Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-03-09
2001-12-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S459000, C435S470000, C435S471000
Reexamination Certificate
active
06331434
ABSTRACT:
The present invention relates to a method of transforming cells in which an appropriate quantity of nucleic acid fragments is introduced into the cells. The nucleic acid fragments are introduced in the form of a nucleic acid composition which comprises, in an appropriate vehicle, at least one nucleic acid fragment and at least one compacting agent comprising a nitrogen-containing silicone, useful for compacting the nucleic acid fragments; the compositions comprise aggregates of nucleic acid fragments and silicones according to the invention,
To transform cells by genetic engineering by introducing a nucleic acid fragment foreign to the said cell (or heterologous) by transfection, it is necessary, beforehand, to compact the nucleic acid fragments. This compacting is carried out by means of hyperbranched polyamine compounds such as spermine or polyethylenimines (Garcia Ramirez et al.,
Biopolymers
, vol. 34, 1984, pp. 285-292; WO 97 06833). It is also known to use monomers of silanes to condense DNA for the purpose of its structural study without, however, seeking to transform cells (Fang Y. and Hoh J: “Surface-directed DNA condensation in the absence of soluble multivalent cations”
Nucleic acids Research
, vol. 26, No. 2, Jan. 15, 1998, pages 588-593).
It is however necessary to find new compacting agents so as to obtain good protection of the nucleic acids and to make it possible to vary the media used for the transfection of cells depending on the applications. The present invention provides a solution to this problem.
The present invention relates to a method of transforming cells in which an appropriate quantity of nucleic acid fragments is introduced into the cells, characterized in that the nucleic acid fragments are introduced in the form of a nucleic acid composition which comprises, in an appropriate vehicle, at least one nucleic acid fragment and at least one compacting agent comprising a nitrogen-containing silicone.
According to the present invention, “nucleic acid fragment” is understood to mean a nucleotide sequence which may be of the DNA or RNA type, preferably of the DNA, in particular double-stranded, type.
Nitrogen-containing silicone is understood to mean according to the invention any compound comprising in its structure at least one silicon atom on which at least one nitrogen-containing hydrocarbon radical comprising at least one nitrogen atom is grafted.
The structure of the nitrogen-containing silicone according to the invention is an oligomer. Oligomer is understood to mean a chain of monomers of at least 2 silanes and less than 30 silanes, advantageously comprising 3 to 20 silanes.
The nitrogen atom in the hydrocarbon radical according to the invention is in the form of a primary, secondary or tertiary amine, preferably a primary amine, optionally in the form of an ammonium salt, or alternatively in the form of functions of the guanidine or amidine type.
Advantageously, the nitrogen-containing hydrocarbon radical according to the invention is an atine-containing hydrocarbon radical comprising at least one amine function as defined above, preferably at least one primary amine function (−NH
2
).
Hydrocarbon radical is understood to mean according to the invention any radical essentially consisting of carbon and hydrogen atoms, the hydrogens being optionally partly or completely replaced by halogens, and optionally comprising oxygen, nitrogen, sulphur or phosphorus atoms.
Preferably, the hydrocarbon radical is chosen from the alkyl, cycloalkyl and aryl radicals and the polycyclic radicals comprising several aliphatic and/or aromatic rings.
Alkyl radical is understood to mean according to the invention preferably any saturated or unsaturated, linear or branched alkyl radical which is optionally interrupted by one or more oxygen atoms, and in the case of several oxygen atoms the latter are not adjacent, which is optionally interrupted by one or more secondary or tertiary amine, carbonyl, carbonyloxy or oxycarbonyl, carbonate, carbamate or urea functions and the likce, which is optionally sustituted with one or more groups chosen from halogens, hydroxyl or primary, secondary or tertiary amine, or cycloalkyl, aryl or polycyclic radicals. Advantageously, the alkyl radical according to the invention comprises from 1 to 10 carbon atoms, preferably at least three carbon atoms, more preferably from 3 to 5 carbon atoms. Advantageously, the alkyl radical is chosen from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and pentyl radicals and the various isomers of pentyl
This definition of alkyl radicals also applies to the cycloalkyl radicals according to the invention, it being understood that the rings comprise at least three elements, comprising in particular the cyclopropyl, cyclopentyl, cyclobutyl or cyclohexyl radicals, as well as monosaccharides, in particular pentoses or hexoses such as the derivatives of glucose.
Aryl radical is understood to mean according to the invention any aryl or heteroaryl radical, optionally substituted with one or more radicals chosen from the alkyl, cycloalkyl or polycyclic radicals and/or with one or more groups chosen from halogens, hydroxyl or primary, secondary or tertiary amine.
Preferably, the amine-containing hydrocarbon radical according to the invention is chosen from the 3-aminopropyl or 3-(2-aminoethylamino)propyl radicals.
Among the amine-containing silicones according to the invention are the organic silicones or the derivatives of silica.
The organic silicones according to the invention comprise at least one silicone unit which follows:
Si R1 O
(3−x−y)/2
R2
x
R3
y
in which,
R1 is a nitrogen-containing hydrocarbon radical as defined above,
R2 represents the R1 radical, a hydroxyl radical or a hydrocarbon radical,
R3 represents R2 or the radical of formula
in which R2 is defined above,
x and y, which are identical or different, are 0, 1 or 2, it being understood that the sum x+y is less than or equal to 3.
According to a specific embodiment of the invention, the amine-containing silicone is chosen from the silicones of general formula I or II which follow:
in which,
R1, R2 and R3 are defined above,
R4 represents a hydrogen atom, a hydrocarbon radical or a radical of formula
in which R2 is defined above,
n is at least equal to 1, m is greater than or equal to 0 and p is greater than or equal to 0, on the condition that the sum n+p is greater than or equal to 3.
Preferably, n is between 1 and 10, more preferably between 5 and 9, and m or p are between 0 and 9.
According to a specific embodiment of the invention, in the silicones of general formula I, R2 represents a hydroxyl radical or a hydrocarbon, in particular alkyl, radical, R3 represents a hydroxyl radical and R4 represents a hydrogen atom, n is between 7 and 9 and m is equal to 0.
The silicones according to the invention are prepared according to the customary methods of the state of the art, which are described in particular in
Organic Chemistry
(vol. 29, Polymer synthese, vol. I, chap. 4, pp. 114-139), by Patai and Rappopor (The chemistry of Silicon Compound, Part 2, pp. 1291-1357), Desachler et al. (Angew. Chem., Int. Ed. Engl., 25, 1986, 236), and in Patent Application JP 9 100 353.
When the amine-containing silicone is a silica derivative, it is advantageously according to the invention a silica support, in the form of particles which are grafted with at least one nitrogen-containing hydrocarbon radical as defined above, Preferably, the particles of silica are nanoparticles having a mean diameter of between 10 and 100 nm, preferably of between 30 and 60 nm, having a specific surface area of about 90 to 100 m
2
/g. The silica support may be a layer of silica which coats a metallic support; it is advantageously according to the invention a tungsten or gold support. The support is advantageously a particle having a diameter ranging from about 0.5 to 2 &mgr;m, preferably of about 1 &mgr;m.
The mean diameter of the particles is defined by size and measured by diffraction of light or by visualization under a transmiss
Decor Rachel
Mioskowski Charles
Schmutz Marc
Wagner Alain
Aventis CropScience SA
Baker & Botts L.L.P.
Ketter James
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