水上 浩明

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.

BACKGROUND: Progesterone rapidly induces ovarian cancer cell death through non-genomic actions mediated by the membrane progesterone receptor (mPR). AIMS: We investigated the combined effects of progesterone and SN38, an active metabolite of irinotecan, on ovarian cancer cells. METHODS AND RESULTS: mPR-positive and PR-negative ovarian cancer cell lines were utilized in experiments. Tumor cells were exposed to SN38 or cisplatin for 48 h following exposure to progesterone for 30 min. The viable cell counts were measured using a colorimetric assay and the expression of topoisomerase I (TOPO-I), the direct target of SN38, was observed with or without exposure to progesterone. Moreover, we investigated the relationship between several types of programmed cell death and the SN38 sensitivity enhancement effect of progesterone using specific cell death inhibitors. The chemosensitivity to SN38 was 8.7- to 26.0-fold higher with the administration of progesterone than that without (p < 0.01), but not to cisplatin in ovarian cancer cells. Progesterone suppressed the expression of TOPO-I mRNA by less than 50% (p < 0.01). Furthermore, among various programmed cell death inhibitors, only the ferroptosis inhibitor attenuated the progesterone-induced SN38 chemosensitivity enhancement effect. CONCLUSIONS: Progesterone increased sensitivity to SN38 by suppressing TOPO-I expression and inducing ferroptosis. The combination of progesterone and irinotecan could be a novel treatment modality for ovarian cancer.

Myelination in the visual pathway is critical for transmitting visual information from retina to the brain. Reducing visual experience shortens myelin sheath length and slows the conduction velocity of the optic nerve. However, the mechanism underlying such experience-dependent myelination is unclear. Here, we found that closing both eyes, binocular deprivation (BD), during the juvenile period less affects the optic nerve myelination than monocular deprivation (MD) via GABA signaling. RNA-seq analysis of optic nerves from MD and BD mice revealed that GABAergic signaling is downregulated on the deprived side of MD compared to the intact side and BD. Inhibition of GABAergic signaling during the juvenile period resulted in myelin sheath shortening and excessive oligodendrocyte generation in normal mice, similar to the changes observed in MD mice. Enhancing GABAergic signaling rescued the myelin sheath shortening and excessive oligodendrocyte generation in the optic nerve of MD mice. Furthermore, we identified novel GABAergic neurons located within the optic nerve, whose neurites form belt-like presynaptic structures with the oligodendrocyte lineage cells, suggesting a potential source of the GABAergic inputs into oligodendrocytes. Our results indicate that the myelination of visual pathway is maintained by binocular visual inputs via intra-nerve GABA signaling.

[This corrects the article DOI: 10.3389/fimmu.2019.00730.].

This study explores a novel therapeutic approach for peritoneal metastasis (PM) using AAV-mediated delivery of tumor suppressor microRNA-29b (miR-29b) to peritoneal mesothelial cells (PMC). AAV serotypes 2 and DJ demonstrate high transduction efficiency for human and murine PMC, respectively. In vitro analysis indicates that AAV vectors encoding miR-29b precursor successfully elevate miR-29b expression in PMC and their secreted small extracellular vesicle (sEV), thereby inhibiting mesothelial mesenchymal transition and reducing subsequent attachment of tumor cells. A single intraperitoneal (IP) administration of AAV-DJ-miR-29b demonstrates robust and sustained transgene expression, suppressing peritoneal fibrosis and inhibiting the development of PM from gastric and pancreatic cancers. Additionally, AAV-DJ-miR-29b enhances the efficacy of IP chemotherapy using paclitaxel, restraining the growth of established PM. While conventional gene therapy for cancer encounters challenges targeting tumor cells directly but delivering miRNA to the tumor stroma offers a straightforward and efficient means of altering the microenvironment, leading to substantial inhibition of tumor growth. AAV-mediated miR-29b delivery to peritoneum via IP route presents a simple, minimally invasive, and promising therapeutic strategy for refractory PM.