Abstract

Recombinant adeno-associated virus (rAAV) is a prominent vector for gene therapy; however, its transduction efficiency is hampered by intrinsic intracellular barriers. This study investigates the regulatory role of the lysosome-resident chloride/proton antiporter CLC-7 in rAAV trafficking and transduction. Using siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that targeted disruption of CLC-7 function significantly enhances rAAV transduction efficiency in multiple in vitro cell models and in vivo murine model. Mechanistically, CLC-7 depletion alters lysosomal chloride homeostasis, leading to selective reduction in the catalytic activity of cathepsins B and L—key proteases involved in rAAV capsid processing —without impacting the activity of the Cl⁻-independent aspartic protease cathepsin D. Consequently, rAAV accumulates in lysosomes with delayed capsid degradation, with facilitates subsequent lysosomal escape of intact virions. Collectively, our findings identify CLC-7 as a critical negative regulator of rAAV transduction through modulation of lysosomal protease activity, providing a novel therapeutic target to optimize rAAV-based gene delivery strategies.