Bigot, K. | bioRxiv Science

bioRxiv2026

SPEAR: Spatial Epitranscriptomic Analysis by Mass Spectrometry Reveals Localized RNA Modification Remodeling During Glioma Progression

RNA modifications shape cell identity and disease, yet their spatial organization within tissues has remained largely inaccessible to existing technologies. NGS-based methods can map a limited number of individual modifications with spatial resolution, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides comprehensive and quantitative coverage of the epitranscriptome from bulk, homogenized tissue. No current approach combines histological resolution, broad modification coverage, quantitative accuracy, and the sensitivity required for microscale clinical specimens. We present SPEAR (SPatial Epitranscriptomic Analysis by mass spectrometRy), a workflow coupling laser microdissection with a redesigned LC-MS/MS pipeline to enable quantitative epitranscriptomic profiling directly from formalin-fixed, paraffin-embedded (FFPE) tissue. This combination enables reproducible detection of dozens of RNA modifications from regions as small as 0.1 mm2, establishing a spatial scale previously inaccessible to mass spectrometry-based approaches. Applied to serial mouse brain sections, SPEAR resolved anatomically defined epitranscriptomic signatures across white and gray matter, establishing its spatial resolution and reproducibility in a controlled system. We then applied SPEAR to human diffuse gliomas containing, within individual specimens, both primary low-grade tumor regions and discrete foci undergoing transformation toward higher-grade malignancy. SPEAR resolved this transition directly, revealing a coordinated decrease in multiple methylated RNA modifications specifically within higher-grade regions, a spatially restricted signal that was masked by bulk analysis and reproducibly observed across patients despite underlying genetic heterogeneity. SPEAR establishes spatial epitranscriptomics as a tractable approach for molecular profiling of clinical tissue, providing a platform for biomarker discovery and molecular stratification based on the spatial organization of RNA modifications.

cancer biology↗

bioRxiv2025

Transcriptional changes in non-human primate tissues after intrathecal delivery of serotype 9 adeno-associated viral vector: insights into organ toxicities

Adeno associated virus (AAV)-based gene transfer has brought transformative therapeutic benefits to patients with otherwise untreatable genetic diseases. However, treatment-related organ toxicities, particularly for high doses, remain a safety concern in the clinic with some translatability in preclinical species. In the present study, we conducted an RNAseq analysis in non-human primates administered intrathecally with scAAV9-CBA-GFP, empty viral capsid particles, or a Promoterless vector. This analysis revealed a broad and long-lasting (4 weeks after dosing) transcriptional impact of the viral transduction/transgene expression on tissues. Liver and DRG, known to be primary sites of toxicity induced by AAV9, had the highest viral load and the most significant transcriptional changes. Our analysis revealed that most of the differentially expressed genes were upregulated, and common gene signatures belonged to immune pathways (innate and adaptive), demonstrating a persistent low-grade immune response up to four weeks post-dosing. Interestingly and across all tissues considered, the impact of empty capsids or of the Promoterless vector was minimal, suggesting that both the presence of the capsid and of a productive viral genome contributed to the observed changes. This study provides a unique insight into the transcriptional responses to AAV9 in key tissues primarily exposed by the vector.

molecular biology↗