Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1x to 100x the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 minutes. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N: P ration of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (< 20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (< 20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)n-Cys were prepared by solid phase peptide synthesis, where Acr is Lys modified on its ε-amine with acridine, X is Arg, Leu or Lys and n is 2, 3 or 4 repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized. Gene transfer experiments in multiple cell lines established polyacridine-melittin peptides mediate expression as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin. The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine peptides as a novel DNA binding anchor peptide.

\ live Asiatic clam Corbicuh mamUnsis. in dcchlorinatcd municipal water containing no added potasphotograph was taken the clam retracted its foot and siphons in response to the light Hash.

We describe 4 patients infected with levofloxacin-resistant pneumococci after therapy for community-acquired pneumonia (CAP). The 4 patients had 15 episodes of CAP; Streptococcus pneumoniae was isolated from blood or sputum samples obtained during 14 of the episodes. The underlying medical condition was Bruton agammaglobulinemia in 3 patients and chronic lymphoid leukemia in the other. The initial episode of CAP in each patient was due to a levofloxacin-susceptible strain. One of 4 reinfections and 5 of 6 relapses were due to levofloxacin-resistant strains. All of these strains had amino acid substitutions in the quinolone-resistance-determining region of the genes parC and gyrA. The time between episodes of pneumonia varied from 1 to 4 months. In immunocompromised patients with suspected or proven pneumococcal infection, it may be prudent not to use fluoroquinolone monotherapy empirically when the patient has a history of fluoroquinolone therapy in at least the past 4 months.

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Cationic condensing peptides and polymers bind electrostatically to DNA to form cationic polyplexes. While many cationic polyplexes are able to achieve in vitro transfection mediated through electrostatic interactions, few have been able to mediate gene transfer in vivo. The present study describes the development and testing of polyacridine PEG-peptides that bind to plasmid DNA by intercalation resulting in electronegative open polyplex DNA. Polyacridine PEG-peptides were prepared by chemically conjugating 6-(9-acridinylamino) hexanoic acid onto side chains of Lys in PEG-Cys-Trp-(Lys) 3, 4, or 5 . The resulting PEG-Cys-Trp-(Lys-(Acr)) 3, 4, or 5 peptides bound tightly to DNA by polyintercalation, rather than electrostatic binding. Unlike polycationic polyplexes, polyacridine PEG-peptide polyplexes were anionic and open coiled, as revealed by zeta potential and atomic force microscopy. PEG-Cys-Trp-(Lys-(Acr)) 5 showed the highest DNA binding affinity and the greatest ability to protect DNA from metabolism by DNase. Polyacridine PEG-peptide DNA open polyplexes were dosed intramuscularly and electroporated in mice to demonstrate their functional activity in gene transfer. These results establish polyacridine PEGpeptide DNA open polyplexes as a novel gene delivery method for in vivo use.

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