HER-2/neu overexpression has been implicated in numerous types of human cancer, and its overexpression often correlates with poor prognosis. The HER-2/neu promoter contains a strategically located purine rich tract that has been successfully targeted in vitro using a triplex forming oligonucleotide (TFO) conjugated at both ends to a DNA alkylating agent. Maximal promoter suppression required alkylation at both ends of the triplex target, possibly due to inhibition of helicase activity. However, modification of both ends of the TFO with an active alkylating agent complicates delivery of the TFO into cells. Peptide nucleic acids (PNAs) are an attractive DNA analogue due to their neutral aminoethylglycine backbone. The neutral backbone decreases protein interactions and is insensitive to nucleases and proteases while allowing high and essentially irreversible target binding affinity and specificity, which may circumvent the need for alkylator conjugates. Two single PNAs separated by a flexible linker (bis-PNA) can invade duplex DNA to form a stable invasion complex. A bis-PNA was designed to target the HER-2 polypurine tract and bound its target in vitro with high affinity and 1000-fold greater specificity than for a 1bp mismatch as demonstrated by non-denaturing EMSA analysis and DNaseI footprinting. Pyrimidine strand displacement was also identified by S1 nuclease digestion, and is indicative of duplex strand invasion by the PNA. In vitro helicase studies indicated the invasion complex may not be an effective substrate for helicase unwinding. The bis-PNA invasion complex formed in vitro using a plasmid target effectively inhibited HER-2/neu promoter activity when transfected into multiple cell types. The decrease in promoter activity was cell-type dependent, ranging from 40% in established cell lines to 20% in a primary cell line. The suppression was maximal at 1uM PNA, and time course analysis demonstrated the suppression was minimal (5–10%) within 48 hours. We are currently determining the ability of the bis-PNA to traverse the nuclear membrane in isolated nuclei and digitonin permeabilized cells, and the subsequent ability of the PNA to target the endogenous promoter. While PNAs may be superior to other DNA analogs in many areas of gene targeting, it is predicted the PNAs will require a carrier to effectively enter the cell and concentrate in the nuclear compartment. Therefore, a PNA-cell penetrating peptide conjugate is being synthesized that can mediate both cell uptake and nuclear localization to determine if intracellular conditions are conducive to invasion complex formation in situ once nuclear localization is achieved.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]