村松 慎一

GM2 gangliosidoses, including Tay-Sachs (TSD) and Sandhoff (SD) diseases, are lysosomal storage disorders with neurological manifestations caused by the excessive accumulation of GM2 ganglioside due to the deficiency of the β-hexosaminidase A (HexA). Although gene therapy approaches are underway, concerns regarding efficacy and safety remain. Here, we evaluate a tyrosine-mutant adeno-associated virus serotype 9 (AAV9/3) vector encoding modified HEXB (modHEXB) wherein nine amino acid residues are substituted from HEXA. The intracerebroventricular administration of AAV9/3-modHEXB in SD mice results in modHexB expression in the brain, reduces GM2 accumulation, and attenuates neuroinflammation. Furthermore, AAV9/3-modHEXB rescues motor function, and longer lifespan in SD mice. In addition, intrathecal administration in non-human primates and rats demonstrates broad biodistribution and an overall favorable safety profile. These findings support the translational potential of AAV9/3-modHEXB as a gene therapy approach for TSD and SD.

Hearing impairment, one of the most prevalent sensory disorders, remains a major risk factor for dementia in the aging population. Although interventions such as hearing aids and cochlear implants provide partial benefit, they do not address the underlying pathology of sensorineural hearing loss. Inner ear gene therapy has attracted significant attention as a promising approach; however, its clinical translation requires minimally invasive and controllable methods for gene activation. We previously developed a photoactivatable Cre recombinase (PA-Cre) system for spatiotemporal regulation of gene expression. In this study, we evaluated the feasibility of irradiating the external auditory canal (EAC) and tympanic membrane (TM) as minimally invasive approaches for activating cochlear gene expression. Tyrosine-mutant AAV9/3 vectors (AAV.GTX) encoding PA-Cre and a Cre-dependent reporter (sfGFP-to-tdTomato) were injected via the round window membrane in 9-week-old C57BL/6J mice. Seven days later, light irradiation was applied using three approaches: (1) Direct cochlear irradiation via postauricular access, (2) TM irradiation with a fiber-optic probe, and (3) noninvasive EAC irradiation through the intact TM. Recombination efficiency in inner hair cells (IHCs) was quantified using whole-mount immunohistochemistry. AAV.GTX efficiently transduced IHCs and drove robust sfGFP expression. In the absence of light, tdTomato expression remained minimal (<5%), indicating low basal Cre leak activity. Direct cochlear irradiation produced strong recombination (conversion rate: 88.4 ± 1.5%), confirming the functionality of PA-Cre in the mouse inner ear. TM and EAC irradiation yielded high conversion efficiencies (95.8 ± 1.7% and 97.6 ± 1.2%, respectively), comparable to direct irradiation, while preserving cochlear integrity. These findings indicate that PA-Cre functions effectively in the mouse cochlea with minimal leak activity and that TM and EAC irradiation enable robust, minimally invasive gene activation. This strategy highlights the light-mediated, noninvasive modulation of cochlear gene expression, informing future translational development.

Niemann–Pick disease type C1 (NPC1) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants of the NPC1 gene that encodes a protein essential for lysosomal cholesterol transport. A deficiency in NPC1 results in the accumulation of unesterified cholesterol and sphingolipids, leading to neurological, psychiatric, and hepatic manifestations from infancy to adulthood. The currently approved treatment is palliative. Although the efficacy of gene therapy has been demonstrated in murine models, reliable biomarkers for evaluating the treatment effects remain unknown. We evaluated adeno-associated virus (AAV) vector-mediated NPC1 gene therapy in Npc1 homo-knockout ( Npc1 ^−/− ) mice, focusing on blood-based biomarkers. An AAV vector carrying human NPC1 under a cytomegalovirus promoter (AAV- hNPC1 ) was administered intraperitoneally on days 6–8 after birth at varying vector doses and analyzed at multiple time points: 1.8 × 10 ¹¹ vector genomes/mouse analyzed at 7 weeks (Low/7w) and 1.0 × 10 ¹² vector genomes/mouse at 4 weeks (High/4w) and 9 weeks (High/9w). hNPC1 is expressed in the brain and liver, and a degree of neuronal cell survival is observed. High-dose AAV treatment improves body weight and rotarod performance. Plasma N -palmitoyl- O -phosphocholine-serine (PPCS) and lysosphingomyelin (lyso-SM) levels were significantly elevated in Npc1 ^−/− mice. PPCS increased with disease progression but was significantly decreased after later points of high-dose AAV treatment (saline-treated Npc1 ^−/− mice: 12.88 ± 3.53 ng/mL, AAV-treated Npc1 ^−/− mice: 7.87 ± 1.67 ng/mL, p = 0.0008). Lyso-SM and oxysterols showed limited changes after therapy. Vector genome analysis revealed higher and more sustained levels in the brain than in the liver, which is consistent with rapid hepatocyte proliferation-reducing vector persistence. These findings demonstrate that systemic AAV- hNPC1 therapy ameliorates motor and neurological deficits but has a limited impact on several cholesterol-related biomarkers. PPCS has been suggested as a sensitive biomarker of therapeutic response and warrants further evaluations in preclinical and clinical NPC1 gene therapy trials.

Shati/nat8l catalyzes the synthesis of N-acetylaspartate (NAA), a precursor for N-acetylaspartylglutamate (NAAG), an endogenous agonist of group II metabotropic glutamate receptor 3 (mGluR3). Although spinal mGluR3 is known to modulate nociceptive signaling, the functional role of Shati/nat8l in pain transmission has remained unclear. In this study, we investigated the involvement of spinal Shati/nat8l in mechanical nociceptive processing and neuropathic pain. We found that Shati/nat8l knockout (Shati-/-) mice exhibited a significantly decreased mechanical pain threshold compared to wild-type controls. This hypersensitivity was reversed by adeno-associated virus (AAV)-mediated expression of Shati/nat8l in the spinal dorsal horn. Intrathecal administration of NAAG-but not NAA-restored mechanical thresholds in Shati-/- mice, and this effect was blocked by the group II mGluR antagonist LY341495. In addition, treatment with LY341495 showed antinociceptive effect in normal mice at higher doses. In a peripheral nerve injury model, expression of Shati/nat8l mRNA in the ipsilateral dorsal horn was significantly decreased. Importantly, AAV-mediated restoration of Shati/nat8l expression in the dorsal horn alleviated neuropathic mechanical hyperalgesia and normalized Shati/nat8l mRNA levels. These findings suggest that downregulation of spinal Shati/nat8l contributes to mechanical hypersensitivity by impairing the NAAG-mGluR3 signaling pathway. Targeting the Shati/nat8l-NAAG-mGluR3 axis may offer a novel therapeutic strategy for the treatment of neuropathic pain.