Targeted gene delivery by AAV

The potential utility for targeted gene addition has been recognized and a number of different approaches are currently under evaluation. A variety of viruses naturally establish latency by integrating their genome into the host genome. The non-pathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene (within the AAVS1 locus). To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved in spite of the resulting rearrangements. A latently infected, Mbs85-targeted mouse embryonic stem (ES) cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multi-lineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a novel viral strategy for targeted genome addition with the apparent absence of functional consequences.