Cationic lipid-based delivery systems such as lipoplexes or aphidicolin exhibited 20-fold lower reporter gene activity stabilized plasmid-lipid particles (SPLP) represent a safer than asynchronous control cells upon incubation with lipoalternative to viral systems for gene therapy applications.plexes. When cells arrested in the G1 phase were allowed We studied the impact of cell cycle status on the efficiency to proceed though the cell cycle in the presence of the lipoof transfection of human ovarian carcinoma tumor cells plex or SPLP, transgene expression was found to coincide using two cationic-lipid based delivery systems. Cells with the transition of cells from the G2/M phase into the arrested in the G1 phase of the cell cycle by treatment with G1 phase of the subsequent cell cycle. In addition, higher aphidicolin were compared with an asynchronous dividing levels of reporter gene expression were observed when the population of cells. Treatment of the cells with aphidicolin cells were incubated with lipoplexes or SPLP during, or just had no effect on the rate of internalization of the lipid forbefore, mitosis. These results suggest that it may be possmulated DNA or on the level of gene expression observible to augment cationic lipid-mediated transfection by able in stably transfected cells. However, cells treated with manipulating the cell cycle status of the target cells.

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.

Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-be generated. The SPLP produced could be isolated from 281) has shown that plasmid DNA can be entrapped in empty vesicles by sucrose density gradient centrifugation, 'stabilized plasmid-lipid particles' (SPLP) containing the and exhibited a narrow size distribution (62 ± 8 nm, as fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), determined by freeze-fracture electron microscopy) and a low levels (5-10 mol%) of cationic lipid, and stabilized by high plasmid-to-lipid ratio of 65 g/ mol (corresponding to a polyethyleneglycol (PEG) coating. The PEG moieties are one plasmid per particle) regardless of the DODAC conattached to a ceramide anchor containing an arachidoyl tent. It was found that isolated SPLP containing 20-acyl group (PEG-CerC 20 ). These SPLP exhibit low trans-24 mol% DODAC resulted in optimum transfection of COSfection potencies in vitro, due in part to the long residence 7 and HepG2 cells in vitro, with luciferase expression levels time of the PEG-CerC 20 on the SPLP surface. In this work comparable to those achieved for plasmid DNA-cationic we employed SPLP stabilized by PEG attached to ceralipid complexes. In vivo studies employing an intraperimide containing an octanoyl acyl group (PEG-CerC 8 ), toneal B16 tumor model and intraperitoneal administration which is able to quickly exchange out of the SPLP, to of SPLP also demonstrated maximum luciferase develop systems that give rise to optimized in vitro and in expression for DODAC contents of 20-24 mol% and sigvivo (regional) transfection. A particular objective was to nificantly improved gene expression in tumor tissue as achieve cationic lipid contents that give rise to maximum compared with complexes. We conclude that SPLP stabiltransfection levels. It is shown that by performing the dialyized by PEG-CerC 8 and containing 20-24 mol% cationic sis procedure in the presence of increasing concentrations lipid are attractive alternatives to plasmid DNA-cationic of citrate, SPLP containing up to 30 mol% of the cationic lipid complexes for regional gene therapy applications. lipid dioleoydimethylammonium chloride (DODAC) could

A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high ,8-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (Ks = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.Pseudomonas putida is a member of the Pseudomonas fluorescens branch (group I) of the family Pseudomonadaceae, a group of organisms that also includes P. aeruginosa (17). The outer membranes of P. putida and related pseudomonads contain a number of proteins which are homologous biochemically, immunologically and genetically to those described in P. aeruginosa (15). However, to date no functional studies of P. putida porins have been described.One interesting class of porins are those outer membrane proteins which are involved in high-affinity substrate transport and contain channels with saturable binding sites for specific substrates. The best studied examples of this class are the maltodextrin-specific porin LamB (5, 6, 21) and nucleotide-specific porin Tsx (18) from Escherichia coli and the phosphate-specific porin OprP (12) and imipenem/basic amino acid-specific porin OprD (26) of P. aeruginosa.LamB is part of a shock-sensitive maltose transport system which mediates the accumulation of maltose into the cell against 100,000-fold concentration gradients, and mutants of E. coli lacking LamB are impaired in maltose uptake (5, 7). LamB reconstituted into black lipid membranes has been shown to bind maltose and maltodextrins up to maltoheptose with Kd decreasing from 10 to 0.067 mM with increasing maltodextrin molecular weight (5, 6). On the basis of this data, a binding site that interacts with up to 5 glucose residues has been proposed (6). In contrast, the phosphate porin OprP appears to have a simple binding site comprising a ring of 3 lysine residues (12).A less well-characterized system for carbohydrate uptake has been described in the pseudomonads. P. aeruginosa and P. putida have common pathways for the dissimilation of glucose (1, 16). Glucose uptake follows either an oxidative pathway in which it is oxidized extracellularly to gluconate or 2-ketogluconate on the cytoplasmic membrane and subsequently transported (1, 10) in which it is transported directly by a high-affinity uptake system (Km = 7 jiM) and subsequently phosphorylated intracellularly (10, 16). High-affinity glucose uptake is induced by growth on glucose (10), and under these condition...

Polyriboinosinic-polyribocytidylic acid [poly(IC ⅐ LC)] was evaluated for its prophylactic and therapeutic efficacies against respiratory influenza A virus infection in mice. Two doses of poly(IC ⅐ LC) (1 mg/kg of body weight per dose) administered intranasally within 12 days prior to infection with 10 50% lethal doses of mouse-adapted influenza A/PR/8 virus fully protected the mice against the infection. Determination of virus titers by hemagglutination and plaque assays showed more than a 2-log 10 decrease in virus titers in lung homogenates of pretreated mice compared with those in the lungs of the nonpretreated group. Treatment of infected mice with poly(IC ⅐ LC) resulted in a modest (40%) survival rate. These results suggest that poly(IC ⅐ LC) provides a highly effective prophylaxis against respiratory influenza A virus infection in mice.