水上 浩明

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.

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.