EXCEED THE SPACE PROVIDED. This proposal explores a novel use of transferrin receptor mediated drug delivery. An oligonucleotide (ODN- based drug will be targeted to brain microvascular endothelial cells, with the goal to inhibit inflammatory responses in these cells. The approach could have broad applicability for diseases affecting the blood-brain barrier and the central nervous system. Here, we focus on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, to demonstrate the feasibility of this vascular targeting strategy. Crucial events in the pathogenesis of that disease affect function and integrity of the BBB. The endothelial cells forming the BBB express adhesion molecules affecting lymphocyte transmigration, and enzymes such as inducible NO-synthase and cyclooxygenase-2 contributing to inflammation. Expression of these proteins is under control of the transcription factor NF-kappaB. Therefore, preventing NF-kappaB activation could be a promising therapeutic strategy. An elegant way to inhibit a transcription factor is the cellular delivery of decoy ODNs, short double stranded ODNs containing the consensus binding sequence of that factor. Unfortunately, decoy ODNs face similar problems as related antisense approaches, namely poor permeability through cell membranes. The receptor-mediated endocytosis provided by the transferrin- receptor system can overcome that obstacle. Here, the 8D3 antibody specific for the mouse transferrin receptor will serve as the vector for delivery of a NF-kappaB decoy ODN. The polyanionic ODN will be bound in a complex by the cationic polymer polyethylenimine, and then coupled to the 8D3 antibody via an avidin-biotin bridge. Polyethylenimine is an attractive carrier for DNA due to its capacity to mediate 'endosomal escape', thus enabling the ODNs to reach their cytosolic or nuclear sites of action.
The specific aims of the project comprise (i) the determination of the cellular uptake and pharmacological activity of the ODN delivery system in an in vitro model with a murine brain endothelial cell line; (ii) determining the in vivo pharmacokinetics of the delivery system in normal mice; and (iii) evaluating in vivo pharmacological effects of vector-mediated ODN delivery in the mouse EAE-model PERFORMANCE SITE ========================================Section End===========================================
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