POLYAMINE OLIGONUCLEOTIDES TO ENHANCE CELLULAR UPTAKE

WE WILL DESIGN AND SYNTHESIZE A NOVEL TYPE OF ANTISENSE PHOSPHOROTHIATE OLIGONUCLEOTIDE MODIFIED TO SPECIFICALLY ENHANCE CELLULAR UPTAKE. ALTHOUGH OLIGONUCLEOTIDE PHOSPHOROTHIOATES ARE KNOWN TO BE TAKEN UP BY CELLS, CONJUGATION WITH POLYAMINES MAY SUBSTANTIALLY ENHANCE CELLULAR UPTAKE AND INFLUENCE INTRACELLULAR DISTRIBUTION OF THE COMPOUNDS. IN THE INITIAL PHASE OF THE PROGRAM, A POLYAMINE CONJUGATED AND UNMODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDE DIRECTED AGAINST THE TAT REGION OF THE HIV GENOME WILL BE SYNTHESIZED. THE TAT, OR TRANS-ACTIVATOR, PROTEIN AND ITS INTERACTION WITH THE TAR REGION WITHIN THE HIV GENOME, ARE VITAL TO THE REPLICATION OF HIV. DISRUPTION OF THIS INTERACTION WILL PROVIDE NOVEL AND SPECIFIC ANTI-HIV THERAPEUTIC AGENTS. THE CELLULAR UPTAKE OF BOTH COMPOUNDS WILL BE EXTENSIVELY CHARACTERIZED USING 35S-RADIOLABELLED OLIGONUCLEOTIDES AND MECHANISMS OF UPTAKE DELINEATED USING SPECIFIC INHIBITORS OF BOTH THE NATURAL POLYAMINE UPTAKE SYSTEM AND INHIBITORS OF KNOWN CELLULAR TRANSPORT PROCESSES. SUBCELLULAR DISTRIBUTION AND CELLULAR PHARMACOKINETICS OF THESE COMPOUNDS WILL BE EXAMINED BY HYPOTONIC LYSIS OF CELLS, FOLLOWED BY DIFFERENTIAL CENTRIFUGATION TO ISOLATE SPECIFIC CELLULAR ORGANELLES. THE POLYAMINE CONJUGATED COMPOUNDS WILL ALSO BE TESTED FOR HYBRIDIZATION AFFINITY, NUCLEASE RESISTANCE AND THE ABILITY TO SPECIFICALLY INHIBIT TAT GENE EXPRESSION IN A CELL CULTURE MODEL SYSTEM.