Novel Targeted Reagents that Modify Oncogene Expression

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DESCRIPTION (provided by applicant): A detailed understanding of the genetic basis of neoplastic diseases has emerged during the past two decades which has encouraged efforts to develop genetically targeted reagents both as experimental tools and as novel anti-cancer drugs. Peptide nucleic acid (PNA), a DNA mimic in which the phosphate deoxyribose backbone of DNA has been replaced by a pseudopeptide polymer, first described in 1991, has attracted particular interest as a gene-targeting reagent, since it is highly stable and binds to complementary RNA and DNA with high affinity and specificity. However, because PNA resists cellular uptake, its potential usefulness as a tool for modifying gene expression in whole animal studies or as a potential therapeutic agent has been limited. In preliminary studies, we have found that Anthrax toxin "protective antigen" (PA), the component of this microbial toxin that mediates cellular delivery, is able to transport ant/sense PNA oligomers into cells. To explore further the feasibility and potential of using Anthrax PA as a vehicle for delivering PNA into cells for the purpose of altering cancer-related gene expression, we propose studies with two specific aims. First, we will define the kinetics and dose limits of PA-mediated cellular delivery of antisense PNA, using PNA oligomers linked to varying polypeptide sequences derived from selected functional domains of toxin proteins. Stably transfected cell lines engineered to express a luciferase gene interrupted by a mutant beta-globin intron-2 (betaIVS2-654) with an aberrant splice site that can be blocked by antisense PNA, thereby allowing luciferase expression, will be used to detect antisense activity and effective cellular delivery of PNA. Second, we will determine whether Anthrax PA permits antisense PNA-peptide constructs to alter Bcl-x L gene expression and induce apoptosis in human cancer cell lines (e.g., PC3 cells) in vitro, and also to limit the growth and survival of these cells in vivo following implantation into nude mice. The potential impact of this research is substantial, not only with regard to the development of experimental tools for modulating cancer-related gene expression selectively and combinatorially in cancer cells in vitro and in vivo, but also with regard to the goal of developing genetically targeted agents for cancer treatment.