Nucleic acid-based drugs have recently emerged as powerful solutions to many of medicine's most enduring problems. These therapeutic agents, and antisense oligodeoxynucleotides (ODN) in particular, possess enormous potential to complement or replace conventional pharmaceutical therapies based traditionally on small molecular weight drugs. With the uncanny ability to selectively downregulate the expression level of a specific protein, antisense ODN can modulate disease states in a highly specialized manner. Recent efforts to enhance ODN stability and advances in the selection method and design of effective ODN sequences have produced ODN molecules that are increasingly potent once delivered to their site of action in the cytosol or the nucleus. Despite the outstanding pharmacological characteristics, their full potential awaits resolution of critical pharmaceutical challenges, due primarily to the low membrane permeability of ODN molecules. Similar to the difficulties facing gene therapy in general, the success of ODN drug therapies relies on delivery to specific cell types at high enough concentrations, followed by efficient cellular uptake and transport into the cytoplasm of the cells. This proposal takes two main approaches to address these delivery problems. First, a listeriolysin O (LLO)-containing liposomal delivery system will be utilized to overcome the membrane barrier for cytosolic delivery. LLO, the hemolysin of Listeria monocytogenes, confers upon the lipsome formulation the mechanism that is utilized by Listeria to escape the endosomal/lysosomal degradation pathway and enter the cytosol. Second, the LLO-liposomes carrying ICAM1 or B7-1/2 specific antisense ODN will be targeted to antigen presenting cells (APC) by conjugating a targeting motif, CTLA4Ig, onto the liposomes. CTLA4Ig, a high affinity, competitive ligand for the B7 molecules on the surface of APC, has been shown to block the essential, CD28/B7-mediated co-stimulatory signal for T cell activation. The effectiveness of B7-specific antisense ODN delivered by this targeted cytosolic delivery vehicle, the CTLA4Ig-conjugated LLO-liposomes, will be assessed for efficient inhibition of alloreactive immune responses using the mixed lymphocyte reaction assay. Several approaches will be taken to optimize various parameters for achieving the special balance of long circulation and targeting of CTLA4Ig-liposomes in vivo. The results will then be extended to in vivo mouse models of transplantation. Efficient blockade of the T cell co-stimulatory signal at multiple levels, as rendered by the ODN delivery systsem characterized and developed in this project, is an ideal modality of immunosuppressive treatments for transplant recipients. Development of such a delivery strategy for ODN can be further generalized beyond immune response modulation to a wide range of diseases mediated by specific proteins.
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