'MAP-GET: Mapping of cellular targets following gene electrotransfer in tumors'

Gene electrotransfer (GET) using plasmid DNA is a promising non-viral strategy for in vivo gene delivery, particularly in oncology and immunotherapy. This project aims to deepen the mechanistic understanding of GET within the tumor microenvironment (TME) by integrating advanced single-cell and spatial analysis approaches. The objective is to establish a pipeline linking gene delivery parameters to the precise identification, spatial localization, and functional characterization of transfected cell populations. While GET offers advantages over viral vectors, such as enhanced safety, reduced immunogenicity, and ability to deliver large genetic constructs, its success is challenged by the complex architecture of the TME. Tumors are not merely clusters of cancer cells but highly organized, dynamic tissues comprising stromal fibroblasts, endothelial cells, immune cells and extracellular matrix components. This heterogeneity affects electric field distribution, plasmid DNA accessibility and cellular uptake. For instance, the extracellular matrix can hinder plasmid diffusion, while densely packed cells and variable local conductivity alter the effective electric field at the cellular level. Experimental models show that while high transfection efficiencies are achievable in 2D cell cultures, they drop significantly in 3D structures like spheroids or solid tumors. Even with uniform permeabilization, gene expression is often limited to peripheral layers, indicating that physical barriers, such as limited DNA penetration and reduced electrophoretic transport, restrict transfection in deeper regions. Additionally, the cellular composition of the TME is highly relevant for therapy. GET can target multiple cell types, including fibroblasts, keratinocytes, endothelial cells, and immune cells like dendritic cells and macrophages. This broad targeting capability is advantageous for immunotherapy, where transfection of antigen-presenting cells can enhance immune activation. However, it also introduces complexity, as different cell populations may respond differently to electric pulses and exhibit distinct DNA uptake, processing, and expression capacities. A critical gap remains: the specific cell populations transfected in vivo, particularly within tumors, are poorly understood. Most studies report overall transfection efficiency or total transgene expression but do not systematically characterize the identity of transfected cells. This is especially relevant for immune cells in the TME, which play central roles in tumor progression and therapeutic response. The extent to which key immune subpopulations, such as T lymphocytes, dendritic cells, macrophages, or myeloid-derived suppressor cells, are directly transfected by GET remains undefined. Furthermore, the functional consequences of transfecting different cell populations can vary substantially. For example, transgene expression in tumor cells may induce direct cytotoxic effects, while transfection of stromal or immune cells may modulate the TME by influencing immune activation, antigen presentation, or cytokine production. In genetic immunotherapy, such as GET of plasmid DNA encoding IL-12, it is crucial to determine whether the therapeutic effect arises from transfected tumor, stromal, or infiltrating immune cells, as this has implications for both efficacy and safety. In summary, plasmid DNA-based GET is a promising and clinically relevant method for in vivo gene delivery. However, its success depends on the complex interplay between physical parameters and the biological context. The heterogeneous composition of the TME can significantly impact plasmid DNA delivery and transgene expression, yet the identity of transfected cell populations, particularly immune cells, remains insufficiently characterized. Addressing this knowledge gap is essential for the rational optimization of GET-based therapies and for understanding their mechanisms of action within tumors. École doctorale : BSB - Biologie, Santé, Biotechnologies Direction : Muriel GOLZIO Financement : MSCA COFUND BEST