The interaction between virus-bound KLF4 and host-bound PARP1 directs the localization of Adeno-Associated Virus Type 2 (wtAAV2) to cellular sites of DNA damage

Wild-type adeno-associated virus type 2 (wtAAV2) is a small, non-pathogenic DNA virus within the family Parvoviridae that is modified to engineer recombinant AAV (rAAV) vectors used in gene therapy. wtAAV2 genomes localize to nuclear domains enriched in DNA Damage Response (DDR) factors through mechanisms that remain unknown. We have discovered that the host transcription factor KLF4 (Kruppel-like Factor 4) and the DDR protein PARP1 (poly-ADP ribose polymerase 1) are key effectors regulating wtAAV2 nuclear trafficking. In-silico analysis revealed that wtAAV2-associated genomic sites are enriched in KLF4 binding motifs. Confocal imaging demonstrated a close spatial association between KLF4 and wtAAV2 nuclear reservoirs, consistent with KLF4s known ability to form nuclear condensates. Mutation of the KLF4 binding site within the wtAAV2 genome and RNAi-mediated knockdown of KLF4 globally reduced the expression of viral Rep68/78 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 (rAAV2) 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 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 DDR proteins. This study advances our understanding of wtAAV2-host interactions and opens new avenues for improving rAAV gene therapy platforms.