Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery.

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Lipopolycationic telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency.

We report on the synthesis of a series of lipopolyamine telomers I-14,n, I-18,n, and II-18,n and on their in vitro gene-transfer capability. Their structure consists of a polyamine polar moiety, including n primary amine functions (from 1 to 70), connected to a hydrophobic moiety, including two hydrocarbon C14 or C18 chains, through a mercaptopropanoyl or mercaptoglyceryl unit and an amide or ether function. They were obtained by telomerization of N-[2-[(BOC)aminoethyl]]acrylamide with N,N-ditetradecyl- and N,N-dioctadecylpropanamide-3-thiol and rac-1,2-dioctadecyloxypropane-3-thiol, respectively, then BOC deprotection. For N/P ratios (N = number of telomer amine equivalents; P = number of DNA phosphates) from 0.8 to 10, these lipopolyamines condensed DNA, with or without the use of DOPE, forming lipopolyplexes or teloplexes of mean sizes less than 200 nm. Some trends, structure-activity and structure-toxicity relationships, were established to achieve both highest in vitro transfection levels and cell viability. Thus, DNA formulations based on telomers I-14,20 and I-18,20 and for N/P ratios lower than 5 led to the most efficient teloplex formulations for plasmid delivery to lung epithelial A549 cells.

A multi-mode chromatographic method for the comparison of the N-glycosylation of a recombinant HIV envelope glycoprotein (gp160s-MN/LAI) purified by two different processes.

The glycosylation pattern of a recombinant gp160s-MN/LAI variant of human immunodeficiency virus type 1 (HIV-1) was studied in relation to two alternative purification techniques one of which involves an immunoprecipitation step. For analysis a multi-mode high performance liquid chromatography (HPLC) method which combines gel permeation chromatography on the RAAM 2000 GlycoSequencer, weak anion exchange chromatography and normal phase chromatography was developed and profiles were obtained for the fluorescently-labelled glycans released from the two gp160s-MN/LAI preparations. Charged glycans accounted for 77 and 80% of the total glycans for the IAP- and SP-purified samples, respectively. The acidic character of these glycans was mainly due to the presence of sialic acids. However, following sialidase treatment, residual charged glycans were still found. No differences were found in the glycan distributions of the two gp160s-MN/LAI preparations either in their degree of sialylation or in their relative proportion of each separated structure. Although this did not reach statistical significance, a lower proportion of large glycan structures regardless of their charge status was found on the gp160s-MN/LAI prepared by the procedure which involved an immunoaffinity chromatography step.

Direct lipid quantitation of cationic liposomes by reversed-phase HPLC in lipoplex preparation process.

The proposed reversed-phase high-performance liquid chromatography method with ultraviolet detection provides a simple and rapid procedure to separate and quantitate lipids from cationic liposomes used in gene transfer. We describe experimental conditions which do not require lipid extraction from liposomes prior to sample analysis. Evaluation of the method reported here showed suitable lipid separation capacity and quantitation accuracy from cationic liposomes composed of either the pentammonio lipid pcTG90 and dioleoyl phosphatidylethanolamine, or 1, 2-dioleoyl-3-trimethylamonium propane and cholesterol. Detection limits were in the range of 0.5-1 microg depending on the lipid. This quantitative method has proven useful in lipoplex formulation processing development and its application may be extended to a wide range of lipid-based gene and drug delivery systems.

Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA.

Cationic polymers have the potential for DNA complexation and it is recognised that they may be useful as non-viral vectors for gene delivery. Highly purified chitosan fractions of < 5000 Da (N1), 5000-10,000 Da (N2) and > 10,000 Daltons (N3) were prepared and characterised in respect of their cytotoxicity, ability to cause haemolysis, ability to complex DNA as well as to protect DNA from nuclease degradation. Also the biodistribution of 125I-labelled chitosans was followed at 5 and 60 min after intravenous injection into male Wistar rats. All chitosan fractions displayed little cytotoxicity against CCRF-CEM and L132 cells (IC50 > 1 mg/ml), and they were not haemolytic (< 15% lysis after 1 and 5 h). Chitosan-DNA interaction at a charge ration of 1:1 was much greater than seen for poly(L-lysine) and complexation resulted in inhibition of DNA degradation by DNase II: 99.9 +/- 0.1, 99.1 +/- 1.5 and 98.5 +/- 2.0% for N1, N2 and N3, respectively. After intravenous injection, all the chitosans showed rapid blood clearance, the plasma levels at 1 h being 32.2 +/- 10.5% of recovered dose for N1 and 2.6 +/- 0.5% of recovered dose for N3. Liver accumulation was molecular mass dependent, being 26.5 +/- 4.9% of the recovered dose for N1 and 82.7 +/- 1.9% of the recovered dose for N3. The observations that the highly purified chitosan fractions used were neither toxic nor haemolytic, that they have the ability to complex DNA and protect against nuclease degradation and that low molecular weight chitosan can be administered intravenously without liver accumulation suggest there is potential to investigate further low molecular weight chitosans as components of a synthetic gene delivery system.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Reaction of mersalyl with mitochondrial proteins under native and denaturing conditions as exemplified by the identification and isolation of the phosphate carrier.

Pig heart mitochondria were incubated with [203Hg]mersalyl and the radioactive pattern was analyzed by fluorography after dodecyl sulfate gel electrophoresis. No differences in the radioactivity distribution were found after labeling with various mersalyl concentrations, at different pH and after labeling in the native or dodecyl sulfate-dissociated state of mitochondria. A redistribution of [203Hg]mersalyl between various proteins in the presence of dodecyl sulfate could directly be demonstrated by mixing labeled membranes with unlabeled matrix proteins, as well as by comparison of the radioactivity patterns of whole mitochondria labeled with irreversibly reacting N-([2-3H]ethyl)maleimide and reversibly binding [203Hg]mersalyl. From these data it is concluded that under native conditions mersalyl is principally bound to the phosphate carrier protein, whereas during dissociation in dodecyl sulfate the organomercurial is redistributed and mainly attached to the ADP/ATP-carrier protein.

Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria.

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.

Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology.

Non-enzymatic deamidation of asparagine and glutamine residues in proteins and peptides are reviewed by first outlining the well-described reaction mechanism involving cyclic imide intermediates, followed by a discussion of structural features which influence the reaction rate. The second and major part describes analytical techniques that allow studying deamidation in proteins using recombinant human growth hormone and recombinant hirudin as examples. Finally, the significance of non-enzymatic deamidation with respect to the production of pharmaceutical proteins is discussed.

Reversed-phase high-performance liquid chromatographic separation and quantitation of phenylthiohydantoin derivatives of 25 amino acids, including those of cysteic acid, 4-hydroxyproline, methionine sulfone, S-carboxymethylcysteine and S-methylcysteine.

A high-performance liquid chromatography system is presented which allows separation and quantitation (in the range 4-1000 pmol) of all common phenylthiohydantoin amino acids, including derivatives of 4-hydroxyproline, methionine sulfone and three differently modified forms of cysteine. By showing the actual solvent gradient during elution (as opposed to the programmed gradient) and by supplying information on the effects of minor changes in solvent-pH, column temperature, flow-rate, and concentration of 2-propanol in the gradient, we make guidelines available for fine-tuning the separation with new Ultrasphere-ODS (C18) columns.

The protein component(s) of the isolated phosphate-transport system of mitochondria.

The ethylmaleimide-sensitive phosphate-transport system of heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. 1. The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4-5 protein components of very similar apparent molecular weight, as shown by gradient dodecylsulfate gel electrophoresis. 2. The amino acid composition of protein 1, 2 and 3(apparent Mr = 34 500, 34 000 and 33 000, respectively) is quite similar although not identical. 3. Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern. 4. Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N'-dicyclohexylcarbodiimide bonds only to protein 1 and 2. 5. N,N'-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. 6. In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. 7. Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation.

Multiple systemic expression of human interferon-beta in mice can be achieved upon repeated administration of optimized pcTG90-lipoplex.

The possibility of achieving multiple systemic expression of human interferon-beta in mice upon repeated intravenous administration of cationic liposome-DNA complex (lipoplex) was investigated. Lipoplexes containing the pentammonio lipid pcTG90 were first optimized by selecting the most efficient ratio of pcTG90 to phosphatidylethanolamine (DOPE) and the N/P ratio of cationic lipid nitrogen to DNA phosphate. Highest levels and reproducibility of gene expression were obtained using pcTG90/DOPE (1:2) liposomes complexed with the IFNB1 gene containing plasmid pTG14169 at a N/P ratio of 10. Following lipoplex administration, an early but transient human interferon-beta expression in serum was observed. Importantly, repeated systemic gene expression could be achieved upon re-administration with a minimal time interval of 14 days between two injections. For an interval period of 6 days, subsequent gene expression was inhibited by a first administration of lipoplexes containing either a luciferase reporter gene plasmid or an empty plasmid, but was not inhibited when free (non-complexed) plasmid pTG14169 was first injected. Multiple injections of pcTG90-lipoplex performed once every other month resulted in three subsequent peaks of systemic IFNB1 gene expression in mice. In conclusion, our study demonstrates the feasibility of expanding the therapeutic window of a cytokine using repetitive intravenous administration of lipoplex.

Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

The phosphate transport protein from beef heart mitochondria has been purified on a large scale by hydroxylapatite chromatography in the presence of sodium dodecyl sulfate and urea. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain), the pure phosphate transport protein preparation consists of two protein bands (alpha and beta, ratio 1:1) with similar mobilities (34 kDa) which display identical peptide maps if fragmented with either CNBr or HCl/dimethyl sulfoxide/HBr. The complete amino acid composition of phosphate transport protein is presented. Quantitative determination of N-terminal amino acids underlines the purity of the preparation and shows for alpha and beta the identical amino-terminals H2N-Ala-Val-Glu-Glu-Glx-Tyr-. Qualitative digestion shows that carboxypeptidase A is able to release at least three amino acids from the C termini of the alpha as well as the beta band of phosphate transport protein. The nature of these two protein bands is discussed. The sum of phosphate transport protein (alpha + beta) per total mitochondrial protein amounts to 2.3% or 1.4 nmol of phosphate transport protein (34 kDa) per nmol of cytochrome b.

Reconstitution of the isolated phosphate-transport system of pig-heart mitochondria.

The phosphate carrier of pig heart mitochondria has been isolated and reconstituted in liposomes. The highest specific activity for [32P]phosphate exchange was obtained with hydroxyapatite eluate from mitochondria, extracted with Triton X-114 in the presence of cardiolipin. This fraction, which is free from the ADP ATP-carrier, had a specific activity of 30 mumol 32Pi x min-1 x mg protein -1. The following conditions were found to inactivate the phosphate carrier irreversibly in the solubilized state: high ionic strength, high detergent concentrations and a high pH. The decrease of the activity by high detergent concentrations can be largely prevented by cardiolipin, present in the extraction buffer, suggesting a specific removal of this lipid by the detergent. After reconstitution in liposomes, the phosphate carrier is rather stable. The Arrhenius plot of the temperature-dependence of the reconstituted phosphate exchange showed different slopes above and below 27 degrees C. Between 0 degrees C and 27 degrees C the EA was 64 kJ . mol-1, between 27 degrees C and 42 degrees C 44 kJ . mol-1. The exchange of Pi followed a first-order kinetic.

Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin.

The binding of iron-loaded human transferrin at the surface of Neisseria meningitidis is mediated by two polypeptides, Tbp1 and Tbp2. Predicted Tbp amino acid sequences from N. meningitidis strains are highly divergent. This variability is particularly pronounced throughout the Tbp2 polypeptide. In this study, a highly structured and extremely stable Tbp2 domain of about 270 to 290 amino acids which is involved in the binding to transferrin and whose position is well conserved has been characterized. The conservation of such a remarkable structure in a very divergent protein domain (there is only 43% amino acid identity within this region) suggests that is plays an essential biological role and raises a number of questions regarding tbp2 evolution.

Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid.

BACKGROUND: One of the main drawbacks of synthetic, non-viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F-PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE. METHODS: To evaluate the contribution of F-PE to lipoplex-mediated gene transfer, the effect of including F-PE in lipoplexes formulated with the lipopolyamine pcTG90 for various pcTG90/DOPE/F-PE molar ratios [1:(1-x): x; 1:(2-y):y] was examined. For the in vitro analyses on human lung carcinoma epithelial A549 cells, the lipoplexes were formulated with the luciferase reporter plasmid pTG11033 using various N/P ratios (from 10 to 0.8, N = number of pcTG90 amines, P = number of DNA phosphates). The in vivo analyses were performed (1) with the luciferase reporter plasmid pCMV-Luc, which gives higher luciferase expression in the lung than pcTG11033; (2) with pcTG90/co-lipid(s) (1:2) lipoplexes which yield higher expression than the (1:1) formulations; and (3) by intravenous (iv) injection into the tail vein of mice. RESULTS: The efficiency of the F-PE lipoplexes to transfect in vitro A549 cells was significantly higher (5-90-fold) than that of DOPE lipoplexes, when formulated in HEPES. However, when formulated in 5% glucose, both co-lipids display a comparable transfection helper potential. Most remarkably, an up to eight-fold increase of luciferase expression could be measured in the lung after iv injection of pcTG90/F-PE (1:2) N/P 5 lipoplexes as compared with the pcTG90/DOPE lipoplexes. It led also to higher luciferase expression than PEI(ExGen500)/pCMV-Luc N/P 10 polyplexes. Besides expression in lung, low levels of luciferase expression were also observed in heart, spleen and liver. CONCLUSION: The present work, showing a higher in vitro and in vivo transfection potential for lipoplexes formulated with a partially fluorinated co-lipid as compared with its analogous DOPE lipoplexes or PEI polyplexes, indicates that 'fluorinated' lipoplexes are attractive candidates for in vivo applications.

Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.