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Several types of environmental damage including UV, hydroxyurea and ionizing irradiation have been shown to augment rAAV transduction. Current hypotheses suggest that these environmental stimuli lead to the enhanced production and/or activation of cellular factors important in the conversion of single-stranded DNA genomes to expressible forms. However, the mechanisms of action are currently unknown. We hypothesized that reactive oxygen intermediates (ROI) may play a common role in the augmentation of rAAV transduction by 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 environmental stimuli, pretreatment with the hydroxyl radical (H0 small middle dot) scavenger, N-acetyl-L-cysteine (NAC), completely blocked augmentation of rAAV transduction. Furthermore, using electron spin resonance spectroscopy (ESR), we demonstrated that both UV and H2O2 treatment of cell lines lead to the induction of H0 small middle dot radicals. Our results demonstrating that NaOV inhibits the augmentation of rAAV transduction following UV and H2O2 treatment, implicate H0 small middle dot radicals as modulators of tyrosine phosphatase pathways involved in rAAV transduction. Alterations in the cellular redox state and subsequent activation of tyrosine phosphatase pathways appear to alter the phosphorylation status of the previously identified single-stranded sequence binding protein (ssD-BP), with reduced phosphorylation correlating with an enhancement in rAAV transduction. In summary, we conclude that the cellular redox state may play an important role in regulating rAAV transduction.

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