Mucosal tissues are composed by a specialized epithelium covered by a mucus layer, and are the main portal of entry of most of pathogens. The induction of local (mucosal) immune response is highly desirable in order to effectively prevent mucosal infection and transmission, especially against rapidly emerging pathogens. The presence in the small intestine of Peyer’s Patches (PP) as inductive sites of the gut-associated lymphoid tissue (GALT) makes the oral route of administration as a desirable approach to mucosal vaccination. However, the oral vaccine delivery needs to overcome some harsh conditions in the gastrointestinal tract (GIT) such as acid pH, degradation by digestive enzymes, low uptake across mucus layer, and immune tolerance. Lipid-polymer hybrid nanoparticles (NPs) allow the modification of surface properties to overcome the mucosal permeation barriers, enhanced physicochemical stability and drug protection during the transit through the GIT. The objective of SpheOrVac project is to conceive a robust mRNA delivery system able to bypass the GI barrier and to permit a mRNA expression, strong enough to induce a mucosal immune response. Previously, we have developed a lipid-polymer hybrid NPs, named spheroplexes (Sphx), as siRNA delivery system. The oral administration of TNF-a siRNA Sphx to mice with ulcerative colitis induced by DSS indicated a disease regression with a decrease in the level of TNF-a in the colon. Our hypothesis is that by using a combination of dedicated lipids and polymers to form Sphx, we could gather mRNA vectorization, adjuvant properties and muco-penetrating property in a unique delivery system able to induce a robust and long-lasting mucosal and systemic immune response following oral administration. Using an automated microfluidic system, we will synthetize mRNA Sphx made with diverse lipid-polymer compositions and evaluate their correlation with formulation stability and interaction with mucus layer. The immunostimulatory properties of the formulation will be evaluated considering the intrinsic adjuvant property of hybrid nanoparticles and the incorporation of adjuvant molecules in the delivery system to overcome the oral tolerance. To analyze the physiological uptake of fluorescent Sphx formulations, we will use an ex-vivo mice loop model, mimicking in vivo situation, and representing the natural environment of intestinal mucosa of living animal. The biodistribution evaluation will be carried out to select mRNA Sphx formulations able to target the small intestine and able to express mRNA in dendritic cells at PP region. Finally, as a proof of concept, mice immunization will be performed with selected Sphx formulated with N1-methylpseudouridine modified mRNA encoding serodominant secreted effector protein B (SseB), which will be used as immunogenic and protective antigen model against Salmonella enterica infection. If our hypothesis is confirmed, SpherOrVac data could be used as a foundation to future mRNA vaccines capable of preventing not only infection but also the transmission of (re)emergent pathogens.