Intervertebral disc degeneration (IVDD) is closely associated with cellular senescence and defective autophagic degradation, but the molecular heterogeneity and functional significance of lysosome-related alterations remain incompletely understood. We integrated bulk transcriptomic datasets (GSE56081 and GSE70362) and single-cell RNA-sequencing data (GSE153066) using consensus clustering, weighted gene coexpression network analysis, machine-learning algorithms, immune-signature analysis, and covariate-adjusted correlation testing. Candidate genes were evaluated in human and rat disc tissues, and PLD3 was further examined by knockdown and overexpression in H2O2-treated nucleus pulposus cells (NPCs). Lysosomal function was assessed using LysoTracker staining, cathepsin activity, and autophagy-related markers, and the preclinical effects of lithocholic acid (LA) were evaluated in cultured NPCs and a rat needle-puncture model. Two lysosome-related molecular subtypes were identified: a senescence/inflammation-enriched subtype and a metabolism-enriched subtype. The lysosomal gene-signature score was positively associated with the senescence score after adjustment for total cellular transcript counts (partial r = 0.422; empirical permutation p = 0.0005), supporting coordinated transcriptional activation rather than enhanced degradative function. HYAL1, MMD, PLD3, and ANK3 were prioritized as candidate hub genes. PLD3 knockdown aggravated H2O2-induced lysosomal impairment, matrix degeneration, and senescence, whereas PLD3 overexpression produced opposing protective effects. LA partially improved acidic lysosomal compartments, cathepsin activity, autophagic degradation, and senescence-associated changes in vitro and attenuated degeneration-associated histological and molecular alterations in vivo. These findings reveal distinct lysosome-related phenotypes in IVDD, functionally support PLD3 as a contributor to lysosomal homeostasis, and suggest that lysosome-modulating interventions may have therapeutic potential.

GNE myopathy is an autosomal recessive disease, associated with skeletal muscle deterioration, which afflicts young adults. GNE plays a pivotal role in sialic acid production. Sialic acid acts as a buffer against reactive oxygen species generated during muscle contraction. Increased oxidative stress may relate to muscle atrophy involving patients with GNE myopathy. GNE[M743T] is the most common mutation leading to GNE myopathy. In our previous work, we demonstrated that a bifunctional plasmid that expresses wild type (wt) GNE and knocks down the GNE[M743T] mutant improves sialic acid production in vitro. Now, we expand evidence of in vivo activity of the bifunctional plasmid using a DOTAP-Cholesterol delivery vehicle and reduced toxic plasmid components using dbDNA conversion. We demonstrate that IV delivery of dbDNA lipoplex (LPX) in murine and rat models shows safety, increased DNA delivery, and improved RNA expression per LPX in skeletal muscle over non db plasmid at equal dose. Sialic acid protein expression was also shown increased in mouse muscle following IV treatment with dbDNA plasmid (pDNA) GNE[wt]/bi-shRNA-GNE[M743T] LPX. These results support further preclinical investigation to justify product IND development towards Phase 1 trial involving patients with GNE myopathy.