Enhanced cytosolic delivery of antisense oligonucleotides using listeriolysin O-containing pH -sensitive liposomes.

Nucleic acid-based drugs such as antisense oligodeoxynucleotides (ODNs) have great potential to be effective therapeutic agents in the treatment of many disease states. However, their success and widespread utilization are limited primarily by the membrane barriers preventing sufficient concentrations of functionally active drugs from accessing the cytosolic site of action. In order for antisense ODN to deliver on the promise as successful therapeutic agents, an effective delivery system must be formulated to circumvent the membrane barriers, thereby allowing the drug to engender a robust antisense effect. The work in this thesis focuses on the development and characterization of a liposomal-based cytosolic delivery formulation utilizing listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODNs from the endocytic compartments into the cytosol. It was demonstrated that LLO-containing liposomes were successful in effectively breaching the endosomal barriers enabling sufficient concentrations of ODNs specific for murine intercellular adhesion molecule-1 (ICAM-1) to be transported to the cytosol. The expression of ICAM-1 was efficiently inhibited in a sequence-specific manner at both the protein and mRNA levels. In addition to demonstrating our delivery strategy, the persistence of antisense activity was estimated for the first time by monitoring the recovery of mRNA levels after bolus administration of phosphorothioate ODNs into the cytosol using LLO-containing liposomes. Furthermore, preliminary groundwork was performed for investigating the mechanism of LLO-induced hemolysis on cholesterol-containing membranes. More studies are required in order to elucidate the mechanism of LLO-induced hemolysis, which will be important in understanding the endosome lysis mechanism of LLO and designing a new strategy for cytosolic delivery of macromolecules. In addition, an allogenic mixed lymphocyte reaction was utilized in order to demonstrate the biological relevance of ICAM-1 down-regulation in bone marrow-derived macrophages. Due to the inability to establish a robust allogenic response in the proposed allogenic model, the assay was not established.PhDHealth and Environmental SciencesPharmaceutical sciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123443/2/3079498.pd