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.
Huang et al. (Sun,) studied this question.