Senataxin (SETX) is a highly conserved helicase involved in a large variety of biological processes, such as transcriptional regulation, pre-mRNA processing and maintenance of genomic integrity. Recently, loss-of-function genetic screening experiments performed at the Garaigorta laboratory (CNB-CSIC) identified SETX as a restriction factor of exogenous DNA expression. In this study we have investigated the role of SETX on rAAV transgene expression in vitro and in vivo.

For the in vitro studies, SETX was first downregulated in HepG2 cells with an shRNA and then cells were transduced with either single stranded (ss) or self-complementary (sc) rAAV3B vectors encoding an mCherry reporter transgene. SETX downregulation significantly increased transgene expression after transduction with both ss and sc vectors, suggesting that SETX may interfere in the AAV transgene expression process after the synthesis of the double strand of the AAV genome. In vivo, we compared SETX expression in the liver and the brain of WT mice upon AAV-mCherry administration to tissues from control mice. Our results showed an increase of SETX in both tissues transduced with AAV compared to control mice SETX expression was mainly nuclear in the brain but showed significant cytoplasmic localization confirming the role of SETX as a regulator of AAV expression in vivo. Further characterization of SETX subcellular localization in the mouse liver showed that it is mostly associated to the cytoskeleton in the cytoplasm and to chromatin in the nucleus.

In summary, SETX is a restriction factor for rAAV expression that is upregulated when AAV is administered in vivo.