Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery.
Adeno-associated virus (AAV) is a widely used vector for gene delivery, yet the host intracellular trafficking barriers often limit its efficacy. Here, we identify microtubule detyrosination--a tubulin post-translational modification--as a key regulator of AAV2 endo-lysosomal processing. Using super-resolution microscopy (SIM/STORM), we show that upon AAV2 endocytosis, the host upregulates microtubule detyrosination via GSK3{beta}-CLASP2 signaling axis. Single particle tracking of the virus reveals that detyrosinated microtubules form a physical and functional barrier, restricting AAV2 motility and promoting lysosomal trapping. Restoring microtubule tyrosination--via tubulin-tyrosine ligase overexpression or pharmacological inhibition of detyrosination with parthenolide--boosted AAV2 endosomal escape, perinuclear accumulation, and gene delivery in cells. Notably, a clinically relevant prodrug of parthenolide, DMAPT, also displayed a similar trend of enhanced AAV2-driven factor IX expression in hemophilia B mouse models. Our findings uncover a host mechanism that reshapes the microtubule landscape to restrict AAV2 trafficking and identify microtubule detyrosination as a novel druggable target to improve AAV2-based gene therapies.