A Breast Cancer Tropic Adeno-associated Virus (AAV)Evolved By DNA Shuffling

Nowadays, traditional therapeutic technology can’t fulfill our demands anymore, but genetic therapy might be a choice for cancer treatment, meanwhile, searching for optimal vector has been an obviously important field.As genetic therapeutic vector, Adeno-Associated Virus(AAV) have those advantages:1) no disease causing possibility brings less safety concern;2) no auto duplicative ability;3) wider host range;4) can long express foreign gene;5) can highly specifically integrate into number19chromatin, and so on. And this makes AAV one of those most important and potential vectors in genetic therapy area. Recently, that AAV benefits genetic therapy for haemophilia, cystic fibrosis, Parkinson, diabetes and some kinds of tumor has been reported.The characteristic of wider host range also makes AAV deficient of specificity to tissue or cell, and this causes the low transfection efficiency of AAV. So improving the transfection efficiency and targeting specificity of AAV are essential for AAV modification. The following methods have been recently reported in AAV modification:1) simple chemical modification;2) capsid gene deletion and insertion;3) exchange the rescue marker areas between different serum types;4) generate chimeric vector, and so on. And taming specified chimeric AAV vector toward one type of tissue or cell has been a hotspot in AAV modification.In this study, we used DNA shuffling method to amplify a capsid gene cap from a AAV mixture of serum type1-9through PCR, then randomized digested by DNaseI, we recycled100-300bp production after electrophoresis to carry out without-primer PCR and with-primer PCR for stochastic chimeric, and connect them to virus packaging vectors to form a series plasmid pAdL of different serum type AAV. By this we established randomized AAV cap gene library. Based on the library, we selected AAV in vitro. After three selections, we found a new type of AAV can specifically target mammary cancer cell sk-br-3, through gene comparion, we confirmed that this AAV chimeric AAV-Y is homologous to AAV1, AAV2, AAV7, AAV8.In further study of its infection trial, we found that although AAV-Y can easily infect mammary cancer cell sk-br-3, AAV-Y has no infectious ability to liver cancer cell BEL-7721at same concentration.