This proposal aims to develop long-circulating polyplexes that target cells and mediate gene expression or siRNA knockdown in remote tissues. The approach utilizes a novel DNA binding peptide, composed of six repeats of (Acr-Lys), where Acr is defined as Lys modified with acridine. The peptide binds DNA or siRNA with high affinity by polyintercalation of six acridines. PEGylated-(Acr-Lys) 6 binds DNA with sufficient affinity to form DNAse stable polyplexes that circulate with a half-life of 3 hours and mediate luciferase expression in mice when triggered by hydrodynamic stimulation. Based on these results, we propose to develop long-circulating multi-component PEGylated PolyAcridine Polyplexes (PPAPs) containing a fusogenic peptide and octreotide as a high affinity ligand to target cells expressing the somatostatin subtype-2 receptor (SS2). Solid phase synthesis and bioconjugation will be used to prepare melittin-(Acr-Lys) 6 and octreotide-(Acr-Lys) 6. Precise add- mixtures of PEG-, melittin- and octreotide-(Acr-Lys) 6 will be used to form multi-component DNA and siRNA PPAPs with defined composition, charge, size and shape. Octreotide containing PPAPs will be used to target stably transformed HEK-SS2 and HEK-SS2-Luc cell lines in vitro and in vivo. Multi-component 125I-DNA or 125I- siRNA PPAPs will serve as tracers to measure SS2 receptor mediated uptake in HEK-SS2 and HEK-SS2-Luc cells. In vitro transfection studies using HEK-SS2, SH-SY5Y and G3 cells will establish optimal ratios of PEG, melittin and octreotide to mediate luciferase expression. Multi-component siRNA PPAPs will be optimized by measuring luciferase knockdown following transfection of HEK-SS2-Luc cells. Multi-component 125I-DNA or 125I-siRNA PPAPs will be dosed i.v. in nude mice possessing hind limb xenographs of SS2-cells. Pharmacokinetic and biodistribution experiments will determine the efficiency of octreotide targeted delivery of PPAPs as a function of xenograph size and cell number, multi-component PPAP composition, charge, dose and route of administration. Scrambled octreotide ligand or SS2 antagonist will be substituted into PPAPs to measure the relative contribution of targeted delivery versus the enhanced permeability and retention effect. Optimal multi-component DNA and siRNA PPAPs will be administered i.v. to xenograph mice and measured for luciferase expression or knockdown using quantitative bioluminescence (BLI) as a function of time, xenograph size and multi-component PPAP composition. These experiments aim to test the hypothesis that multi-component combinations of polyacridine peptides possessing PEG, ligand and fusogenic peptide form functionally active PPAPs that target DNA and siRNA to remote tissue sites in animals following i.v. dosing. The proposed studies are innovative in their use of short, high affinity polyacridine peptides to simultaneously bind PEG, melittin and octreotide to DNA and siRNA. The studies are further innovative in the proposed novel use of octreotide as a gene targeting ligand. The successful execution of the proposed studies will establish fundamental parameters to incorporate in the design of i.v. dosed nonviral gene delivery polyplexes targeted to cells outside the liver. These studies are essential to advance nonviral gene delivery toward long-circulating polyplexes that target to remote tissue sites.