Thèse 'Map-Get Mapping Of Cellular Targets Following Gene Electrotransfer In Tumors' H/F Doctorat.Gouv.Fr

Établissement : Université de Toulouse École doctorale : BSB - Biologie, Santé, Biotechnologies Laboratoire de recherche : IPBS - Institut de Pharmacologie et Biologie Structurale Direction de la thèse : Muriel GOLZIO ORCID 0000000274703708 Début de la thèse : 2027-09-01 Date limite de candidature : 2026-11-23T23:59:59 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. Gene electrotransfer (GET) using plasmid DNA has emerged as a versatile non-viral strategy for in vivo gene delivery, particularly in oncology and immunotherapy. Building on current joint research, this cooperative project aims to deepen the mechanistic understanding of GET within the tumor microenvironment (TME) by integrating advanced single-cell and spatial analysis approaches. Together, the doctoral thesis project seeks to establish a fully integrated pipeline linking gene delivery parameters to the precise identification, spatial localization, and functional characterization of transfected cell populations.
Gene electrotransfer (GET) relies on the application of controlled electrical pulses to transiently permeabilize cell membranes, allowing plasmid DNA to enter cells and induce transgene expression. It offers several advantages over viral vectors, including improved safety, reduced immunogenicity, and the ability to deliver large genetic constructs, making it particularly attractive for repeated local treatments such as cancer gene therapy or DNA vaccination. In addition to GET, electrical pulses are also used to administer chemotherapeutic agents like bleomycin in tumors, enhancing their cytotoxic activity. In this context, the approach is called electrochemotherapy and is already used in clinical practice.
A major obstacle to the success of GET in vivo is the complex architecture of the tumor microenvironment (TME). Tumors are not merely composed of cancer cells but are highly organized and dynamic tissues, including cancer cells, stromal fibroblasts, endothelial cells, immune cells, and extracellular matrix components. This structural and cellular heterogeneity affects both the distribution of the electric field and the accessibility and mobility of plasmid DNA within the tissue. For example, the extracellular matrix can hinder plasmid DNA diffusion and limit its interaction with target cells, while tightly packed cells and variable local conductivity alter the effective electric field at the cellular level.
Experimental models also highlight the importance of tissue organization. While high transfection efficiencies can be achieved in cell suspensions or 2D cultures, they drop significantly in 3D structures such as spheroids or solid tumors. Even when cells are uniformly permeabilized, gene expression is often limited to peripheral layers, indicating that physical barriers-such as limited DNA penetration and reduced electrophoretic transport-play a dominant role in restricting transfection in deeper regions. These findings show that the success of in vivo gene electrotransfer depends not only on cellular susceptibility but also on the spatial organization and composition of the surrounding tissue.
The cellular composition of the TME is also highly relevant from a therapeutic perspective. In vivo studies have demonstrated that GET can target multiple cell types within a tissue, including fibroblasts, keratinocytes, endothelial cells, and immune cells such as dendritic cells and macrophage-like populations. This broad targeting capability is particularly advantageous for immunotherapy approaches, where transfection of antigen-presenting cells can enhance immune activation. However, this also introduces complexity, as different cell populations may respond differently to electric pulses and exhibit distinct DNA uptake, processing, and expression capacities.
Nevertheless, a critical gap persists in this field: there is poor understanding of which specific cell populations are transfected in vivo, particularly within tumors. Most studies report overall transfection efficiency or total transgene expression but do not systematically characterize the identity of transfected cells. This is particularly 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 bone marrow-derived suppressor cells-are directly transfected by GET remains poorly defined. Given that these cells differ significantly in their membrane properties, localization, and endocytic activity, their susceptibility to gene electrotransfer is likely heterogeneous.
Moreover, 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 tumor microenvironment by influencing immune activation, antigen presentation, or cytokine production. In the context of genetic immunotherapy, such as GET of plasmid DNA encoding IL-12, it is particularly important 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 gene electrotransfer 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 tumor microenvironment 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. The primary objective of this project is to provide a comprehensive and quantitative characterization of the specific cell populations within the tumor microenvironment (TME) that are transfected after in vivo GET.
This project will determine which cells within tumors are primarily transfected following gene electrotransfer (GET) using plasmid DNA. By combining spatial imaging with single-cell analysis using spectral flow cytometry, it will be possible to distinguish transgene expression in tumor cells, stromal compartments, and various immune subpopulations.
This knowledge will address a critical gap in the field by moving beyond global transfection efficiency measurements toward a mechanistic understanding of cellular targeting. Most importantly, these findings will establish a foundation to determine whether the therapeutic effects of GET-based interventions primarily arise from the direct modification of tumor cells or from the modulation of stromal and immune components. We will use B16-F10 murine melanoma models, subcutaneously induced in C57Bl/6 mice and expressing the tdTomato fluorescent protein. Once tumors reach a volume of 50-60 mm³, we will perform gene electrotransfer using plasmid DNA encoding enhanced green fluorescent protein (EGFP). This involves an intratumoral injection of EGFP plasmid DNA, followed by the application of electrical pulses via stainless steel plate electrodes. Other tumor models, more compact and containing a higher number of immune cells than the B16 model (such as CT26), could also be considered.
Two electrical pulse protocols will be tested:
A clinical electrochemotherapy protocol: eight square wave pulses at 1,300 V/cm, each with a 100 µs duration and a 5 kHz repetition frequency.
An effective in vitro and in vivo transfection protocol: eight square wave pulses at 600 V/cm, each with a 5 ms duration and a 1 Hz repetition frequency.
At 24 and 48 hours after electrotransfer, tumors will be excised. One half of each tumor will be fixed in 4% PFA and embedded in O.C.T. compound for frozen section preparation, while the other half will be processed into single-cell suspensions for spectral flow cytometry analysis. Frozen sections will allow us to map the spatial distribution of EGFP expression within the tumor, whereas spectral flow cytometry will identify and quantify transfected cells, distinguishing between tumor and immune populations.
The tdTomato fluorescence in tumor cells will enable their distinction from other cells in the tumor microenvironment (TME), allowing us to determine the percentage of cells expressing the EGFP transgene. Fluorescence-labeled antibodies will also be used to identify additional TME cell populations, such as immune cells, fibroblasts, and endothelial cells.
A fluorescently labeled plasmid will be used to assess its spatial distribution within the tumor using intravital microscopy. Finally, we will test a therapeutic plasmid encoding an immunotherapy candidate gene, such as IL-12 or another cytokine, to validate the therapeutic potential of this approach.