• cancer
• cancer vaccines
• immunotherapy
• Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that, when properly attenuated, has emerged as a promising vector for cancer immunotherapy. Although attenuated strains lose their virulence, they retain the ability to induce potent immune responses, including the activation of cytotoxic immune cells via the presentation of immunogenic peptides. Owing to its intracellular lifestyle and strong immunostimulatory properties, Listeria has also been extensively used as a delivery system for therapeutic proteins, drugs, and foreign antigens in tumor models.
Within this context, the aim of the laboratory where this thesis was conducted is to develop an engineered Listeria monocytogenes strain capable of delivering CRISPR-based systems into tumor cells. To support this long-term objective, two preliminary steps were carried out as part of the project:
1. Engineering Listeria to express a fluorescent reporter gene, enabling visualization and monitoring of the bacterium within tumor cells.
2. Generating a tumor cell line stably expressing a destabilized GFP, which will serve as a reporter system to assess the functional delivery and gene-editing activity of the CRISPR system transported by Listeria.
To achieve the first goal, a reporter gene will be integrated into the Listeria genome using an integrative plasmid containing a Listeria-specific promoter and the coding sequence of the protein. The generated Listeria strain will allow the real-time study of bacterial infectivity and intracellular behavior, both in vitro and in vivo.
As for the second objective, the destabilized GFP-expressing tumor cell line will be compared to a control line expressing standard GFP. A siRNA targeting the GFP sequence will be used to evaluate silencing efficiency, providing insights into the expected outcomes when Listeria is engineered to deliver a CRISPR system carrying a specific sgRNA against GFP.
This thesis contributes to the development and validation of these dual reporter systems, laying the groundwork.