Chitosan-DNA nanoparticles: Synthesis, characterization, subcellular transport and oral delivery of genetic vaccines

The ultimate success of gene therapy depends on the development of safe and efficient DNA carriers. Current delivery vehicles, mostly viral or lipid based systems, have several drawbacks including toxicity and immunogenicity. Although historically, gene therapy has been conceived as treatment to replace defective or missing genes, in recent years considerable research has focused on genetic immunization. Successful immunization with “DNA vaccines” has been demonstrated through different routes but little success has been achieved through oral delivery. The overall goal of this work was to study the use of a natural biopolymer, chitosan, as carrier for DNA in gene and vaccine therapy with particular emphasis on oral immunization.; DNA carrying nanoparticles were successfully synthesized using chitosan and plasmid DNA. Their physico-chemical properties, ligand conjugation, encapsulation efficacy etc. were characterized in detail using biochemical, microscopic and light scattering techniques.; In-vitro transfection efficacy was evaluated in various cell-lines and under different culture conditions. The nanoparticles were effective in transfecting mammalian cells in culture with marker and therapeutic genes. A study on the transport of these nanospheres across a Peyer's patch model of intestinal Caco-2 cells showed that particles are efficiently and actively transported by intestinal M cells.; In order to identify rate-limiting steps in transfection, detailed kinetic and mechanistic study of cellular uptake, intracellular transport and nuclear trafficking were performed using various biochemical and microscopic techniques. The study indicated that particle uptake in cells were non-specific and inefficient compared to lipid based vehicles. Furthermore intracellular release of plasmid from these particles were slow.; In-vivo oral gene transfer efficiency of these nanoparticles was evaluated using a murine model of peanut allergy. A DNA vaccine, coding for the major peanut allergen Arah-2 was orally delivered using chitosan nanospheres. The immunization protocol resulted in partial protection against anaphylactic challenge and altered the immune response towards a protective Th1 phenotype.; In conclusion, we successfully developed a polymeric non-viral carrier for DNA that is non-toxic, less immunogenic and is effective for oral delivery in an animal model. The scientific and clinical implication of this system can be potentially far-reaching especially for oral delivery of genetic vaccines.