using the three-plasmid co-transfection method to obtain the AAV-PHP.eB-Syn-QPCT-EGFP virus. The AAV-PHP.eB-Syn-QPCT-EGFP virus was injected into the tail vein of mice, and the QC transgenic mouse model was obtained 4-8 weeks after the injection.

2. The method for constructing the QC transgenic mouse model according to claim 1, characterized in that, The AAV-PHP.eB-Syn-QPCT-EGFP virus contains a Thr BBB shuttle peptide that is non-covalently coupled.

3. The method for constructing the QC transgenic mouse model according to claim 1, characterized in that, The injection volume of the AAV-PHP.eB-Syn-QPCT-EGFP virus is 100µL-200µL per mouse.

4. The method for constructing the QC transgenic mouse model according to claim 1, characterized in that, The mice were C57BL / 6J, 6-8 weeks old, male, and weighed 20-22g.

5. The application of a QC transgenic mouse model obtained by the construction method according to any one of claims 1-4 in the study of AD pathological mechanisms.

6. The application of a QC transgenic mouse model obtained by the construction method according to any one of claims 1-4 in the in vivo biological function evaluation and mechanism of action study of QC inhibitors.

7. The application of a QC transgenic mouse model obtained by the construction method according to any one of claims 1-4 in the discovery of anti-AD lead drugs.

8. The application of a QC transgenic mouse model obtained by the construction method according to any one of claims 1-4 in the study of the mechanism of action of anti-AD drugs.

9. The application of a QC transgenic mouse model obtained by the construction method according to any one of claims 1-4 in pharmacokinetic studies of anti-AD drugs.

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