The Libraries Building And Screening Of Chimeric AAV Targeting Liver Cancer Cells

Adeno-associated virus (adeno-associated virus, AAV) belongs to the parvovirus families, and itsgenomic DNA is single, linear molecule which contains inverted terminal repeats (ITRs, about145bps long) that contain the same structure at each end. It is between ITRs that there are twoopen reading frame containing Rep gene and Cap gene respectively. Now more than100serotypes of AAV have been identified, and AAV1-9are most commonly used.The traditional methods of cancer treatment mainly includes the radiation and chemotherapy,surgical resection, and with the development of modern biological science and technology, wehave developed virus therapy, gene therapy and combination virus-gene therapy. A largenumber of scientists are working in these areas. Traditional treatment technique (except forsurgical resection) have targeting problems, and virus therapy, gene therapy and combinationvirus-gene therapy have the same problem. Shuffling technology is promising in solving thetargeting problem. Not only it can make point mutation in gene sequence, but also allowinsertion, deletion, inversion and integretion, even shuffling the genes shuffled again and again,which makes shuffling have great advantage over other technologies.The targeting of AAV depends on its Cap protein coding by Cap gene, and we can get kinds ofchimeric cap genes by shuffling cap genes, which code diversified proteins that have verydifferent targeting properties. Consequently, we may get a kind of Cap genotype targeting acertain cell from the Cap gene pool built by shuffling. Among all the AAV serotypes, AAV2hasbeen studied deepest than others and its biological properties are clearest, so it is safest to use itsRep gene as the common Rep gene of the recombant AAV. Based on these principles, we madeour studies as follows:(1) We made pAdB vector with AAV2ITR and Rep2gene by reforming pAAV-MCS vector. Thegenes of AAV1-9Cap werr cut into100-300bp fragments randomly by DNaseI, then they wererebuilt by PCR into chimeric Cap sequences. In the following, pAdB vector and the chimericCap gene were linked into pARC vectors after digested by the same restriction enzyme.Repeating the progress again and again, we’ve made the pool of pARC vector containingchimeric Cap genes.(2) Chimeric AAV viruses were packaged by the cotransfection of293cells with Ad5and pARC vectors. The reason we added Ad5into293cells is that Ad5is essential for the replication ofAAV.Then we used chimeric AAV virus and Ad5to coinfect BEL7402liver cancer cells to getthe chimeric AAV virus that can effectively infect BEL7402cells by three rounds of coinfection.3) In the end, we tested the efficiency of the chimeric AAV we’d gotten.First, we linkedRep2-Chimeric Cap genes to pMD18-T vector to consturct pARC-T vector; then we cotransfect293cells with pARC-T plasmid, AAV-EGFP plasmid and Ad5，and got the AAV virus includingEGFP gene and coated AAV Cap capsid to infect BEL7402cells to express the EGFP proteins.EGFP gene were expressed, which proved that we had screened the chimeric AAV virustargeting BEL7402cells.This study showed that the method and the technology are feasible, and provided beneficialexploration for further expansion of the Chimeric AAV pool and deeper research.