IN VITRO ANALYSIS OF AAV TARGETED INTEGRATION

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Adeno-associated virus has been round to integrate preferentially at a specific locus on human chromosome 19 (the preintegration site). The primary objective of this fellowship proposal is to characterize the targeted integration of AAV by using in vitro systems. The specific goals of this project are divided into two aims: 1. to develop a cell-free system with which we can analyze the targeted integration reaction of adeno-associated virus in vitro; and 2. to determine the minimal cis and trans-acting elements from AAV and the preintegration locus required for the in vitro integration event. To accomplish these aims, we will establish an in vitro integration system for AAV by using two plasmid DNA substrates: one containing a minimal cis-element from AAV (a modified terminal repeat), and the second containing a cloned 2.6 kb fragment of DNA from the chromosome 19 preintegration site. These plasmids will be used in conjunction with host cell extracts and recombinant AAV Rep protein(s) to reconstitute the targeted integration reaction in vitro. AAV Rep proteins are believed to be required as trans-acting factors for targeted integration of AAV. Genetic selection assays will be developed to detect and characterize the products of the integration reactions. The experiments proposed for Aim 2 will establish the minimal cis and trans- acting elements required from both AAV and the preintegration site for targeted integration in vitro by employing the assays developed in Aim 1. The long term objectives of this proposal are to identify the factors from AAV, the preintegration site, and the host cell required for the targeted integration of AAV into the locus on human chromosome 19 and to determine a possible mechanism for this unique reaction.