Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofection) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated-polylysine, polylysine, DNA, and the dimeric peptide (E5-WYGG)2-KA was less cell-type dependent than other peptide-based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%).

Amino Acid Sequence↗

PubMedDate not supplied

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

BACKGROUND: We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes. METHODS: and results Here, we have analyzed the ability of histidylated polylysine to transfer reporter or CFTR genes into immortalized cystic fibrosis airway surface epithelial cells (sigmaCFTE29o- cells) and airway gland serous cells (CF-KM4 cells) which are both important targets for cystic fibrosis gene therapy. The luciferase reporter gene expression measured after gene transfer with histidylated polylysine into both cell lines was quite high and similar to that obtained with commercially available vectors. In addition, the level of expression was not dependent on the presence of a membrane disrupting agent such as chloroquine. Histidylated complexes were present in slightly acidic non-lysosomal cellular compartments as shown by a cytological approach using biotinylated plasmid, lysosome-specific antibodies and confocal microscopy. Histidylated complexes appeared to be of small size when prepared at low ionic strength and formed aggregates upon increasing the ionic strength. However, aggregate formation was prevented by the addition of 10% fetal bovine serum. Gene transfer efficiency varied with the size of the complexes and decreased when small particles were used. CONCLUSIONS: These results suggest that histidylated polylysine may be an efficient non-viral vector for gene transfer into cystic fibrosis airway surface epithelial cells and airway gland serous cells.

Animals↗

PubMedDate not supplied

Endocytosis mediated by monocyte and macrophage membrane lectins--application to antiviral drug targeting.

Sugar receptors, or membrane lectins, have been evidenced at the surface of various normal and tumour cells using fluoresceinylated neoglycoproteins (glycosylated bovine serum albumin (BSA]. By flow cytometry we have shown that macrophages bind and internalize mannosylated and 6-phosphomannosylated ligands in acidic compartments. Freshly isolated monocytes and U937, a promonocytic cell line, lack a mannose-specific receptor, but express mannose-6-phosphate (Man-6P) membrane lectin. Neoglycoproteins are potent drug carriers: muramyl dipeptide (MDP), an immunoactivator, when bound to Man-BSA or Man-6P-BSA, is 100 times more efficient than free MDP in activating macrophages; in vivo, it enables eradication of lung metastases in mice. Recently, neutral glycosylated biodegradable and nonimmunogenic polymers, were synthesized and found to be as efficient as neoglycoproteins. Antiviral drug conjugates were more active than the free drug, inhibiting the multiplication of virus (herpes) in human macrophages in vitro.

Animals↗

PubMedDate not supplied

Cellular vaccines.

This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient's own tumour. (1) Autologous cellular vaccines are prepared by processing circulating blood mononuclear cells outside of the patient's body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation. Tolerance and biologic effects are documented in primates. (4) The first clinical trials on cancer patients are taking place in 1998 on melanoma and prostate cancer to validate the concept. Specialized cell processors with dedicated software and standardized controls are being developed and used for the preparation of cellular vaccines. (5) The evaluation of new non-viral vectors and the validation of new non-viral transfection methods of mononuclear cells with marker genes is in progress and will lead to the ex vivo transfection of genes coding for immunostimulating cytokines or for tumour antigens in MD-APCs. Efficiency will be validated in vitro and in animal models. The ex vivo and animal model studies validate the clinical relevance of this new cellular immunotechnology. Clinical validation of individual autologous cellular vaccines in specific indications for which no treatment is presently available will allow the development of cellular and gene immunotherapy for other types of cancers.

Animals↗

PubMedDate not supplied

Production of macrophage-derived cytotoxic factor by N-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins.

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethyl)thio]propionyl residues are potent macrophage activators.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMedDate not supplied

Activation of mouse macrophages by muramyl dipeptide coupled with an anti-macrophage monoclonal antibody.

A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues were prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules. Mouse peritoneal macrophages, incubated for 24 h with MDP-mAb3A33 conjugate became cytostatic against P815 mastocytoma cells, whereas unconjugated mAb3A33 and MDP-bound to a nonspecific rat IgG2a were ineffective. An enhancement of the cytostatic activity induced by MDP-mAb3A33 conjugate was obtained in the presence of gamma-IFN. These results show that several tens of MDP molecules can be linked to a macrophage-specific monoclonal antibody by using a neutral intermediate polymer without impairing the binding antibody capacity and that this type of MDP conjugate can efficiently activate macrophages and therefore could be the basis of the development of new antitumor therapy.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMedDate not supplied

Uptake by macrophages of a biotinylated oligo-alpha-deoxythymidylate by using mannosylated streptavidin.

Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(alpha-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis (alpha-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.

Animals↗

PubMedDate not supplied

Glycosylated polylysine/DNA complexes: gene transfer efficiency in relation with the size and the sugar substitution level of glycosylated polylysines and with the plasmid size.

A DNA delivery system based on the use of polylysine substituted with small recognition signals, such as carbohydrate moieties specifically recognized by membrane lectins present in a given cell line, has been developed [Midoux et al. (1993) Nucleic Acids Res. 21, 871-878]. Human hepatoma (HepG2) cells which express a galactose-specific membrane lectin are efficiently transfected in the presence of chloroquine with pSV2Luc plasmid complexed with a lactosylated polylysine. The optimization of the parameters involved in the formation of DNA/glycosylated polylysine complexes leads to the following conclusions: a high gene transfer efficiency is reached when (i) DNA/glycosylated polylysine complexes are completely retarded when subjected to electrophoresis and when (ii) 31 +/- 4% or 40 +/- 8% of the amino groups of a polylysine having a degree of polymerization (DP) of 190 are substituted with lactosyl or beta-D-galactosyl residues, respectively. In addition, carbohydrate residues bound to polylysine decrease the electrostatic strength between plasmid DNA and glycosylated polylysine, suggesting that the strength of the electrostatic interactions between the plasmid and the glycosylated polylysine plays an important role in the efficiency of the gene expression. The optimal lactosylated polylysine conjugate (polylysine DP 190 substituted with 60 lactosyl residues) transfers a 5 kb and a 12 kb plasmid with a similar efficiency.

Carcinoma, Hepatocellular↗

PubMedDate not supplied

Synthesis and antiviral activity of peptide-oligonucleotide conjugates prepared by using N alpha-(bromoacetyl)peptides.

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression. In order to direct oligonucleotides to specific compartments within the cell, we have investigated the possibility of coupling them to a signal peptide Lys-Asp-Glu-Leu (KDEL). This sequence should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located. On this basis we prepared peptide-oligonucleotide conjugates by coupling, in a single step, a Nalpha-bromoacetyl peptide with an oligonucleotide bearing a thiol group, through a thioether bond. This paper deals with the definition of the optimal pH and temperature conditions leading to an efficient synthesis of peptide-oligonucleotide conjugates: the reaction was quantitative at pH 7.5 within few hours. This method was first set up using a 5',3'-modified dodecanucleotide and a (bromoacetyl)pentapeptide as a conjugation model. Then a 5',3'-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond has a higher antiviral activity than the peptide-free oligonucleotide and the conjugate linked via a disulfide bond.

Amino Acid Sequence↗