Development of anionic liposomal carriers containing listeriolysin O and red blood cell ghosts as plasmid DNA delivery platforms.

Gene therapy is often cited as the future of therapeutics. DNA has the potential to revolutionize disease treatment through correction of genetic disorders, enzyme replacement, and modulation of the immune system. Despite numerous inventive concepts for therapeutic use of genetic material, the progress toward developing these therapies is impeded by limitations in the effective delivery of DNA to its site of action. Physical and enzymatic barriers exist at multiple stages along the route to DNA delivery due to the large size and anionic nature of plasmid DNA molecules. Thus, effective delivery of DNA remains a critical area of need toward the next generation of pharmaceuticals. This thesis focuses on the development of lipid-based anionic DNA delivery vehicles using two different carriers: a protamine-condensed plasmid DNA particle that has been augmented to overcome the cytosolic membrane barrier and a red blood cell ghost carrier for plasmid DNA. The protamine-condensed DNA in the former vehicle is modified for overcoming the plasma membrane barrier through electrostatic complexation with anionic liposomes containing listeriolysin O (LLO). LLO is the endosomolytic hemolysin from Listeria monocytogenes, which mediates escape of the Listeria from the endosomal compartment and into the cytosol. LLO was capable of facilitating transfection of P388D1 cells following incorporation of LLO into pH-sensitive liposomes that were complexed to the protamine-DNA condensate. Effective encapsulation of the plasmid DNA was the focus of the research on the latter red blood cell ghost delivery vehicle. While red blood cells have long been proposed as carriers for DNA due to their promising natural characteristics such as long circulation times, low immunogenicity, and biocompatibility, an effective method of DNA encapsulation in these physiological vehicles has been elusive. To overcome electrostatic interactions between cationic DNA condensing agents and the red blood cell membrane, a neutral method for plasmid DNA condensation was utilized involving polyethylene glycol as a condensing agent. DNA condensed through this neutral method was then encapsulated in red blood cells ghosts. This study aimed to develop platform technology for DNA delivery using red blood cell ghosts, and this work presents promising technologies which can be further optimized.Ph.D.Health and Environmental SciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124467/2/3138209.pd