AAV vectors have limited impact on host chromatin accessibility and nuclear architecture

Recombinant adeno-associated virus (AAV) vectors are widely used for gene therapeutics, yet their early effects on host chromatin remodeling and nuclear organization remain insufficiently characterized. In contrast, several DNA viruses are known to remodel host chromatin accessibility, for example HSV-1 in mammalian cells and baculoviruses in insect systems. Here we evaluated genome accessibility and nuclear organization of cultured primary human cells at 24 and 48 hours after infection with wildtype AAV2, single-stranded (ss) and self-complementary (sc) recombinant AAV2 in primary human fibroblasts, as well as ssAAV-DJ in human lung carcinoma cells. Genome-wide ATAC-seq showed no detectable change in host chromatin accessibility at either time point. A DNase I digestion assay at five candidate loci supported this observation. Although host accessibility remained stable, immunofluorescence-based single-cell analyses uncovered modest changes in histone abundance, RNA polymerase II associated signals, nuclear morphology, and the organization of liquid-liquid phase-separated nuclear compartments. These effects were generally mild in primary fibroblasts but showed greater dependence on vector type, cell cycle state, and transgene expression in carcinoma cells. Collectively, at the tested doses and times, AAV-based vectors did not remodel chromatin accessibility at scale and induced only small changes in polymerase associated readouts and nuclear architecture. This data is consistent with limited nuclear perturbation under these conditions.