CN116716351B - Composition for constructing cynomolgus monkey Alzheimer's disease model, application and construction method thereof - Google Patents
Composition for constructing cynomolgus monkey Alzheimer's disease model, application and construction method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of non-human primate experimental animal disease models, in particular to a composition for constructing a cynomolgus monkey Alzheimer disease model, and application and a construction method thereof. The composition comprises: viral vectors AAV-P301L-TAU and fibrotic Abeta; wherein the viral vector AAV-P301L-TAU is obtained by inserting a gene encoding the P301L-TAU protein into an AAV viral vector. In the present invention, a model of cynomolgus monkey Alzheimer's disease can be obtained rapidly by introducing the viral vectors AAV-P301L-TAU and fibrotic Abeta into the prefrontal cortex of the cynomolgus monkey brain. The cynomolgus monkey Alzheimer's disease model is an AD monkey model with greatly reduced cognitive ability and maintained at low level and accompanied by two pathological changes of Abeta and Tau.
Description
Technical Field
The invention relates to the technical field of non-human primate experimental animal disease models, in particular to a composition for constructing a cynomolgus monkey Alzheimer disease model, and application and a construction method thereof.
Background
Alzheimer's Disease (AD) is a central nervous system degenerative disease that occurs in the elderly and in the pre-senile stages and is characterized by progressive cognitive dysfunction and behavioral impairment, the most common type of dementia. Neurofibrillary tangles formed by extracellular deposition of amyloid β and aggregation of hyperphosphorylated Tau protein in nerve cells are typical changes in AD histopathology.
Over 5500 tens of thousands of people in 2019 have dementia, and by 2050, the number of patients reaches as much as 1.39 million people, and the cost for AD is as high as 6040 million dollars each year worldwide, thus bringing a heavy economic burden to the world. 1507 cases of dementia patients exist in 60 years old and above people in 2020, wherein 983 cases of AD patients have prevalence and mortality slightly higher than the global average level. In recent years, due to the continuous increase of the number of people suffering from AD, the social and economic burden is increasingly obvious, and the AD has become a serious disease and social problem seriously harming the health of people in China. The existing clinical commonly used AD therapeutic drugs can only delay the progress of diseases and improve symptoms, cannot radically treat the diseases, and basically have serious side effects. For many years, global pharmaceutical enterprises have invested in enormous costs for developing related drugs, but most end up with failures. For this reason, it is important that the commonly used rodent models not mimic the patient's reality, resulting in many drug candidates failing in clinical trials. Therefore, the construction of an animal model capable of simulating the real situation of a patient is always a major problem to be solved in the field of mechanism research and drug development.
Rodents have the characteristics of low price, wide resources, high survival rate, clear genetic background, mature gene operation, low raising cost and the like, and are widely used in research. The method has the advantages that the pathogenesis of the transgenic animals is determined, the pathological symptoms are known, the research of AD mechanism and the screening of prevention and treatment drugs are facilitated, and the method is the most widely used animal model in the research of AD. However, a single type of transgenic model cannot simultaneously express all typical symptoms of AD, and the transgenic animal model has the defects of difficult repetition, unstable exogenous gene expression, low reproductive capacity, poor disease resistance and the like. Furthermore, rodent central nervous anatomy and physiological system are greatly different from human, and typical AD pathological symptoms such as aβ amyloid deposition and Tau abnormal phosphorylation, even neurofibrillary tangles, etc. do not occur spontaneously. As such, rodent AD models are unable to mimic the reality of human patients, and thus new drug clinical outcomes validated using their models are difficult to successfully transform.
The brain structure of the non-human primate is similar to that of human, particularly the prefrontal cortex and the sea horse are developed, the non-human primate has advanced brain function, can complete the complex behavioural test commonly used in clinic, and has high result interoperability. The amino acid sequence of the Aβ protein of a non-human primate is 100% identical to that of human, the amino acid sequence of the Tau protein is 98% identical to that of human, and Aβ amyloid deposits and phosphorylates Tau protein are present in the brain of an aged monkey. Researchers found that aged monkeys developed spontaneous aβ and Tau pathological changes, and that cognitive performance was reduced, but these changes did not reach the severity of AD, and were minimal in number. While the research on AD monkey model has been reported at home and abroad. Studies have shown that neither expression of Tau protein in the monkey brain by AAV-tool virus nor direct large-dose injection of Abeta oligomers can result in monkey models that simultaneously exhibit reduced cognitive ability, altered blood, cerebrospinal fluid and brain AD indices (e.g., abeta and Tau pathology, glial activation, neuronal loss, and synaptic damage, etc.).
Therefore, the model of Alzheimer's disease in which typical pathological changes such as Abeta and Tau occur in blood plasma, cerebrospinal fluid and brain tissues is established, and the model is of great significance to the evaluation of novel therapies.
Disclosure of Invention
Currently, rodents are widely used in the construction of models of alzheimer's disease. However, rodent central nervous anatomy and physiological systems vary greatly from human to human, and the clinical outcome of new drugs validated using their models is difficult to successfully transform. Less reports are given on the study of the Alzheimer's disease monkey model, and none of the reported monkey models can simultaneously show cognitive decline and AD index changes (such as Abeta and Tau pathological changes, glial cell activation, neuron loss, synaptic injury and the like) in blood, cerebrospinal fluid and brain, and can not completely simulate the typical symptoms of AD patients. It is therefore an object of the present invention to provide compositions for and methods of constructing models of cynomolgus monkey alzheimer's disease.
In order to achieve the above object, according to one aspect of the present invention, there is provided a composition for constructing a model of cynomolgus monkey alzheimer's disease, comprising: viral vectors AAV-P301L-TAU and fibrotic Abeta;
wherein the viral vector AAV-P301L-TAU is obtained by inserting a gene encoding the P301L-TAU protein into an AAV viral vector.
In a second aspect the invention provides a kit for constructing a model of Alzheimer's disease in cynomolgus monkeys, said kit comprising a composition as hereinbefore described.
The third aspect of the invention provides the composition for constructing the cynomolgus monkey Alzheimer's disease model or the application of the kit for constructing the cynomolgus monkey Alzheimer's disease model in screening medicines for preventing or treating Alzheimer's disease.
In a fourth aspect, the present invention provides a method for constructing a model of cynomolgus monkey alzheimer's disease, the method comprising the steps of:
(1) Selecting male cynomolgus monkeys with ages of 14-19;
(2) Injecting viral vectors AAV-P301L-TAU and fibrotic aβ into the prefrontal cortex of the cynomolgus monkey described in step (1);
wherein the viral vector AAV-P301L-TAU is obtained by inserting a gene encoding the P301L-TAU protein into an AAV viral vector.
In a fifth aspect, the invention provides a cynomolgus monkey Alzheimer's disease model constructed by the method described above.
In the present invention, a model of cynomolgus monkey Alzheimer's disease can be obtained rapidly by introducing the viral vectors AAV-P301L-TAU and fibrotic Abeta into the prefrontal cortex of the cynomolgus monkey brain. The cynomolgus monkey Alzheimer's disease model is an AD monkey model with greatly reduced cognitive ability and maintained at a low level and accompanied by two pathological changes of Abeta and Tau, and simultaneously changes of AD biomarkers in cerebrospinal fluid and blood.
Compared with the prior art, the invention has the following advantages:
1. the model built by the invention is a cynomolgus monkey model, the brain structure of the cynomolgus monkey is similar to that of a human being, the model has advanced brain function, can complete the complex behavioural test commonly used in clinic, and has high result interoperability; the amino acid sequence of the Abeta protein of the cynomolgus monkey is 100 percent consistent with human, the amino acid sequence of the Tau protein is 98 percent consistent with human, and the cynomolgus monkey is found in the aged monkey.
2. According to the cynomolgus monkey Alzheimer's disease model, the cognitive ability level is greatly and obviously reduced after modeling for 60 days, and the model can be maintained for a long time.
3. The AD typical biomarkers such as Abeta and Tau in cerebrospinal fluid, blood plasma and brain tissues after modeling show great change in the cynomolgus monkey Alzheimer disease model.
Drawings
FIG. 1 is a graph showing the results of the change in WGTA correct response rate of cynomolgus monkey before and after molding in example 1 of test example 1.
FIG. 2 is a graph showing the results of changes in the levels of cerebral spinal fluid and plasma Aβ in cynomolgus monkeys before and after molding in example 1 of test example 1.
FIG. 3 is a graph showing the results of the changes in the levels of cerebral spinal fluid, plasma and frontal lobe brain tissue NfL of cynomolgus monkeys before and after molding in example 1 of test example 1.
FIG. 4 is a graph showing comparison of the levels of TAU protein in brain tissue at the injection side of the forehead lobe AAV-P301L-TAU of a model cynomolgus monkey and a control cynomolgus monkey in example 1.
FIG. 5 is a graph showing comparison of the levels of Tau protein in brain tissue at the injection side of the forehead lobe fibrosis Abeta of a model cynomolgus monkey and a control cynomolgus monkey in test example 1.
FIG. 6 is a graph showing comparison results of Tau protein levels of hippocampal brain tissues of model cynomolgus monkeys in test example 1 and cynomolgus monkeys in a control group in accordance with example 1.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In one aspect, the invention provides a composition for constructing a model of cynomolgus monkey alzheimer's disease, comprising: viral vectors AAV-P301L-TAU and fibrotic Abeta;
wherein the viral vector AAV-P301L-TAU is obtained by inserting a gene encoding the P301L-TAU protein into an AAV viral vector.
In the present invention, AAV viral vectors represent adeno-associated viral vectors.
In the present invention, the gene encoding the P301L-Tau protein differs from the normal gene encoding the Tau protein in that the second C of the three bases encoding amino acid 301 is changed to T, resulting in proline to leucine at that position. The gene sequence of the coding P301L-Tau protein is shown as SEQ ID NO: 1.
SEQ ID NO：1
CCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAAT
AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC
GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC
CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA
TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC
CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATT
GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA
CCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT
ATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGC
GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATG
GGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA
ATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG
AGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAATTAACCATG
GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACA
AGGATGACGATGACAAGCTTGCGGCCGCGAATTCaATGGCTGAGCCCC
GCCAGGAGTTCGAAGTGATGGAAGATCACGCTGGGACGTACGGGTTG
GGGGACAGGAAAGATCAGGGGGGCTACACCATGCACCAAGACCAAGA
GGGTGACACGGACGCTGGCCTGAAAGAATCTCCCCTGCAGACCCCCA
CTGAGGACGGATCTGAGGAACCGGGCTCTGAAACCTCTGATGCTAAGA
GCACTCCAACAGCGGAAGATGTGACAGCACCCTTAGTGGATGAGGGA
GCTCCCGGCAAGCAGGCTGCCGCGCAGCCCCACACGGAGATCCCAGA
AGGAACCACAGCTGAAGAAGCAGGCATTGGAGACACCCCCAGCCTGG
AAGACGAAGCTGCTGGTCACGTGACCCAAGCTCGCATGGTCAGTAAA
AGCAAAGACGGGACTGGAAGCGATGACAAAAAAGCCAAGGGGGCTG
ATGGTAAAACGAAGATCGCCACACCGCGGGGAGCAGCCCCTCCAGGC
CAGAAGGGCCAGGCCAACGCCACCAGGATTCCAGCAAAAACCCCGCC
CGCTCCAAAGACACCACCCAGCTCTGGTGAACCTCCAAAATCAGGGG
ATCGCAGCGGCTACAGCAGCCCCGGCTCCCCAGGCACTCCCGGCAGCC
GCTCCCGCACCCCGTCCCTTCCAACCCCACCCACCCGGGAGCCCAAGA
AGGTGGCAGTGGTCCGTACTCCACCCAAGTCGCCGTCTTCCGCCAAGA
GCCGCCTGCAGACAGCCCCCGTGCCCATGCCAGACCTGAAGAATGTCA
AGTCCAAGATCGGCTCCACTGAGAACCTGAAGCACCAGCCGGGAGGC
GGGAAGGTGCAGATAATTAATAAGAAGCTGGATCTTAGCAACGTCCAG
TCCAAGTGTGGCTCAAAGGATAATATCAAACACGTCCTGGGAGGCGGC
AGTGTGCAAATAGTCTACAAACCAGTTGACCTGAGCAAGGTGACCTCC
AAGTGTGGCTCATTAGGCAACATCCATCATAAACCAGGAGGTGGCCAG
GTGGAAGTAAAATCTGAGAAGCTTGACTTCAAGGACAGAGTCCAGTC
GAAGATTGGGTCCCTGGACAATATCACCCACGTCCCTGGCGGAGGAAA
TAAAAAGATTGAAACCCACAAGCTGACCTTCCGCGAGAACGCCAAAG
CCAAGACAGACCACGGGGCGGAGATCGTGTACAAGTCGCCAGTGGTG
TCTGGGGACACGTCTCCACGGCATCTCAGCAATGTCTCCTCCACCGGC
AGCATCGACATGGTAGACTCGCCCCAGCTCGCCACGCTAGCTGACGAG
GTGTCTGCCTCCCTGGCCAAGCAGGGTTTGTGAGGATCCCGGGTGGCA
TCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCAC
TCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTG
TCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTAT
GGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTC
TATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCA
ATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGT
TGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTT
TTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTC
CTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTA
CAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTAAAATAAC
TATACCAGCAGGAGGACGTCCAGACACAGCATAGGCTACCTGGCCATG
CCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACTGTCCTCTCATG
CGTTGGGTCCACTCAGTAGATGCCTGTTGAATTGGGTACGCGGCCAGC
TTGGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTC
CCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC
CAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAA
GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCC
CATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGC
TGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGAATTGATCAGC
TTGGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAA
CAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTA
TTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCC
GTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACC
GACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCT
ATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGT
TGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGG
GGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCAT
CATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGC
CCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGG
ATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAG
GGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCC
CGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAA
TATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGG
CTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATA
TTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT
ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT
TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAA
GCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCT
ATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGA
TCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCGGGC
TCGATCCCCTCGCGAGTTGGTTCAGCTGCTGCCTGAGGCTGGACGACC
TCGCGGAGTTCTACCGGCAGTGCAAATCCGTCGGCATCCAGGAAACCA
GCAGCGGCTATCCGCGCATCCATGCCCCCGAACTGCAGGAGTGGGGAG
GCACGATGGCCGCTTTGGTCGACCCGGACGGGACGCTCCTGCGCCTGA
TACAGAACGAATTGCTTGCAGGCATCTCATGAGTGTGTCTTCCCGTTTT
CCGCCTGAGGTCACTGCGTGGATGGAGCGCTGGCGCCTGCTGCGCGAC
GGCGAGCTGCTCACCACCCACTCGCCAAGCTGGAACCGTAAAAAGGC
CGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCGATCATAATCAGCCATA
CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCC
CCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTT
ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCA
CAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACT
CATCAATGTATCTTATCATGTCTGGATCAATTCCCTATAGTGAGTCGTATT
AAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT
CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTG
GGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG
CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC
GGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT
TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG
GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG
GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA
GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC
CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA
AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC
CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG
CCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAG
GTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC
GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC
TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA
CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT
TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG
GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA
GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG
TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT
CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA
CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGA
TCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG
TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT
CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG
TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA
TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA
ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTA
TCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTA
GTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCAT
CGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCC
CAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG
GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA
GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT
GCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCA
TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAA
TACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT
TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT
GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA
TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAA
AATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT
CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT
CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG
GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGC
GGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCT
ACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT
TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT
CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAA
CTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGG
TTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT
GTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGAT
TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT
TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT in a preferred embodiment, the serotype of the viral vector AAV-P301L-TAU is type 9;
in a preferred embodiment, the promoter of the viral vector AAV-P301L-TAU is CMV.
In a preferred embodiment, the fibrillated aβ is fibrillated aβ1-42.
In a specific embodiment, the preparation process of the fibrosis A beta 1-42 comprises the following steps: the human Abeta 1-42 monomer is diluted to 2ug/ul by using physiological saline and is placed at 37 ℃ for 7 days.
In a second aspect the invention provides a kit for constructing a model of Alzheimer's disease in cynomolgus monkeys, said kit comprising a composition as hereinbefore described.
The third aspect of the invention provides the composition for constructing the cynomolgus monkey Alzheimer's disease model or the application of the kit for constructing the cynomolgus monkey Alzheimer's disease model in screening medicines for preventing or treating Alzheimer's disease.
In a fourth aspect, the present invention provides a method for constructing a model of cynomolgus monkey alzheimer's disease, the method comprising the steps of:
(1) Selecting male cynomolgus monkeys with ages of 14-19;
(2) Injecting viral vectors AAV-P301L-TAU and fibrotic aβ into the prefrontal cortex of the cynomolgus monkey described in step (1);
wherein the viral vector AAV-P301L-TAU is obtained by inserting a gene encoding the P301L-TAU protein into an AAV viral vector.
In a preferred embodiment, the serotype of AAV in the viral vector AAV-P301L-TAU is type 9;
in a preferred embodiment, the promoter of the viral vector AAV-P301L-TAU is CMV.
In a preferred embodiment, the fibrillated aβ is fibrillated aβ1-42.
In a preferred embodiment, the injection may be a stereotactic injection.
In a preferred embodiment, in step (2), the viral vector AAV-P301L-TAU is injected to one side of the prefrontal cortex and the fibrosing aβ is injected to the other side of the prefrontal cortex. For example, in one specific embodiment, the viral vector AAV-P301L-TAU is injected into the dorsal prefrontal cortex of one half brain, followed by injection of fibrosing aβ into the dorsal prefrontal cortex of the opposite half brain.
In a preferred embodiment, the viral titer at the time of injection of the viral vector AAV-P301L-TAU is 10 12 vg/ml。
In the method, a virus vector AAV-P301L-TAU and fibrosis Abeta can be introduced into the prefrontal cortex of the cynomolgus brain by adopting a conventional mode in the field to obtain the model of the cynomolgus Alzheimer's disease. There is no special requirement for selecting cynomolgus monkey, and the introduction mode can adopt a stereotactic injection method common in the field. Based on the method, the Alzheimer's disease model which is more similar to the human state can be successfully established, a plurality of models can be simultaneously established, and the method has important significance in the research of new drugs aiming at AD.
In a fifth aspect, the invention provides a cynomolgus monkey Alzheimer's disease model constructed by the method described above.
The present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.
The cynomolgus monkey used in the examples is all from Hubei Tian Du biotechnology limited company, and the purchasing procedure accords with the requirements of the law and regulation of the national laws of the people's republic of China, and is approved by the forestry hall of Hubei province and Guangdong province. The experimental monkeys pass through physical examination, and all indexes meet the local inspection and quarantine standards;
the AAV-P301L-TAU is obtained by providing P301L-TAU plasmid information by the inventor, and packaging the AAV by entrusting and metabiotechnology (Shanghai) stock company;
prepared by the company Mimo Biotechnology (Shanghai) Inc.;
the human A beta 1-42 monomer is purchased from Shanghai blaze.
Example 1
(1) 7 male cynomolgus monkeys with ages of 14-19 years old are selected;
(2) Constructing a packaging humanized phosphorylated Tau protein virus expression vector AAV-P301L-TAU, and synthesizing and preparing fibrosis A beta 1-42;
(3) Injecting a virus vector AAV-P301L-TAU (experimental group) and fibrosis Abeta 1-42 (experimental group) into the prefrontal cortex of the cynomolgus monkey described in the step (1) by a cynomolgus monkey craniocerebral localization injection technology, wherein the virus vector AAV-P301L-TAU is injected into the prefrontal cortex of one side half brain dorsal side and the fibrosis Abeta 1-42 is injected into the prefrontal cortex of the opposite side half brain dorsal side; the control group is respectively injected with AAV empty vector and Abeta menstruum;
the virus vector AAV-P301L-TAU is obtained by inserting a gene encoding P301L-TAU protein into an AAV virus vector, and the gene sequence encoding the P301L-TAU protein is shown in SEQ ID NO:1 is shown in the specification; the serotype of AAV in the AAV-P301L-TAU is type 9; the promoter of the virus vector AAV-P301L-TAU is CMV; the viral titer at the time of injection of the viral vector AAV-P301L-TAU was 10 12 vg/ml；
The preparation process of the fibrosis A beta 1-42 comprises the following steps: diluting the human Abeta 1-42 monomer to 2ug/ul by using physiological saline, and standing at 37 ℃ for 7 days; the injection amount of the fibrotic Abeta 1-42 was 100 ug/each monkey.
Test example 1
Performing WGTA evaluation on the cynomolgus monkey before and after modeling; plasma and cerebrospinal fluid Aβ1-42, Aβ1-40 and NfL levels at different time points before and after molding were measured using the Elisa method; respectively extracting total proteins of the forehead leaves and the sea horses of a control cynomolgus monkey and a cynomolgus monkey 30 days after AD modeling, and detecting the levels of AT8, thr181, ser404, ser396, HT7, tau5 and beta-actin of the forehead leaves and the sea horses of the control cynomolgus monkey and the cynomolgus monkey 30 days after AD modeling by using an immunoblotting method; detecting the forehead leaf NfL level of the control cynomolgus monkey and the cynomolgus monkey 30 days after AD modeling by using an Elisa method;
wherein, the delayed response test of the Wisconsin universal tester (Wisconsin general testing apparatus, WGTA) is a behavioural test which has long application and simple operation and is used for detecting the learning and memory of primates; the detection establishes two trap boxes, favorite foods are randomly placed in one trap box, animals are blocked from observing the trap boxes by setting a certain delay time, then the animals are allowed to freely select, if the trap boxes with the foods are selected, the animals are considered to respond correctly, and the correct response rate of the animals under the longest delay time is calculated, so that the cognitive ability of the animals is evaluated.
As shown in FIG. 1, pre-AD represents the test result before molding of the cynomolgus monkey in example 1, pro-AD-60-Day represents the test result 60 days after molding of the cynomolgus monkey in example 1, pro-AD-90-Day represents the test result 90 days after molding of the cynomolgus monkey in example 1, and FIG. 1 shows the results of comparison of the correct response rates of WGTA of the model for 90 days before molding and 60 days after molding. ns represents P > 0.05, P < 0.05.
As shown in fig. 2, pre-AD represents the test result before molding of the cynomolgus monkey in example 1, pro-AD-30-Day represents the test result 30 days after molding of the cynomolgus monkey in example 1, pro-AD-60-Day represents the test result 60 days after molding of the cynomolgus monkey in example 1, wherein fig. 2A shows the change of the level of the model cerebrospinal fluid aβ1-42 before molding and after molding for 30 days, fig. 2B shows the comparison of the level of the model cerebrospinal fluid aβ1-42 before molding and after molding for 60 days, fig. 2C shows the comparison of the level of the model cerebrospinal fluid aβ1-40 before molding and after molding for 60 days, fig. 2D shows the comparison of the model cerebrospinal fluid aβ1-42/aβ1-40 before molding and after molding for 60 days, and ns shows P > 0.05, P < 0.05.
As shown in FIG. 3, pre-AD represents the test results before molding of the cynomolgus monkey in example 1, pro-AD-30-Day represents the test results 30 days after molding of the cynomolgus monkey in example 1, pro-AD-60-Day represents the test results 60 days after molding of the cynomolgus monkey in example 1, pro-AD-90-Day represents the test results 90 days after molding of the cynomolgus monkey in example 1, WT-PFC-AAV represents the test results of empty vector control group or wild monkey frontal lobe, AD-PFC-AAV represents the test results of the injection side of the model frontal lobe AAV-P301L-TAU of the model 30 days after molding of example 1, AD-A betse:Sub>A represents the test results of the injection side of the model frontal lobe fibrosis A betse:Sub>A of the model 30 days after molding of example 1, FIG. 3A represents the change in the level of model fluid NfL between the model anterior and the model 30 days after molding, FIG. 3B represents the comparison of the levels of the model anterior lobe and the model anterior lobe of the model, and 3C 25 days after molding and the comparison of the results of the model of the plasmse:Sub>A lobe concentration of the model peak AAV-P301L-TAU in comparative side 35L-35. ns represents P > 0.05, P < 0.05.
As shown in FIG. 4, WT-PFC represents the results of the test of the frontal lobe of the wild-type control monkey, and AD-PFC-AAV represents the results of the test on the injection side of the frontal lobe AAV-P301L-TAU of the monkey model 30 days after molding in example 1. FIG. 4A shows the results of immunoblotting assays of control monkey forehead leaves and AD model monkey forehead leaf AAV injection side AT8 (recognizing Tau protein phosphorylated by Ser202 and Thr 205), thr181, ser404, HT7 (recognizing humanized Tau), tau5 (recognizing total Tau) and beta-actin (internal control); FIGS. 4B, C and D are comparison of AT8, thr181 and Ser404 phosphorylated Tau protein levels on the injection side of AAV in the forehead leaves of control monkeys and in the AD model monkeys; FIGS. 4E and F are results of comparison of HT7 and TAU5 protein levels at the injection side of control monkey forehead lobe and AD model monkey forehead lobe AAV-P301L-TAU; FIGS. 4G, H and I are results of comparison of the ratio of the levels of phosphorylated TAU protein to total TAU (TAU 5) AT the injection side of AT8, thr181 and Ser404 for both control and AD model monkey forehead leaves AAV-P301L-TAU; * P < 0.01, P < 0.001, P < 0.0001; ns represents P > 0.05.
As shown in FIG. 5, WT-PFC shows the results of the control monkey forehead lobe test, and AD-PFC-Abetse:Sub>A shows the results of the test on the injection side of the day monkey model forehead lobe fibrosis Abetse:Sub>A after molding 30 in example 1. Wherein, fig. 5A is immunoblotting detection results of Thr181, ser404, HT7 and β -actin on the injection side of fibrosis aβ in the forehead leaves of control monkeys and AD model monkeys; FIGS. 5B, C and D are comparison results of the levels of phosphorylated Tau protein of Thr181, anti-Thr181 and Ser404 on the injection side of fibrosis A beta of the forehead leaves of control monkeys and of the forehead leaves of AD model monkeys; fig. 5E is a comparison of HT7 protein levels on the injection side of control monkey forehead lobe and AD model monkey forehead lobe fibrosis aβ. * P < 0.05, P < 0.01, P < 0.001, ns represents P > 0.05.
As shown in FIG. 6, WT-HIP represents the test results of the control monkey hippocampus, and AD-HIP represents the test results of the model monkey hippocampus model after molding 30 in example 1. Wherein, fig. 6A is immunoblot detection results of control monkey forehead leaves and AD model monkey hippocampus Ser396, ser404 and Thr 181; FIGS. 6B, C and D are comparison results of the levels of phosphorylated Tau protein of the hippocampus Ser396, ser404 and Thr181 in control monkey forehead leaves and AD model monkeys. ns represents P < 0.01 and P < 0.001.
As can be seen from fig. 1, the correct WGTA response rate of the cynomolgus monkey model obtained in example 1 before modeling was significantly higher than that of the model 60 days and 90 days after modeling, and the model monkey cognitive ability after modeling was significantly reduced (P < 0.05, n=3).
As can be seen from fig. 2, the concentration of the cerebral spinal fluid aβ1-42 in the model of the cynomolgus monkey after molding continuously rises, the concentration of the cerebral spinal fluid aβ1-42 in the model of the cynomolgus monkey before molding is significantly lower than that in the model of the cynomolgus monkey after molding for 60 days (P < 0.01, n=5), the concentration of the cerebral spinal fluid aβ1-40 in the model of the cynomolgus monkey before molding has no significant difference from that in the model of the cynomolgus monkey after molding for 60 days (P > 0.05, n=5), and the concentration of the cerebral spinal fluid aβ1-42/aβ1-40 in the model of the cynomolgus monkey before molding is significantly lower than that in the model of the cynomolgus monkey after molding for 60 days (P < 0.05, n=5).
As can be seen from fig. 3, the concentration of the cynomolgus monkey model cerebrospinal fluid NfL continuously rises after molding, the concentration of the cynomolgus monkey model cerebrospinal fluid NfL is significantly raised (P < 0.05, n=6) after molding, the average concentration of the cynomolgus monkey model cerebrospinal fluid NfL is 4 times (n=6) before molding, the concentration of the cynomolgus monkey model plasma NfL is significantly raised (P < 0.05, n=4) after molding, the average concentration of the cynomolgus monkey model plasma NfL is 4.5 times (n=4) before molding after molding, the AAV-P301L-TAU injection side NfL protein concentration of the anterior lobe of the model for 30 days of molding is significantly higher than that of the anterior lobe of the control monkey by 2 times before molding, and the protein concentration of the anterior lobe fibrosis aβ injection side NfL of the model for 30 days of molding is not significantly different from that of the control monkey.
As can be seen from fig. 4, the injection side AT8 (Ser 202, thr 205), thr181 and HT7 levels of the model of cynomolgus monkey AT 30 days in the model of cynomolgus monkey obtained in example 1 were significantly higher than those of the control cynomolgus monkey AT the forehead (P < 0.0001, P < 0.001, n=3), the injection side of the model of cynomolgus monkey AT 30 days in the model of cynomolgus monkey AT Ser404 and TAU5 (total TAU protein) levels (P > 0.05, n=3) were not significantly different from those of the control cynomolgus monkey AT the forehead (P > 0.05, n=3), whereas the levels of Ser404/TAU5 (P > 0.05, n=3) were significantly higher than those of the model of cynomolgus monkey AT8/TAU5 AT 30 days in the model of cynomolgus monkey AT 301L-TAU injection side AT 181/TAU5 and AT thre 181/TAU 5. In summary, the cynomolgus monkey model forehead AAV-P301L-TAU injection side lobe phosphorylating TAU and human TAU levels were greatly increased after 30 days of modeling.
As can be seen from FIG. 5, the injection side Thr181, ser404 and HT7 levels of the model of 30 days in model of cynomolgus monkey obtained in example 1 were significantly higher than those of the control cynomolgus monkey forehead lobe (P < 0.001, P < 0.05, P < 0.01, n=3), wherein the antibody Thr181 of the model group developed a positive band at-180 kDa in addition to the development of the 55-70 kDa region compared to the control group, suggesting that the model group may have Tau protein accumulation. In summary, injection-side phosphorylated Tau and humanized Tau levels were greatly elevated in the model 30 days of modeling in cynomolgus monkey model with prefrontal leaf fibrosis aβ, suggesting the presence of Tau protein accumulation.
As can be seen from fig. 6, the levels of hippocampus Ser396, ser404 and Thr181 in the model of cynomolgus monkey obtained in example 1 were significantly higher than those of the control cynomolgus monkey (P < 0.001, P < 0.01, n=3), and in summary, the level of hippocampal phosphorylation Tau was significantly increased in the model of cynomolgus monkey obtained in example 1 for 30 days.
The data show that the model similar to the human cynomolgus monkey Alzheimer's disease can be successfully obtained by adopting a virus vector, a chemical inducer and a craniocerebral positioning injection mode.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (4)
1. A composition for constructing a model of cynomolgus monkey alzheimer's disease comprising: viral vectors AAV-P301L-TAU and fibrotic Abeta 1-42;
wherein the viral vector AAV-P301L-TAU is produced by introducing into a host cell a sequence of SEQ ID NO:1, and the gene encoding the P301L-Tau protein is inserted into an AAV viral vector.
2. The composition of claim 1, wherein the serotype of the viral vector AAV-P301L-TAU is type 9.
3. The composition of claim 2, wherein the promoter of the viral vector AAV-P301L-TAU is CMV.
4. A kit for constructing a model of cynomolgus monkey alzheimer's disease, characterized in that the kit comprises a composition according to any of claims 1-3.
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| CN202310350444.7A CN116716351B (en) | 2023-03-30 | 2023-03-30 | Composition for constructing cynomolgus monkey Alzheimer's disease model, application and construction method thereof |
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