綿野 亮太

Ovarian mucinous carcinoma (OMC) is a rare subtype of ovarian cancer characterized by frequent KRAS mutations and a poor response to platinum- and taxane-based chemotherapy, underscoring the need for novel therapeutic strategies. MRTX1133, a recently developed non-covalent and selective KRAS (G12D) inhibitor, has demonstrated potent antitumor activity in pancreatic and colorectal cancers; however, its efficacy in OMC remains unexplored. In the present study, the antitumor effects of MRTX1133 in the KRAS (G12D)-mutant OMC cell line MCAS and its interactions with conventional chemotherapeutic agents were evaluated. Cell viability was assessed using WST-1 assays, ERK phosphorylation was evaluated by western blotting and gene expression levels were analyzed by reverse transcription-quantitative PCR. Results indicated that MRTX1133 suppressed MCAS cell proliferation in a concentration-dependent manner, whereas proliferation of KRAS wild-type and KRAS (G12S)-mutant cells was not significantly inhibited. Treatment markedly inhibited ERK phosphorylation, suggesting suppression of the MAPK pathway. MRTX1133 reduced the mRNA expression of Ki-67 and numerous cyclins (D1, A2 and B1), suggesting attenuation of proliferative signaling. When combined with cytotoxic agents, including paclitaxel, SN38, gemcitabine and cisplatin, MRTX1133 reduced the sensitivity to cell cycle-dependent chemotherapeutic agents, namely paclitaxel, SN38 and gemcitabine, while not affecting the activity of the non-cell cycle-dependent agent cisplatin. The present study therefore provided preclinical evidence for the potential utility of KRAS (G12D)-targeted therapy in OMC and highlights the importance of sequential rather than concurrent scheduling of MRTX1133 with cell cycle-dependent chemotherapy to optimize therapeutic efficacy.

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.

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.

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.