CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin [CUT&Run]

Recombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV2) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of rAAV transgenes require large doses, leading to sporadic toxic side effects. We have identified a novel cis-acting element in wtAAV2 bound by the cellular architectural protein CTCF (CCCTC-binding Factor). This CTCF binding element is necessary for efficient wtAAV2 gene expression and is sufficient to enhance the efficiency of rAAV vector’s ability to express reporter transgenes. Chromatin profiling studies reveal that this CTCF binding element regulates the chromatin landscape of the virus and viral vectors. Our discovery of adding merely 19bp of AAV’s CTCF binding element has generated a promising new rAAV gene therapy platform with enhanced transgene expression without impacting vector packaging capacity.