INTEGRATION OF AAV2 GENOME TO MUSCLE-SPECIFIC DNA REGION

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DESCRIPTION (Copied from Applicant Abstract): Adeno-associated virus type 2 (AAV) is unique in the ability to integrate its genome site-specifically into the human genome in a region on the q-arm of human chromosome 19 designated AAVSI. Most intriguingly, only one viral gene product, the Rep protein, is required to mediate targeted integration, presumably in concert with host cell factors. This characteristic of AAV has made possible a novel concept for therapeutic gene delivery: the targeted integration of a transgene into a defined locus within the human genome. Prerequisite for the usefulness of this approach is the characterization of the human target region as well as of the rearrangements resulting from Rep-mediated AAV DNA integration. We have found that the gene encoding the slow skeletal muscle troponin T (TNNTI) is located Ca. 14 kb telomeric to AA VS1 Furthermore, we show that TNNTI can be disrupted as a result of site-specific AAV DNA integration. These data allow us to answer the following questions: 1. Is transcriptional activity in the region of AAVSI required for and/or does it enhance the AAV integration efficiency? 2. Does transcription from this region, i.e. from the TNNTJ gene, affect expression of a transgene inserted into AA VSI. 3. Does targeted insertion of AAV or rAAV DNA into AAVS1 potentially lead to phenotypic changes in the infected cell due to disruption of the expression of a proximal gene (now shown to be TNNT1). To date, skeletal muscle has been one of the most successful tissues for AAV mediated gene transfer. We propose to answer these questions in order to establish if skeletal muscle might serve as a suitable tissue for site-specific gene delivery into AAVS1.