Preparation And Activity Analysis Of Recombinant Adeno-Associated Virus(rAAV) Expressing Single-Chain Antibody Against P-Tau

Alzheimer’s disease(AD)is a progressive neurodegenerative disease and one of the most common forms of dementia.According to reports,the number of AD patients worldwide is expected to reach 152 million by 2050,with an average of one AD patient appearing every 3seconds.At present,the prevalence of AD in the elderly over 65 years old in China is 5.9%,and there are about 7 million patients in China currently.The incidence of AD increases by years,and the development trend of AD is not optimistic.This disease has devastated the physical and mental health of countless patients,and brought a huge burden to the medical and health systems all over the world.Studies have shown that neuronal tangles formed by the highly phosphorylated microtubule-binding protein Tau(phosphorylated Tau,p-Tau)may be the main pathogenic substance of AD.Therefore,using passive immunotherapy which input therapeutic antibodies targeting p-Tau into patients is a promising method to delay the progression of AD disease.At present,many passive immunotherapies targeting p-Tau have entered clinical trials,and the results are anticipated.However,the application of this immunotherapy also has certain limitations,such as the large molecular weight of the whole antibody and the low blood brain barrier permeability,which limits its therapeutic effect.And the Fc segment of the antibody may activate immune cells in the brain,resulting in non-infectious encephalitis.In addition,since AD treatment is a long-term continuous process,how to reduce the number of injections and to improve patient compliance are also problems need to be solved.In order to solve these problems,this study used recombinant adeno-associated virus(r AAV/BBB)which can penetrate the blood-brain barrier as a vector,and expressed a single-chain antibody(AT8-sc Fv)targeting p-Tau.We first designed and constructed a single-chain antibody AT8-sc Fv targeting p-Tau,then expressed in E.coli expression system,purified by nickel-column affinity chromatography system,and its binding ability to antigen was detected by ELISA and immunohistochemistry;we constructed and packaged a blood-brain barrier-penetrating recombinant Adeno-associated virus(AAV/BBB-AT8-sc Fv-m Cherry)capable to express AT8-sc Fv,and using 293 T cells and Balb/c mice as models,respectively,to study the in vitro and in vivo activities of the recombinant adeno-associated virus by Biophotonic imaging,immunohistochemistry,ELISA and other techniques.The results show that AT8-sc Fv constructed in this study has a high affinity for p-Tau and can bind to neuronal tangles in the AD model mouse brain.In addition,the recombinant adeno-associated virus expressing the single-chain antibody can penetrate the blood-brain barrier,infect neuronal cells,and stably express AT8-sc Fv in the mouse brain for up to 22 weeks.In this study,a recombinant adeno-associated virus capable of expressing p-Tau-specific single-chain antibody was constructed,and its in vitro and in vivo activities were studied,providing new ideas for AD immunotherapy.