Localised miRNA profiling in the CNS: development of a chimeric HIV-1 Gag VLP toolbox for high-resolution cell- and compartment-specific analysis

The mammalian brain is a complex network of over 3,000 neuronal and non-neuronal cell types, each exhibiting unique gene expression patterns shaped by their anatomical location, connectivity and physiological properties. This complexity is even more evident in neurons, where subcellular compartments such as dendrites and axons possess unique mRNA profiles that are locally translated to support rapid, energy-efficient, and input-specific plasticity events at synapses.In this intricate landscape, microRNAs (miRNAs) have emerged as critical post-transcriptional regulators, finely tuning local protein availability. Like protein-coding transcripts, miRNAs display highly patterned expression across brain regions and cell types, influencing processes such as progenitor proliferation and lineage commitment. Furthermore, the pre- and post-synaptic compartments in neurons exhibit distinct miRNA profiles, essential for controlling activity-dependent protein synthesis. Disruptions in miRNA biogenesis or function are increasingly linked to altered brain development, excitability, and plasticity, underscoring these molecules as potential biomarkers for neurological disorders.Despite their clinical significance, brain miRNAs remain challenging to profile in a cell- and compartment-specific manner. Techniques like single-cell miRNA sequencing struggle with non-coding RNA sensitivity, while compartment-specific profiling approaches -such as synaptosomal or neuropil preparations- are limited by transcript contaminations from nearby cells or compartments. Comprehensive insights into miRNAs' regulatory roles in the CNS in both health and disease states require advances in sensitivity and spatial resolution for miRNA profiling across different cells and compartments.To overcome current limitations in miRNA profiling, this thesis introduces a novel miRNA export system based on HIV-1 Gag virus-like particles (VLPs). Building on the inherent miRNA-binding properties of the Gag polyprotein, we engineered this system with functional modules to enable (i) enhanced miRNA packaging, (ii) postsynaptic miRNA detection, and (iii) cell-type-specific miRNA export.Results presented throughout this work establish the feasibility of Gag VLPs packaging in unconventional eukaryotic models, namely neuronal and glial cells, achieving particle yields comparable to gold-standard HEK-293T cells. Having optimised VLP production, we confirmed miRNA enrichment in wild-type Gag VLPs compared to controls across all models tested. Nevertheless, we achieved target export up to saturation by fusing Gag with double-strand RNA-binding domains from the TAR RNA-binding protein (TRBP), which elevated miRNA packaging in a sequence-independent manner with minimal impact on VLP assembly or cell viability. This observation was validated in HEK-293T cells and further extended to mouse stem-cell-derived neurons, highlighting the synergistic effect of multiple modules for spatially restricted neuronal miRNA capture.To achieve postsynaptic-specific miRNA export, we incorporated a dendritic localization signal (DLS) derived from PSD95 mRNA into the Gag sequence. This modification directed VLP assembly and buddying to postsynaptic sites in primary and stem cell-differentiated neurons. Progressive optimization, particularly in human cortical glutamatergic neurons, enabled Gag-DLS VLPs to selectively capture postsynaptic miRNAs while excluding presynaptic species. The spatial resolution achieved by Gag-DLS VLPs surpassed traditional synaptosomal miRNA profiling, which retained axonal contaminants to a greater extent. Postsynaptic targeting efficiency increased with neuronal maturation, suggesting our system’s potential to distinguish neuronal differentiation stages through miRNA content.Finally, to achieve lineage-restricted miRNA export, we regulated Gag expression using promoters with defined cell lineage specificity, namely CaMKIIα for neurons and Iba1 for glia. This strategy facilitated targeted miRNA export from neuronal Ht22 and microglial BV-2 cells in a co-culture setting, demonstrating the feasibility of effective, cell-specific miRNA capture within heterogeneous cellular environments.In conclusion, our work positions the chimeric Gag VLP toolbox as a powerful tool for high-resolution, spatially precise miRNA profiling in the CNS. Its non-destructive nature offers potential for future longitudinal miRNA profiling in living animal models. Coupled with next-generation sequencing, this platform presents an opportunity to generate comprehensive miRNA datasets for comparative studies between health and disease, ultimately assisting the identification of spatially resolved miRNA signatures. The modular adaptability of the Gag VLP system also provides future possibilities for profiling other non-coding RNA species in a cell- or compartment-specific manner, extending its utility beyond the CNS to diverse tissues with an unprecedented level of detection sensitivity and spatial accuracy.