Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci

Abstract

Adeno-associated Viral vectors (AAVs) have been successful exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector but it has been shown to integrate within the host-cell genome following generation of double-strand DNA breaks or nicks. While AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T cell progenitors and long-term T cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T-cells as compared to liver and brain tissue from treated mice. Notably, we found that while AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T-cells were clustered within the T cell receptor α, β and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAG) during V(D)J recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.