The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of wild-type adeno-associated virus type 2 (wtAAV2) to cellular sites of DNA damage.

genes and attenuated the localization of the viral genome to cellular DDR sites. Chemical inhibition of the KLF4-interacting protein PARP1 with olaparib decreased the ability of wtAAV2 genomes to localize to cellular DDR sites and transcribe viral genes. Ectopic expression of wild-type PARP1, but not its KLF4-binding-deficient mutant, rescued wtAAV2 gene expression in PARP1-deficient cells. These findings define a novel mechanism by which wtAAV2 exploits the interaction between host transcription factors and DNA repair machinery to establish a persistent nuclear niche. Insertion of KLF4-binding elements into recombinant AAV2 gene therapy vectors is sufficient to enhance transduction of target cells, providing a framework for engineering vectors with improved nuclear targeting and transcriptional activity.IMPORTANCEWild-type adeno-associated virus type 2 (wtAAV2) has emerged as the preferred platform for engineering gene therapy vectors due to its non-pathogenic nature and ability to persist in host cells long term. However, limited understanding of how wtAAV2 genomes navigate the nuclear environment to establish viral reservoirs has hindered the development of efficient recombinant AAV (rAAV) vectors. We demonstrate that the protein KLF4 bound to the wtAAV2 genome recruits the virus to cellular KLF4 sites bound by PARP1. Disruption of either KLF4 binding, PARP1 activity, or KLF4-PARP1 interaction significantly impairs wtAAV2 localization and transcription, highlighting the importance of their function in the non-replicative wtAAV2 life cycle. KLF4 binding sites are sufficient to improve the expression of transgenes from rAAV vectors and increase their association with cellular DNA damage response proteins. This study advances our understanding of wtAAV2-host interactions and opens new avenues for improving rAAV gene therapy platforms.