Conditional site-specific integration into human chromosome 19 by using a ligand-dependent chimeric adeno-associated virus/Rep protein

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integrati on site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genom e and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the onl y viral elements necessary and sufficient for site-specific integration. Ho wever, it is also known that unrestrained Rep activity may cause nonspecifi c genomic rearrangements at AAVS1 and/or have detrimental effects on cell p hysiology. In this paper we describe the generation of a ligand-dependent f orm of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncate d form of the hormone binding domain of the human progesterone receptor, wh ich does not bind progesterone but binds only its synthetic antagonist RU48 6. The activity of this chimeric protein, named Rep1-491/P, is highly depen dent on RU486 in various assays: in particular, it triggers site-specific i ntegration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner , as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.