Construction Of AAV Libraries And Screening Of Neural Cell-targeting AAV Vectors Based On DNA Shuffling Strategy

Background The nervous system is the most complex system in human body in terms of structure and function,and it is still difficult to treat neurological diseases with traditional drugs and surgical procedures.High-throughput library establishment and targeted enrichment screening can obtain novel AAV capsids for efficient targeted delivery to the central nervous system,and this modification strategy has been widely used.Objective By interrupting ten serotype Cap genes of AAV1,AAV2,AAV5,AAV6,AAV7,AAV8,AAV9,AAV10,AAV-DJ and AAV9.37 after amplification and further random recombination,a chimeric library was successfully obtained and then screened in three rounds in the cerebral cortex of female BALB/c mice and U87 cell line,respectively,to finally AAV serotypes highly targeted to the central nervous system were obtained.Methods(1)Molecular cloning to construct the core vector of the library virus.(2)DNA Shuffling technique to construct a chimeric Cap gene library.(3)Molecular cloning to construct a chimeric plasmid library pAAV-SCAP.(4)Plasmid cotransfection of 293 T cells to package AAV-WK virus.(5)Tail vein injection of virus into BALB/c mice to complete screening at the individual level.(6)AAV-WK infected U87 cell line to complete the screening at the cellular level.(7)Sanger sequencing technique was used to analyze the screening sequences.Results(1)The core vector of the library virus was successfully constructed.(2)The chimeric Cap gene library was successfully constructed by DNA Shuffling technique.(3)The chimeric plasmid library pAAV-SCAP was obtained.(4)The AAV-WK virus was successfully packaged.(5)Successfully completed two rounds of screening experiments on BALB/c mice.(6)Successfully completed four rounds of screening experiments on U87 cell line.(7)Sanger sequencing technology was used to analyze the screening sequences.Conclusion In this study,we initially established an AAV chimeric library constructed based on DNA Shuffling strategy,and identified library candidate viruses by sequencing.(1)The pAAV-SCAP plasmid library was successfully constructed.(2)AAV-WK virus library was successfully packaged.