The Study Of Anti-A? Antibody Fusion Protein Delivery Via Adeno-associated Virus For Treatment Of Alzheimer's Disease

Alzheimer's disease is a neurodegenerative disease of the central nervous system characterized by progressive cognitive obstacle.The main neuropathological features are extracellular A? deposition,plaque formation,and intracellular neurofibrillary tangles.At present,the hot spot of drug research and development is antibodies targeting A?,however,the antibody drug treatment needs long-term repeated administration,which will bring heavy burden to the patients.This study explored an innovative drug design program aiming to achieve long-term stable expression of scFv fusion protein targeting A? mediated by AAV9.The results showed that: 1.Three anti-A? antibody fusion proteins with secretory expression,scFv-SUMO3,scFv-Fc-SUMO3 and scFv-Fc,were confirmed,among the eukaryotic transient expression of 7 sequences.The specific binding ability with A? and the biological activities of scFv-Fc-SUMO3 and scFv-Fc were demonstrated by functional evaluation.The purified proteins were prepared as the standard for subsequent content detection,which laid a foundation for subsequent virus packaging and evaluation.2.Three recombinant viruses,AAV9/scFv-SUMO3,AAV9/scFv-Fc-SUMO3 and AAV9/scFv-Fc,were successfully packaged,and high purity recombinant viruses with the titer up to 1.00E+13 vp/ml were gained by production,purification and identification of the viruses.This work made a base for the following efficacy evaluation.3.The recombinant viruses can be expressed effectively in vitro and in vivo.4.The therapeutic effect of neutralization and elimination of A? mediated by AAV9/sc Fv-Fc-SUMO3 and AAV9/scFv-Fc was preliminarily demonstrated by the pharmacodynamic study both in vitro and in vivo.In the process of the project,it was reported that IL-5 can protect A?-induced PC12 cell apoptosis and Tau phosphorylation,thus we supposed that the IL-5 might become a potential therapeutic strategy for Alzheimer's disease.At the same time,IL-5/IL-5R? therapeutic monoclonal antibody for the treatment of eosinophilic asthma is a research hotspot.An innovative reporter gene assay(RGA)was developed and valided based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements.After careful optimization,we demonstrate the excellent specificity,precision,accuracy and linearity of the established RGA.These results show that the novel RGA can be employed in the bioactivity determination of IL-5 and anti-IL-5/anti-IL-5R? biotherapeutics.The establishment of the noval RGA not only has important significance for the screening and quality control of IL-5/IL-5R? monoclonal antibodies,but also provide a valuable tool for the pharmacodynamic evaluation of the potential IL-5 strategy for the treatment of Alzheimer's disease...