Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Patent
1999-09-27
2000-12-12
Ketter, James
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
4353201, 530399, C12N 1564
Patent
active
061597376
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of enhancing the rate of transfection of cells, to a kit containing the components therefore and to applications in gene therapy.
The rate of uptake of DNA into cells, and hence the efficiency of transfection of cells, is dependent on presenting the DNA in a suitably packaged form for uptake, traditionally by co-precipitation with calcium phosphate, and the ability of the cells to incorporate the packaged DNA into their cytoplasm.
A paper by Bottger et al, Biochemical et Biophysica Acta 950 (1988) 221-228, describes condensation of vector DNA by chromosomal protein HMG1. The method resulted in enhanced transfection.
It is an aim of the present invention to provide alternative methods of enhancing transfection which methods could, as well as being of benefit for transfection of cells in culture, give rise to applications in gene therapy.
The applicants have determined that increased efficiency of transfection can be obtained by stimulating the cell membrane with a growth regulating agent, leading to receptor mediated endocytosis, and that the increase in the rate of transection is at least as great as that resulting from enhanced packaging of DNA with HMG1.
According to a first aspect of the present invention there is provided a method of enhancing the rate of transfection of cells comprising stimulating the cells to be transfected with a growth regulating agent prior to transfection.
Preferably the growth regulating agent is a growth promoting agent, more particularly still a growth factor.
An advantage of using a growth regulating agent is that they can be selected to be specific or non-specific.
Thus, Epidermal Growth Factor (EFG), in spite of its name, will stimulate almost any cells of endothelial or epithelial origin, Fibroblast Growth Factor (FGF) stimulates cells of mesothelial origin and Nerve Growth Factor (NGF) has a very narrow specificity for neuronal cells. The specificity is dictated by the presence of specific receptors for the growth factors on the cell membrane.
This specificity may be particularly advantageous when it comes to consider gene therapy applications.
This is because a major problem to be overcome with gene therapy is how to achieve high rates of transfection with some specificity. Some present approaches can achieve physical localisation of the delivered gene, for instance to a specific organ. The methodology of the invention would, however, allow the selective targetting of a particular cell type within that organ, in addition to enhancing the overall level of transfection.
Other advantages over prior art methods are the cost benefit and the possibility of avoiding introducing "foreign" matter (e.g. viral matter)
As well as growth factors, other growth regulating agents such as, for example, Peanut Agglutinin and Mushroom Lectin could be used.
Lectin Peanut Agglutin is, for example, thought to promote the growth of cells by interaction with surface glycoproteins. In contrast Mushroom (Agaricus bisporis) Lectin, which shares the specificity of peanut agglutinin, is inhibiting to the growth of cells. Mushroom Lectin is found to be carried within the cell to the surface of the nuclear membrane and may possibly carry the DNA with it though the cell membrane. It is thus envisaged that, for example, a combination of a growth factor such as EGF with mushroom lectin would allow the enhancing effects of the growth promotion to be used for gene therapy without the possible disadvantage of the growth promoting effect in vivo.
In accordance with a further aspect of the present invention there is provided a method of inserting genetic material into a cell in gene therapy comprising stimulating the cell into which genetic material is to be inserted with a growth regulating agent in the presence of a growth inhibitor.
According to yet a further aspect of the present invention there is provided a kit for the methods of the invention comprising at least one growth regulating agent; and a buffer.
The Growth regulating agent should be present in an effe
REFERENCES:
Boettger et al, "Condensation Of Vector DNA by the Chromosomal Protein HMG1 Results in Efficient Transfection" Biochimica Et Biophysica Acta, 950(2):221-228 (1988).
Boettger et al., "Transfection by DNA-Nuclear Protein HMG1 Complexes: Raising the Efficiency And Role of DNA Topology", Archiv Fuer Geschwulstforschung, 60(4):265-270 (1990).
Cristiano et al., "Epidermal Growth Factor Mediated DNA Delivery Into Lung Cancer Cells Via the Epidermal Growth Factor Receptor", Cancer Gene Therapy, 3(1):4-10 (1996).
Kaneda et al., "Increased Expression of DNA Cointroduced With Nuclear Protein in Adult Rat Liver", Journal of Molecular Medicine, 73(6):289-297 (1995).
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