pH/ROS dual-responsive SmartGel-Cas9/TRPV4 for autophagy regulation in intervertebral disc degeneration

This study developed a pH/ROS dual-responsive SmartGel platform for local CRISPR-Cas9 RNP delivery and TRPV4 inhibition in intervertebral disc degeneration (IVDD). The GelMA/PBA-based SmartGel responded to acidic and oxidative microenvironmental cues and released Cas9/sgRNA under disease-mimicking conditions. In nucleus pulposus (NP) cells, SmartGel-Cas9/TRPV4 lowered ROS accumulation, restored mitochondrial membrane potential (ΔΨm), and coordinated apoptosis, senescence, Ca 2+ homeostasis, CaMKKβ-dependent AMPK-mTOR signaling, and autophagic flux. CaMKKβ knockdown weakened AMPK-mTOR activation and autophagic-flux restoration, supporting CaMKKβ as a required link in this mechanism. In the rat IVDD model, the system preserved disc architecture, improved hydration status, reinforced extracellular matrix (ECM) homeostasis, and restrained downstream pathological signaling. Exploratory scRNA-seq and proteomic analyses further indicated treatment-associated shifts in Ca 2+ /autophagy/MAPK/Wnt-related programs. Overall, SmartGel-Cas9/TRPV4 may attenuate IVDD by restoring CaMKKβ-associated autophagic degradation and ECM balance while limiting aberrant signaling activation.