Yun-Ru Chen

Background: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, resulting in α-galactosidase A (α-Gal A) deficiency and progressive accumulation of globotriaosylceramide (Gb3). Current therapies, such as enzyme replacement and chaperone therapy, have limitations, including incomplete biodistribution and mutation-specific efficacy. Gene therapy using adeno-associated virus (AAV) vectors presents a promising alternative. Methods: In this study, we assessed the dose-dependent effects of AAV8 and AAV9 vectors encoding human GLA in Gla knockout (Gla^−/y) mice by measuring α-Gal A activity and monitoring safety. To evaluate therapeutic efficacy, symptomatic Fabry mice (G3S^Tg/+Gla^−/y) were used. Results: AAV9-GLA produced significantly higher and more sustained enzyme activity than AAV8-GLA across plasma, liver, heart, and kidney. In symptomatic mice, AAV9-GLA achieved superior reductions in serum Gb3 and lyso-Gb3 levels, greater Gb3 clearance in heart and kidney tissues, and improved proteinuria. Anti-GLA IgG titers remained below threshold for the first four weeks and increased modestly by week eight, indicating a limited humoral immune response. No significant clinical signs or weight loss were observed in Gla^−/y mice over the 3.5-month study period, supporting the favorable safety profile of AAV-mediated gene therapy. Conclusions: These findings demonstrate that AAV9 provides enhanced biodistribution and therapeutic efficacy compared to AAV8, supporting its potential for the treatment of Fabry disease.

Background: Fabry disease (FD) is a lysosomal storage disorder impacting multiple organs, including the heart. We investigated whether early-stage globotriaosylceramide (Gb3) accumulation, before occurrence of inclusion bodies, could cause significant stress and irreversible damages of the cardiomyocytes in FD patients. To assess the cellular stress and irreversible damage of cardiomyocytes in FD during early-stage Gb3 accumulation before the occurrence of typical pathology. Methods: Immunofluorescent (IF) staining or Western blotting were performed on fibroblasts from FD patients and myocardial biopsies from G3Stg/GLAko mice and FD patients. Notably, all biopsies exhibited detectable Gb3 accumulation under IF but lacked typical FD (Gb3 inclusion body) pathology. Staining targeted nuclear factor-κB (NF-κB), interleukin-18 (IL-18), phospho-p42/44 mitogen-activated protein kinase (MAPK), and inducible nitric oxide synthase (iNOS) as inflammatory and oxidative stress markers. Alpha-smooth muscle actin (α-SMA) IF staining was conducted to detect myofibroblasts. Results: Fibroblasts from FD patients, in conjunction with cardiomyocytes from both G3Stg/GLAko mice and FD patients, exhibited significant accumulation of inflammatory markers such as NF-κB IL-18 and phospho-p42/44 MAPK, as well as the oxidative stress marker iNOS. Despite the absence of typical FD pathology, the presence of fibrosis was confirmed in myocardial biopsies from these patients through strong positive staining of α-SMA. Conclusions: Significant cellular stress and even irreversible damage may occur before the onset of typical pathological changes in cardiomyocytes of FD. Based on our findings, treatment should be initiated much earlier than we currently thought to prevent irreversible damage and improve the prognosis of FD patients.

Background Fabry disease (FD) is a rare X-linked lysosomal storage disorder that affects multiple organs including the heart. In this study, we investigated if early-stage globotriaosylceramide (Gb3) accumulation, before occurrence of inclusion bodies, could cause significant stress and irreversible damages of the cardiomyocytes in FD patients. Methods Immunofluorescent (IF) staining was performed on fibroblasts from FD patients with IVS4 mutation and myocardial biopsies from G3Stg/GLAko mice and IVS4 FD patients. The staining targeted interleukin-18 (IL-18), nuclear factor-κB (NF-κB), and inducible nitric oxide synthase (iNOS) as inflammatory and oxidative stress markers. Remarkably, the mouse and human myocardial biopsies selected for this study showed detectable Gb3 accumulation under fluorescence but did not exhibit typical FD (Gb3 inclusion body) pathology. Additionally, alpha-smooth muscle actin (α-SMA) IF staining was conducted to detect fibrosis. Results The results showed that fibroblasts and cardiomyocytes of mice and patients had significant accumulation of inflammatory markers IL-18 and NF-κB, and oxidative stress marker iNOS. The presence of fibrosis was confirmed in these myocardial biopsies through strong positive staining of α-SMA, despite the absence of typical FD pathology. Conclusions This study suggests that significant cellular stress and even irreversible damages could exist before the typical pathological changes (inclusion bodies formation) in cardiomyocytes of FD. Based on our findings, it is evident that to prevent irreversible damage and improve the prognosis of patients with FD, treatment should be initiated much earlier than we currently thought. What Is New? Evidence suggests significant cellular stress and potential irreversible damage may precede inclusion body formation in Fabry cardiomyocytes. Inclusion body formation likely represents a later stage event following intracellular Gb3 accumulation past a critical threshold. What Are the Clinical Implications? These findings indicate a need to re-evaluate conventional Fabry diagnostic criteria. There is an emphasis on early therapeutic intervention to mitigate progressive cellular dysfunction, inflammation, and fibrosis.

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