Transient Expression Of REP Portein Mediates The Interest Gene To Intergrate Into The AAVS1Region In A Site-specific Manner

Background&Objective: Gene thrapy means target cells to the introduction of thenormal function of the gene in order to correct or compensate for the arising of defectsso as to achieve the purpose of treatment of disease. The most critical step in genetherapy is how the corresponding position of the exogenous gene introduced into a hostcell chromosome accurate and secure in order to achieve the purpose of correctingdeficiencies gene.The adeno-associated virus (AAV) genome can be stably integratedinto the AAVS1region of human chromosome19(19q13.4-qter) with the assistance ofRep68/78protein. In the current models of AAV integration in a locus-specific manner,the foreign genes were randomly inserted into the AAVS1region, which containsseveral functional genes. As random integration in this region may lead to insertionmutations and disrupt normal gene expression or critical signaling pathways of the hostcells, Rep is assisted AAVS1regional integration indispensable ingredient, Reprandomly integrated into Genomic DNA excluding the AAVS1region, causingmutations,the Rep plasmid transfection method may lead to a sustained and excessiveexpression of the gene,leading to cell toxicity and lead to cancer risk, so the in AAVS1area to find a precise integration sites and how to regulate the expression of Rep is veryimportant,it is necessary to find a precise insertion site in the AAVS1region.Homologous recombination is the most accurate and versatile mechanism for suchsite-specific integration, but the recombination rate is very low, this article by thebuilding constructed vector pTAVEN with Rep mRNA transfected into Hela cells to regulate the expression of the REP protein, to improve the homologous recombinationthe rate of AAVS1region, which would be beneficial to improve the safety andeffectiveness of the gene therapy.Methods: To investigate site-specific integration in the AAVS1region, a targeted vectorcontaining two homologous arms derived from AAVS1and a reporter gene was transfct-ed into HeLa cells with or without Rep68/78mRNA, then con-transfection into Helacells, The time and the expression of Rep protein expression detected by Western blot,cell of GFP+cells sorted by fluorescence-activated cell sorter, G418screening positivecell clones, PCR detection of the genomic DNA of the homologous recombination ofcell clones, south blot to Streaming surgery by fluorescence cell sorting cells screenedpositive cell clones, and then southern blot after the experimental and control groups forhomologous recombination fragment detection, comparing the two homologous recomb-ination rate differences.Results: Rep protein transiently expressed only and reduces the cytotoxicity. RepmRNA of transfection can better promote the site-specific integration of the host cell, inpTAVEN and Rep mRNA co-transfected into HeLa Cells,29.4%cell clones, theexogenous gene into AAVS1specific sites. Far higher than that of the control group,indicating the Rep protein regulation in ch19q13.4-ter AAVS1region homologousrecombination. The results indicated that transient expression of Rep68/78in HeLa cellsimproved integration of the gene of interest at the AAVS1locus in a site-specificmanner. The site-specific integration may minimize the risk associated with randomDNA integration in the AAVS1region.