THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OR AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEND ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE ABSTRACT BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. DESCRIPTION: The applicant states that antisense DNA technology may prove to be the """"""""magic bullet"""""""" for inhibiting the function of any disease-related gene. Since the natural form of DNA does not have the requisite properties for use as a therapeutic agent, many analogs of DNA, such as phosphorothioate and methylphosphonate, are currently under intensive study. The purpose of the applicant's project is to investigate a novel form of antisense DNA, the cationic peptide- oligonucleotide cyclic conjugate. In the cyclic conjugates, the 5' terminus of the oligonucleotide is covalently linked to the C-terminus of the peptide and the 3' terminus of the same oligonucleotide is linked to the N-terminus of the same peptide. So far, the applicant has shown that appended cationic peptides can markedly increase the avidity of binding of an antisense DNA to its complementary DNA or RNA. The applicant has also developed a process for synthesizing cyclic conjugates, and now wishes to demonstrate and optimize the physicochemical and pharmacologic properties required for an antisense DNA therapeutic agent, namely strong and selective binding to the designated complementary target, in vivo stability (i.e. resistance to degradation) and enhanced cellular uptake and subcellular localization. The applicant proposes to use a model 12-mer DNA sequence to prepare cyclic conjugates that incorporate different cationic peptide motifs. One proposed series includes isopeptides containing different combinations and permutations of lysine and ornithine residues linked via their side chain amino groups. Another motif comprises peptides with different combinations and permutations of leucine and lysine groups. Another series involves arginine residues spaced by glycine or b-alanine residues. The applicant explains the rationale for these choices. The applicant will measure binding strength by gel shift analysis using radiolabeled complementary and single-base mismatched RNA targets. The property of RNase H stimulation of target strand cleavage, which the applicant says may be critical to in vivo antisense efficacy, will be evaluated. Cellular uptake and subcellular distribution will be monitored using radiolabeled cyclic conjugates. Stability of radiolabeled cyclic conjugate in culture medium and in cell extracts will be determined by chromatographic and electrophoretic procedures. The applicant states that the successful outcome of the proposed experiments would be the development of a cyclic conjugate displaying a dramatic improvement in the physicochemical and pharmacologic properties required for an antisense DNA therapeutic agent.
