瀬原 吉英

BACKGROUND: Fabry disease is a hereditary disorder caused by a deficiency of α-galactosidase A, leading to the accumulation of globotriaosylceramide (GL-3) in multiple cell types throughout the body. Terminally differentiated non-dividing cells, such as podocytes, are particularly susceptible to such accumulation and therefore require effective therapeutic intervention. Gene therapy is an ideal therapeutic intervention for replacing the deficient enzyme; however, one of the major challenges is maintaining long-term expression of episomal transgenes during cell division. In this regard, podocytes, as non-dividing cells, represent an ideal target for gene therapy in Fabry disease. Nevertheless, it has not been confirmed yet whether gene therapy vectors can transduce podocytes and reduce GL-3 accumulation especially in podocytes. METHODS: Male Fabry disease model mice (TgG3S/GLA knockout mice) received an intravenous injection of 2 × 1012 vector genomes of AAV9 encoding human GLA at 6 weeks of age. Kidney tissues were analyzed 8 weeks after administration by electron microscopy (EM) and immunogold EM. RESULTS: In AAV9/hGLA-treated mice, GL-3 accumulation was markedly reduced in most podocytes, endothelial cells, and tubular epithelial cells, whereas it was evident in untreated mice. Virus-like particles were detected only in treated mice. Furthermore, immunogold EM confirmed the presence of AAV9 particles in the podocytes of treated mice. CONCLUSIONS: AAV9/hGLA gene therapy may reduce podocyte GL-3 accumulation in a Fabry disease mouse model, potentially through AAV9 transduction of podocytes; however, given the technical limitations of our approach, the precise cellular mechanisms remain to be determined.

Gene therapy using adeno-associated virus (AAV) vectors is currently expanding to broad clinical applications. As the presence of a neutralizing antibody (NAb) against AAV capsids significantly restrains their efficacy, an accurate evaluation of NAb status is crucial for selecting appropriate candidates for gene therapy. Notably, cell-based NAb assays may not be sufficiently sensitive for detecting low-titer NAb, and few assays can evaluate multiple AAV serotypes using a commonly available cell. In this study, we developed a sensitive NAb assay against various AAV serotypes using commonly available HEK293 and Huh-7 cells. We found that adding glucose efficiently enhanced transgene expression across various AAV serotypes without causing cell damage. In addition, by combining a highly sensitive reporter gene, NanoLuc, the necessary dose of AAV vector was significantly reduced. The reduction of AAV dose resulted in the increased sensitivity of NAb detection as low as 100 vector genomes/cell. At the lower vector doses, sensitivity improvement was not observed regardless of serotypes, suggesting the limit of assay sensitivity of the cell-based NAb assay. These findings provide a highly sensitive methodology for assessing NAb titers and offer insights into conditions to attain maximal sensitivity in the cell-based NAb assay.

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

BACKGROUND: Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A) encoded by the GLA gene. The symptoms of FD occur as a result of the accumulation of globotriaosylceramide (Gb3), comprising a substrate of α-Gal A, in the organs. Adeno-associated virus (AAV)-mediated gene therapy is a promising treatment for FD. METHODS: α-Gal A knockout (GLAko) mice were injected intravenously with AAV2 (1 × 1011 viral genomes [vg]) or AAV9 (1 × 1011 or 2 × 1012 vg) vectors carrying human GLA (AAV-hGLA), and plasma, brain, heart, liver and kidney were tested for α-Gal A activity. The vector genome copy numbers (VGCNs) and Gb3 content in each organ were also examined. RESULTS: The plasma α-Gal A enzymatic activity was three-fold higher in the AAV9 2 × 1012 vg group than wild-type (WT) controls, which was maintained for up to 8 weeks after injection. In the AAV9 2 × 1012 vg group, the level of α-Gal A expression was high in the heart and liver, intermediate in the kidney, and low in the brain. VGCNs in the all organs of the AAV9 2 × 1012 vg group significantly increased compared to the phosphate-buffered-saline (PBS) group. Although Gb3 in the heart, liver and kidney of the AAV9 2 × 1012 vg was reduced compared to PBS group and AAV2 group, and the amount of Gb3 in the brain was not reduced. CONCLUSIONS: Systemic injection of AAV9-hGLA resulted in α-Gal A expression and Gb3 reduction in the organs of GLAko mice. To expect a higher expression of α-Gal A in the brain, the injection dosage, administration route and the timing of injection should be reconsidered.