Inhibition of DNA-dependent protein kinase catalytic subunit boosts rAAV transduction of polarized human airway epithelium

Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PK(cs) inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PK(cs) inhibitors worked as a “booster” to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PK(cs) inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.