Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-05-03
2002-04-16
Nguyen, Dave T. (Department: 1633)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S069100, C435S320100, C435S325000, C424S486000
Reexamination Certificate
active
06372499
ABSTRACT:
The invention relates to new complexes of nucleic acids and polymer substituted by residues which cause destabilization of cell membranes.
The introduction of a foreign gene into a cell is the basis of genetic treatment. The transfer of genes can be achieved using either a modified viral material (vaccine virus, retrovirus, adenovirus or herpes virus) or using non-viral vectors (cationic lipids, liposomes). The former, although effective, have safety problems. As regards the latter, the effectiveness is greatly reduced in the presence of serum, and as a result their use is restricted to in vitro or ex vivo.
Polylysine, which can form stable electrostatic complexes with a plasmid DNA is the basis for development of non-viral vectors for transfer of genes in animal cells.
Complexes of DNA and unsubstituted polylysine generally are not effective for transfection of cells because of the very high stability of the complexes (and therefore weak dissociation and salting out of the DNA) under physiological conditions as a consequence of a very high co-operativity of polycation-polyanion interactions.
The transfection efficiency can be improved if the number of charges present on the polypeptide is decreased in order to reduce the interactive forces between the DNA and the polylysine. For example, if 40% of the &egr;-NH
3
+
functions of the lysine residues of the polylysine are partly neutralized by polyhydroxyalkanoyl derivatives, such as &dgr;-gluconolactone, the DNA/partly gluconylated polylysine complexes are more effective than DNA/polylysine complexes in transfection of cells.
The polylysine can be substituted by specific receptor ligands which are present on the surface of cells and are capable of inducing specific endocytosis of complexes with a plasmid DNA by target cells.
Conjugates obtained by substituting polylysine by asialoorosomucoid, transferrin, insulin, immunoglobulin and growth factors have been proposed as plasmid guide vectors. However, these protein ligands render the complexes highly immunogenic.
The polylysine can be substituted by low molecular weight ligands which are less immunogenic than the osides and oligosides recognized by specific membrane receptors (membrane lectins) on the surface of target cells. Glycosylated polylysine has been proposed as non-viral vectors perfectly defined for transfer of genes.
Numerous animal cells have membrane lectins which recognize oligosides of various structures and which induce endocytosis of their ligands. For example, the membrane lectin of cells of the hepatic parenchyma recognize glucidic structures carrying a galactose residue in the terminal position, which is the case for the desialylated serum glycoproteins. The specificity of membrane lectins depends on the cell type and the state of differentiation of the cells.
The transfection efficiency of DNA/glycosylated polylysine complexes depends on the level of substitution of the polylysine by osides: The most effective transfections are obtained if 30 to 40% of the &egr;-NH
3
+
functions of the lysine residues of the polylysine are substituted by mono- or disaccharides.
In French Patent no. 2,719,316, it has been shown that the use of partly gluconylated polylysine carrying an already reduced number of positive charges allows the number of osides required for bonding on the polymer to obtain a good transfection efficiency of DNA/glycosylated and gluconylated polylysine complexes to be decreased by a factor of 5 to 10. The use of partly gluconylated polylysine allows the solubility of complexes to be increased and their size to be reduced to about 50 nm.
The transportation of plasmids by non-viral vectors which can be recognized specifically by compounds of the plasma membrane of cells is dependent on a step which imitates the mechanism of entry of viral genetic material into a cell. In all the cases described, the DNA/polycationic polymer complexes are carried into endocytosis vesicles, into endosomes and probably into other deeper intracellular compartments removed from the plasma membrane.
The transmembrane passage of plasmid DNA is consequently a critical stage with respect to the release of the said DNA into the cytosol for its passage into the nucleus where the gene will be expressed.
In all the cases described, transmembrane passage auxiliaries are used to promote passage of the DNA into the cytosol. These are:
chloroquine
defective adenoviruses
permeabilizing and/or fusiogenic peptides
a) Chloroquine is a weak base used in an amount of 50 &mgr;M or 100 &mgr;M in culture in vitro and for some cells these concentrations are toxic. Chloroquine, which permeates, crosses the membrane and accumulates in the acid compartments because it carries amines of low pK which capture protons; protonated chloroquine is cationic and less permeating. Acidification of endosomes and lysosomes is caused by a membrane enzyme which injects H
+
from cytosol into vesicles; to re-establish electroneutrality, this proton accumulation is accompanied by an entry of chloride ions Cl
−
. To the extent that chloroquine accumulates, protons and chlorides also accumulate, which causes an increase in the intravesicular ionic force, which induces the arrival of water, resulting in swelling of the vesicles and their destabilization. The intracellular concentration of chloroquine can be more than 100 times higher than its concentration in the medium after a few hours. It can thus exceed 10 mM. This phenomenon is comparable to that which occurs in persons who use a daily dose of 300 mg chloroquine per day. After a few days, the plasma concentration is about 0.7 &mgr;M and the tissue concentration is 200 to 700 times higher, that is to say 140 to 500 &mgr;M. Inside the cells, the acid compartments can reach concentrations several tens of times higher. It is furthermore known that chloroquine concentrations of 10 mM (concentration obtained a few hours after having used an initial chloroquine concentration of 100 &mgr;M) promote dissociation of DNA/polylysine complexes.
Chloroquine in combination with DNA/polylysine complexes in gene transfer can be used only in applications in vitro or ex vivo, because of its toxicity and its rapid dilution after injection into the individual. In fact, in vivo, to achieve the high concentrations mentioned above, several days are necessary. It has thus been found in vitro that in cells pretreated with chloroquine, expression of the transferred genes was very low. In addition, if the chloroquine is added more than three hours after the incubation of the cells in the presence of the complexes, the transfection is very low. For these reasons chloroquine, which is a very good auxiliary in vitro, is not effective in vivo.
b) The fusiogenic properties of defective adenovirus particles in an acid medium are used to promote passage of DNA into the cytosol from endocytosis vesicles. Adenoviruses have fusion proteins which are active in a slightly acid medium. Defective adenoviruses can be used in the free form or bonded to DNA/polylysine complexes.
However, the use of even defective viral particles presents safety problems. Adenoviruses induce a very strong immune response after injection with the complexes.
c) Peptide which are permeabilizing and/or fusiogenic in a slightly acid medium are used as auxiliaries to promote passage of DNA into the cytosol. These are mainly peptides of 20 amino acids derived from virus fusion proteins, such as, for example, the N-terminal peptide of the sub-unit HA2 of the haemagglutinin of the influenza virus, or synthetic peptides, such as GALA (SEQ ID NO:1), an oligomer containing several recurring units of Glu-Ala-Leu-Ala (SEQ ID NO:1). These peptides are most often used in the free form (that is to say not covalently bonded) with the DNA/polylysine complexes. The efficiency of peptides is greatly reduced in the presence of serum in the cell culture medium, which restricts their use to experiments in vitro or to ex vivo. Some peptides covalently bonded to DNA/polylysine complexes are still effective in promoting transmem
Midoux Patrick
Monsigny Michel
I.D.M. Immuno-Designed Molecules
Nguyen Dave T.
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