Construction,Characterization And Screening Of Recombinant Adeno-associated Virus For Animal Model Of AD

Alzheimer's disease(AD)is a progressive neurodegenerative disease.The main pathological features include A? plaques formed by the excessive deposition of ?-amyloid and the formation of hyperphosphorylated tau protein(pTau).Neuron fiber tangles and loss of neurons.In recent years,scientists have tried many treatment strategies for AD,but the AD drugs approved so far can only alleviate the disease to a certain extent,and cannot cure or block the development of AD.Therefore,the clinical demand for new drugs to prevent and treat AD is extremely urgent.Immunotherapy is the focus of research and development of new AD drugs.However,the research on AD vaccines targeting A? or tau in the past decade has rarely been successful,because the safety of vaccines in humans is lacking and the induced immunogenicity is not strong enough.In addition,the A? vaccine or pTau vaccine only acts on one AD pathogenic factor and makes the efficacy poor.Therefore,the development of a vaccine that simultaneously targets the A ? protein and pTau protein in the brain of AD patients is a potential strategy to further improve the therapeutic effect of AD.In order to further develop the double-targeted AD vaccine against A? protein and pTau protein,an animal model capable of expressing two pathological proteins at the same time is very necessary for the evaluation of the therapeutic effect of the vaccine.At the same time,this animal model is used to explore the A? and tau in vivo The interaction is also very important.However,in the three transgenic mice that have been able to express two pathological proteins at the same time,the expression time of A ? protein and pTau protein has a long interval,which is not conducive to the study of dual-targeted AD vaccines,so constructing a method that can simultaneously express A? Animal models of protein and pTau protein are imminent.However,with the continuous development and enrichment of researchtechnology,recombinant adeno-associated virus(rAAV)vectors have been used in the construction of various disease models.Adeno-associated viruses have the advantages of strong transduction ability,high safety,low immunogenicity,and can mediate long-term gene expression,and are used in the treatment of many diseases.Therefore,using the ability of rAAV to efficiently transduce genes to achieve the expression of AD pathological proteins in animals has become a potentially effective method for constructing AD animal models.Studies have shown that different serotypes rAAV have tissue tendencies in animal tissues,so that the transduction efficiency of the target gene is optimized in specific tissues.At present,10 serotypes have been discovered.According to the research on the affinity of each serotype to different tissues and organs cells,rAAV2 has a better affinity for the nervous system,and rAAV8 has a better affinity for the liver,muscle,eye,and nervous system.All have good affinity.RAAV9 also shows good affinity in many organs,such as lung,liver,muscle,heart,and nervous system.Therefore,the three viral serotypes can achieve the transduction of the target gene in the nervous system.Some previous studies have shown that rAAV8 has better transduction efficiency in the nervous system than rAAV2,and rAAV9 is also highly hoped,but Which serotype is more suitable for the nervous system still needs further study.RAAV viruses of the same serotype will have different expression effects on foreign genes under the action of different promoters,so the difference in promoters will also affect the gene expression efficiency.The types of promoters commonly used in the process of transducing foreign genes with the rAAV in the nervous system include hSyn,mecp2,TUBA1 A,CaMK??a,Iba1,CNP,etc.Among them,CaMK??a is abundant in the brain and is involved in the transmission of nerve excitation between protrusions,and It can specifically initiate gene expression in excitatory neurons.On the contrary,the CAG promoter is a broad-spectrum promoter.Studies have shown that the CAG promoter can mediate gene expression in most cells.In neurodegenerative diseases,nerve cells are extensively damaged.If the CAMK ? ? a promoter can mediate specific expression of foreign genes in nerve cells,will the CAMK??a promoter accelerate the construction of AD animal models compared withthe CAG promoter?,Further investigation is needed.The main purpose of this paper is to find the optimal rAAV serotype that can express the gene of interest in a large amount in the mouse brain.We use two types of promoters,namely the CAG promoter and the CAMK??a promoter,and three different rAAV serotypes,including rAAV2,rAAV8,and rAAV9,and package three serums under the action of two different promoters.Type rAAV.By packaging under the same promoter,three different serotype viruses expressing the RFP gene at the same time compare the affinity of different serotype viruses in the mouse brain;by packaging the same virus expressing the GFP target gene under different promoters Serotype viruses compared the CAG promoter and CAMK??a promoter gene transduction efficiency in mouse brain.In order to optimize the method of animal model construction,this paper also carried out a series of methodological establishments,including the optimization of rAAV packaging method,the selection of animal model construction materials and the use of virus injection methods.Our results show that compared to rAAV2 and rAAV8,rAA9 has a more prominent ability to transduce and express the gene of interest in the mouse brain.At the same time,the in vivo virus infectivity test under the action of the CAG promoter and the CAMK??a promoter showed that the CAMK??a promoter did not specifically express foreign genes in nerve cells.Therefore,the use of CAG promoter and type 9 rAAV virus in the construction of animal models is more conducive to the induction of gene transduction and expression in brain tissue.We will also use the CAG promoter and type 9 rAAV to construct AD model mice that overexpress A? and pTau.