ADENOASSOCIATED VIRUS REP78 PROTEIN AND TERMINAL REPEATS ENHANCE INTEGRATION OF DNA-SEQUENCES INTO THE CELLULAR GENOME

Two adeno-associated virus (AAV) elements are necessary for the integr ation of the AAV genome: Rep78/68 proteins and inverted terminal repea ts (ITRs), To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration effici encies of three different plasmids containing a green fluorescent prot ein (GFP) expression cassette, In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter ge ne (integration cassette), and a third plasmid consisted of an integra tion cassette plus a Rep78 expression cassette, One day after transfec tion of 293 cells, fluorescent cells were sorted by Bow cytometry and plated at 1 cell per well, Two weeks after sorting, colonies were moni tored for stable expression of GFP, Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent coloni es, Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid conta ining both the integration cassette and Rep78 expression cassette prod uced stable fluorescent colonies at a frequency of 47% +/- 7.5%. South ern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVS1 site in more than 50% of the clones analyzed , Some clones also showed tandem arrays of the integrated GFP cassette , Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.