Engineered DNA viruses are increasingly used as delivery vectors for gene therapy. Among these, the most promising viral vectors are those that do not naturally integrate into the cellular genome but instead persist as episomes, such as adeno-associated viral vectors (rAAV) and integration-deficient lentiviral vectors (IDLV). In this study, we characterize the role of replication protein A1 (RPA1), a subunit of the RPA heterotrimer, in regulating gene expression from mCherry-expressing rAAV and IDLV vectors. RPA1 is a single-stranded DNA (ssDNA)-binding protein involved in DNA repair and metabolism. Silencing RPA1 in cells led to increased reporter gene expression from both vector systems, suggesting that this protein negatively regulates viral gene expression. Ectopic expression of wild-type RPA1 reduced reporter expression, confirming that the phenotype was not due to off-target effects. Deletion and point mutant versions of RPA1 were employed to further investigate the genetic and functional requirements for the restriction phenotype observed in rAAV-transduced cells. Our results indicate that the restriction occurs in the nucleus, and depends on the ability of RPA1 to bind ssDNA. Similar results were obtained with self-complementary AAV and IDLV vectors. Furthermore, RPA1 depletion increased cellular DNA damage, while RPA1 overexpression restored basal damage levels in a ssDNA binding activity dependent manner. Importantly, the increase in reporter expression correlates with elevated DNA damage, implying that episomal vector DNA may integrate at double-strand breaks generated upon RPA1 loss. Ongoing studies aim to directly confirm this mechanism. Overall, this work identifies RPA1 as a key negative regulator of episomal vector expression, linking DNA repair with the control of transgene integration and expression.