Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-03-06
1999-10-12
Mertz, Prema
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 701, 435 703, 435325, 435357, 4353201, C12N 1563, C12N 516
Patent
active
059654040
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for introducing nucleic acid into higher eukaryotic cells.
There is a need for an efficient system for introducing nucleic acid into living cells particularly in the field of gene therapy. This involves locking genes into cells in order to achieve in vivo synthesis of therapeutically active gene products.
Standard transfection methods used, inter alia, calcium phosphate, cationic lipides or liposomes, whilst in order to improve the transfection efficiency of some of these methods, it has been proposed to carry out osmotic shock treatment of the cells with glycerol or with dimethylsulphoxide (Chen and Okayama, 1988; Parker and Stark, 1979; Okada and Rechsteiner, 1982).
The technologies which are currently most advanced for the use of nucleic acids in the field of gene therapy make use of retroviral systems to transfer genes into the cell (Wilson et al., 1990; Kasid et al., 1990).
Alternative strategies for gene transfer are based on mechanisms which the cell uses for the transportation of macromolecules. One example of this is the introduction of genes into the cell by the route of receptor-mediated endocytosis (e.g. Wu and Wu, 1987, Wagner et al., 1990, and EP-A1 0388 758).
For gene transfer with DNA/polycation-complexes by means of receptor-mediated endocytosis, an improvement has been proposed which envisages using components on the basis of their ability to release the contents of endosomes, e.g. adenoviruses or fusogenic peptides. The use of the endosomolytic components causes an increase in the efficiency of gene transfer by avoiding breakdown of the DNA complexes internalised in the cell in the lysosomes (Curiel et al., 1991; Curiel et al., 1992a; Zatloukal et al., 1992; Cotten et al., 1992; Wagner et al., 1992; Curiel et al., 1992b; Wo 93/07283). It has been proposed, inter alia, to modify the adenoviruses by binding to polylysine. The adenovirus-polylysine conjugates may be complexed with DNA together with conjugates of transferrin-polylysine, thereby producing ternary transferrin-polylysine/adenovirus-polylysine/DNA-complexes (Wagner et al., 1992). The complexes bind to transferrin and adenovirus receptors on the target cells. After the endocytosis the adenovirus causes the endosomes to break open, resulting in the release of the material from the endosome into the cytoplasm. This technique is more reliable than conventional viral techniques (Cotton et al., 1992).
The aim of the present invention was to provide a process for introducing nucleic acid complexed with polycations into the cell, which constitutes a further improvement in terms of the simplicity of the method and reliability thereof.
The objective was achieved by a means of a process for introducing nucleic acids/polycation complexes into higher eukaryotic cells in which the complexes are placed on the cells in the presence of ethyleneglycol and/or glycerol.
Unlike the treatments with glycerol or dimethylsulphoxide, which are proposed for the calcium phosphate precipitation or DEAE-Dextran method, carried out as an after-treatment subsequent to the introduction of the transfection components, thereby inducing the desired osmotic shock, in the process according to the invention, the polyhydric alcohol is present in the medium throughout the entire transfection time. The polyhydric alcohol is conveniently present as a component of the transfection medium. In the light of the results of the experiments carried out within the scope of the present invention, it can be assumed that the polyhydric alcohol has no osmotic activity but triggers another mechanism.
The optimum concentration of glycerol and/or ethyleneglycol depends on various factors particularly the cell type; it can be determined by titration experiments. Within the scope of the present invention, as a result of such titrations, a concentration of about 8 to 15% (v/v), especially about 8 to 13%, based on the transfection medium, has proved favourable.
In addition to glycerol and/or ethyleneglycol, substances which prevent acidification o
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Buschle Michael
Wagner Ernst
Zauner Wolfgang
Boehringer Ingelheim International FmbH
Mertz Prema
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