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

•Intrathymic delivery of AAV results in vector integration within TCR genes at RAG-induced DNA breaks produced during V(D)J recombination.•This “targeting” approach opens therapeutic avenues for long-term AAV gene transfer in dividing cells without any toxic conditioning. [Display omitted] Adeno-associated virus (AAV) vectors have been successfully 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 after the generation of double-strand DNA breaks or nicks. Although 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 and compared these with liver and brain tissue from treated mice. Notably, we found that although 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 (RAGs) during variable, diversity, and joining 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. In a novel gene therapy approach, Calabria et al delivered adeno-associated virus vectors (AAVs) by direct injection into mouse thymus in vivo and demonstrate that over 90% of the transgene integration sites were within T-cell receptor (TCR) gene loci. Integration occurs through exploitation of TCR gene rearrangement during variable, diversity, and joining segment rearrangement through the activity of recombination activation genes. Whether this can be translated into human gene therapy remains to be established.