VIRUS LIKE PARTICLES AS MICROCARRIERS OF MOLECULES WITH BIOLOGICAL FUNCTIONALITY

THE USE OF VIRAL-BASED NANOPARTICLES HAS GAINED A LOT OF INTEREST, AS THEIR INHERENT NATURAL TRAITS OFFER HIGH POTENTIAL TO BE USED IN VACCINES AND NOVEL THERAPEUTICAL DRUG DELIVERY SYSTEMS. AMONG THEM, VIRUS LIKE PARTICLES (VLPS) ARE NANOSTRUCTURES THAT RESEMBLE THE CONFIGURATION OF A NATIVE VIRUS, BEING COMPOSED OF ONE OR SEVERAL VIRAL PROTEINS BUT LACK THE VIRAL GENETIC MATERIAL AND HENCE CANNOT REVERT INTO THE VIRULENT FORM OF THE VIRUS, WHICH MAKES THEM SAFER FOR PRODUCTION OPERATORS AND PATIENTS. THE REPETITIVE SUBUNITS AND TRIDIMENSIONAL STRUCTURE OF VLPS ELICIT HIGH CELLULAR AND HUMORAL IMMUNE RESPONSES. FURTHER CHEMICAL AND GENETIC MODIFICATION OF THE OUTER SURFACES OR INTERNAL PARTS OF VLPS ENABLE THE PREPARATION OF NEW SMART MATERIALS BASED ON NATURAL BIOLOGICAL MATERIALS FOR DRUG DELIVERY THE CORE VLP STRUCTURES PROPOSED ARE BASED ON THE GAG PROTEIN FROM HIV-1, PRODUCED IN HEK 293 CELLS CULTURE, IN WHICH THE TEAM HAS EXTENSIVE EXPERIENCE. AFTER SELF-ASSEMBLY OF GAG PROTEIN INSIDE THE HOST CELLS, HEK 293 IN THIS PROPOSAL, THE GAG CORE IS EXPORTED OUTSIDE THE CELLS BY BUDDING, IN WHICH THE PARTICLE IS WRAPPED IN A LIPID LAYER OF THE HOST CELL (ENVELOPED VLPS). TWO DIFFERENT APPROACHES ARE PROPOSED TO FURTHER FUNCTIONALIZE THE GAG VLPS WITH MOLECULES OF BIOLOGICAL INTEREST. IN ONE OF THEM, THE TARGET MOLECULES TO BE DISPLAYED ON THE NANOCARRIER PARTICLES ARE FUSED WITH THE CD81, A KEY TETRASPANIN MEMBRANE PROTEIN IN INFECTIOUS PROCESSES, THAT IS RECRUITED AT BUDDING SITES DURING HIV-1 EGRESSION. BY THIS, WHEN THE GAG VLPS ARE FORMED INSIDE THE CELL AND BUD OUT, THEY ARE FUNCTIONALIZED BY THE MOLECULES OF INTEREST BEING EXPOSED AT THE MEMBRANE. THE SECOND FUNCTIONALIZATION APPROACH IS BASED ON TWO STEPS, COMBINING THE PRODUCTION OF GAG VLPS IN HEK 293 CELLS AND THEIR CHEMICAL FUNCTIONALIZATION BY BOUNDING THE MOLECULES OF INTEREST TO SPECIFIC GROUPS OF THE VLPS LIPIDIC MEMBRANE. TO SUPPORT ALL THIS WORK, THE PROPOSAL ADDRESSES THE PRODUCTION PLATFORM TO OBTAIN THE VLPS, BASED ON HEK 293 CELLS CULTURE, LOOKING INTO ITS IMPROVEMENT AND OPTIMIZATION, BOTH FROM THE ANGLE OF CELL ENGINEERING AND BIOPROCESS INTEGRATION (UP-STREAM PRODUCTION BIOREACTOR AND DOWN-STREAM PURIFICATION SYSTEM). THE PROPOSED PROOF OF CONCEPT OF USING VLPS AS A PLATFORM FOR NANOCARRIERS FUNCTIONALIZED WITH MOLECULES OF BIOLOGICAL FUNCTIONALITY IS FOCUSED ON TUBERCULOSIS (TB). AN INNOVATIVE APPROACH IS PROPOSED TO BLOCK THE FORMATION OF MYCOBATERIUM TUBERCULOSIS BIOFILMS IN THE DEVELOPMENT AND PROGRESSION OF TB BY NANOCARRIER PARTICLES EXPOSING PEPTIDES INTERFERING AT MOLECULAR LEVEL WITH THE PROCESS, LEADING TO POTENTIAL APPLICATIONS EITHER FOR VACCINES OR TREATMENT OF TB. IN THE FINAL PROOF OF CONCEPT, 3 DIFFERENT PEPTIDES ARE TARGETED TO FUNCTIONALIZE GAG VLPS WITH THE TWO PROPOSED METHODOLOGIES. ALL THE OBTAINED CANDIDATES WILL BE COMPLETELY CHARACTERIZED AND THEN USED IN ANIMAL MODEL TESTS FOR TB, TO DEMONSTRATE THE CAPACITY OF THE FUNCTIONALIZED NANOPARTICLES IN PREVENTING TB INFECTION AND BLOCKING ITS PROGRESS.