Cellular redox state alters recombinant adeno-associated virus transduction through tyrosine phosphatase pathways

Several types of environmental damage including UV, hydroxyurea and ionizin g irradiation have been shown to augment rAAV transduction. Current hypothe ses suggest that these environmental stimuli lead to the enhanced productio n and/or activation of cellular factors important in the conversion of sing le-stranded DNA genomes to expressible forms. However, the mechanisms of ac tion are currently unknown. We hypothesized that reactive oxygen intermedia tes (ROI) may play a common role in the augmentation of rAAV transduction b y these environmental stimuli. Our results demonstrate that treatment with hydrogen peroxide can give equivalent or greater levels of augmentation in rAAV transduction as that seen by hydroxyurea or UV irradiation. For all en vironmental stimuli, pretreatment with the hydroxyl radical (HO)scavenger, N-acetyl-L-cysteine (NAC), completely blocked augmentation of rAAV transduc tion. Furthermore, using electron spin resonance spectroscopy (ESR), we dem onstrated that both UV and H2O2 treatment of cell lines lead to the inducti on of HO radicals. Our results demonstrating that NaOV inhibits the augment ation of rAAV transduction following UV and H2O2 treatment, implicate HO ra dicals as modulators of tyrosine phosphatase pathways involved in rAAV tran sduction. Alterations in the cellular redox state and subsequent activation of tyrosine phosphatase pathways appear to alter the phosphorylation statu s of the previously identified single-stranded sequence binding protein (ss DBP), with reduced phosphorylation correlating with an enhancement in rAAV transduction. In summary, we conclude that the cellular redox state may pla y an important role in regulating rAAV transduction.