Prion diseases are caused by an infectious protein (20,25). These invariably fatal illnesses cannot be cured using routine antimicrobial agents, and materials contaminated with prions cannot be disinfected by conventional methods. Therefore, it is important to identify compounds that can be used either as therapeutic or disinfecting reagents for prion diseases. Ongoing epidemics of new variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (BSE) in the United Kingdom highlight the urgency of this task.We recently reported that branched polyamines could purge scrapie-infected neuroblastoma (ScN2a) cells of PrP Sc , the disease-causing isoform of the prion protein (33). The ability of these compounds to eliminate PrP Sc from ScN2a cells depended upon a highly branched structure and a high surface density of primary amino groups. The most potent compounds identified were generation 4.0 polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers. Dendrimers are branched polyamines manufactured by a repetitive divergent growth technique, allowing the synthesis of successive, welldefined "generations" of homodisperse structures. In the current study, we demonstrate that branched polyamines cure prion-infected cells and identify the site and mechanism of polyamine-mediated prion clearance. We also demonstrate that these compounds can be employed in a rapid and simple assay to discriminate between different prion strains in vitro. MATERIALS AND METHODSChemical compounds. High-molecular-weight polyethyleneimine (PEI) was purchased from Fluka. SuperFect transfection reagent was purchased from Qiagen. All other polyamines were purchased from Sigma-Aldrich. Fluoresceinlabeled PPI was synthesized by mixing 30 mg of fluorescein isothiocyanate (FITC) with 1 mg of PPI generation 4.0 in 2 ml of ethanol overnight at 4°C. Labeled PPI was separated from residual, unreacted FITC using a Sephadex P-2 column.Cultured cells. Cultures of ScN2a cells were maintained as described previously (33). Cytotoxicity after treatment with polyamines was assessed in ScN2a cells by the following four methods: (i) examination of morphology under phase contrast microscopy, (ii) observation of growth curves and cell counts for 3 weeks after treatment, (iii) vital staining of living cells with 0.4% trypan blue (SigmaAldrich), and (iv) assay of dehydrogenase enzymes with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). For ScN2a cells treated with either PAMAM or PPI generation 4.0 continuously for 1 week, the 50% toxic dose was ϳ50 g/ml.To prepare samples for infectivity assays, 100-mm-diameter plates (Falcon) of confluent cells were washed three times with 5 ml of phosphate-buffered saline, scraped into 2 ml of phosphate-buffered saline, and homogenized by repeated extrusion through a 26-gauge needle. Prion infectivity was determined by intracerebral inoculation of 30 l of cell homogenate into Tg(MoPrP)4053 mice. Mice were observed for clinical signs of scrapie, and a subset of diagnoses were confir...

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We introduce a lung inflation-fixation protocol to examine the distribution and gene transfer efficiency of fluorescently tagged lipoplexes using fluorescence confocal microscopy within thick lung tissue sections. Using this technique, we tested the hypothesis that factors related to lipoplex distribution were the predominant reason that intravenous (i.v.) administration of lipoplex was superior to intratracheal (i.t.) administration for gene transfer in the murine lung. Lipoplex distribution was analyzed using digitized images of overlapping fields, reconstructed to view an entire lung lobe. Intravenously administered lipoplexes were confined to the capillary network and homogenously distributed throughout the lung lobe. In contrast, i.t. administration resulted in regional distribution of lipoplex, concentrated around bronchioles and distal airways. Not all the bronchioles were stained with lipoplex, suggesting that the airway-administered solution became channeled through certain bronchiolar pathways. A fluorescent oligonucleotide was used as a marker for cytoplasmic release of nucleic acids. Quantification of the

It was recently suggested that intravenously administered plasma as determined by an in vitro transfection assay. In lipoplexes serve as a depot for the extracellular release of spite of this high level of transcriptionally active DNA, there naked DNA and it is the naked DNA that mediates gene was no significant gene expression in the lung or any other delivery in the lung. If this is the mechanism responsible organ tested. In addition, when lipoplex containing a for gene expression, we reasoned that continuous infusion reporter gene was injected, followed by an infusion of nonof plasmid DNA should also result in significant lung coding plasmid DNA as a potential competing molecule for expression in the absence of lipoplexes. Moreover, the DNA released from the lipoplex there was no effect on infusion of non-coding plasmid DNA should inhibit gene gene expression. These experiments indicate that the catdelivery by lipoplexes. Infusion of plasmid DNA at a rate ionic lipid component of the lipoplex functions in an active of 80 g/min into the tail vein of a mouse resulted in a DNA capacity beyond that of a simple passive release matrix for serum concentration of 800 g/ml. This was equivalent to plasmid DNA. a transcriptionally active DNA concentration of 120 g/ml Keywords: DNA; infusion; mechanism; nonviral Lipoplex-mediated gene delivery 1,2 in vivo was demonstrated by the pioneering work of Brigham and colleagues. 3 Since then, many cationic lipid-based gene delivery protocols have entered clinical trials. [4][5][6][7][8][9] Despite this progress, the mechanism(s) by which lipoplexes deliver genes in vivo has not been elucidated.Recently, Song et al 10 injected cationic liposomes intravenously (i.v.) 5 min before an i.v. injection of a reporter gene. Surprisingly, the level of transgene expression in the lung was the same as that obtained following injection of the preformed lipoplex. Based on these data, Song et al suggested that extracellular DNA released from the lipoplex is the transfectionally active agent, and that the cationic lipid component serves only to increase pulmonary retention of DNA following systemic administration. 10 Given that naked plasmid DNA is capable of transfecting striated muscle 11,12 , the thyroid gland 13 , the liver 14 , the lung 15 , solid tumors 16 and synovial tissue 17 , we found Song's observations intriguing and believed the hypothesis to be an important conjecture. If the proposal is correct, then any sustained release system for DNA should mediate transfection in the lung. Potentially, one could obtain gene expression without the adverse effects associated with the cationic lipid.We repeated Liu's experiments and confirmed the observations. The pre-injection of cationic liposomes, followed 5 min later by a luciferase reporter gene, yielded We hypothesized that if free DNA is the active transfection agent in pulmonary gene delivery following i.v. lipoplex injection, then establishing a relatively high steady-state plasma concentration of transcriptionally active DNA would yie...

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