Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-24
2002-12-24
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091400, C435S320100, C435S325000
Reexamination Certificate
active
06498011
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to the transformation of eukaryotic cells, particularly animal cells, with exogenous nucleic acids and to the generation of transgenic organisms generated from such cells. More particular, the present invention relates to a method for introducing nucleic acids into cells for producing transiently transfected/transformed or stably transformed cells by using a specifically designed nucleic acid/protein complex, as well as to cells transfected or transformed thereby.
BACKGROUND OF THE INVENTION
Several methods have been developed for introducing exogenous DNA molecules into eukaryotic cells in order to take advantage of the widespread benefits arising from the application of recombinant DNA technology to the production of transiently transtected/transformed cells as well as to transgenic cells and organisms generated from such cells. These methods include physical, non-biological systems such as electroporation, microinjection, calcium phosphate or polyethylene glycol (PEG) mediated DNA uptake or cell fusion, and microprojectile bombardment, and modified biological systems such as Agrobacterium-mediated T-DNA transfer to plant cells (for a general overview, see chapters 2 and 3 of “Plant Genetic Transformation and Gene Expression, A Laboratory Manual”, ed. by Draper, J. et al., pub. by Blackwell Scientific Publications (1988); see also Potrykus, et al., “Direct Gene Transfer: State of the Art and Future Potential”,
Plant Mol. Biol. Rep.
3: 117-128 (1985)).
The methods which have been developed have allowed the stable transformation of a wide variety of cells and organisms with exogenous DNA. In particular, the development of physical techniques such as “biolistics” has overcome apparent host-range limitations imposed by biological systems. However, a common deficiency of these physical methods is that they do not provide any means for ordered integration of the delivered nucleic acid into the cell genome. Consequently these methods must depend upon uncontrolled integration of the delivered nucleic acid by poorly understood mechanisms, causing exogenous DNA to be integrated as multiple copies of random fragments usually at a single site in the cell genome.
Improving the predictability of stable transformation events arising from the physical introduction of exogenous nucleic acid into the cell would significantly improve the utility and overall efficiency of these processes for producing genetically stable transformed cells or organisms exhibiting stable expression of transgenes. One approach which has been taken to accomplish this goal has been to combine proteins which promote transformation and/or integration in biological systems with non-biological delivery techniques. In order to achieve the desired effect, it has been considered necessary to associate the proteins themselves with the exogenous DNA molecules prior to delivery to the transformation target cell, thus mimicking as closely as possible the biological system from which the proteins are derived (see, e.g. international application no. PCT/EP94/02566 to Hohn et al. published Feb. 23, 1995 as WO 95/05471; international application no. PCT/US95/07543 to Conary, J. et al. published Dec. 21, 1995 as WO 95/34647).
The Agrobacterium plant transformation system mentioned above is widely used for the stable transformation of higher plants. In this system genes to be transferred are carried by the T-DNA, a well-defined region of the Agrobacterium Ti plasmid. The Ti plasmid also contains a virulence (vir) region, which encodes proteins involved in the transformation via Agrobacterium of plant cells. At least one of these proteins, VirD2 is involved in targeting to the plant nucleus and integration into the plant genome (Tinland et al. (1992)
Proc. Natl. Acad. Sci. USA
89: 7442; Tinland et al. (1995)
EMBO J.
14: 3588-3595). WO 95/05471, the contents of which is herewith incorporated by reference, discloses a method for producing stably transformed plant material, including phenotypically normal looking and preferably fertile plants, which method does not involve Agrobacterium transformation. In particular, it discloses a specifically adapted nucleic acid/protein complex comprising a chimeric recombinant nucleic acid, which may comprise, for example, an expressible DNA or an oligonucleotide operably linked to suitable plant expression signals involving promoter and termination sequences and covalently associated therewith a VirD2, and, optionally, VirE2 protein units. However, the teaching does not mention potential applicability of this transformation technique to the field of animal cells. Furthermore, it does not concern using specifically designed oligonucleotides as nucleic acid component of said complex in an antisense- , antigene- or oligozyme-approach for (transient) transfection/transformation of eukaryotic cells.
Since there exists a continuous need for further techniques which are useful for the introduction of nucleic acids into animal and plant cells, e.g. oligonucleotides for antisense- or antigene-approaches, or for the permanent transformation of animal cells, the object of the present invention is therefore to provide a new method for introducing nucleic acids into eukaryotic cells.
SUMMARY OF THE INVENTION
The present invention provides an improved method for delivering nucleic acids as nucleic acid/protein complex to eukaryotic cells, e.g. oligonucleotides or exogenous DNA for stably transforming or transiently transfecting/transforming animal, preferably mammalian cells. This improved method for example generally comprises providing to the cell targeted for transfection/transformation a specifically designed nucleic acid/protein complex comprising nucleic acids such as e.g. exogenous DNA or oligonucleotide desired to be introduced and, if desired, to be integrated in the later transformant.
For example, the present invention particularly provides an improved method for transiently transfecting/transforming or for stably transforming animal cells with exogenous nucleic acids such as e.g. DNA, which combines positive attributes of
Agrobacterium tumefaciens
mediated T-DNA transfer such as high-efficient nuclear targeting and integration, with non-biological delivery methods. This aspect of the invention e.g. comprises providing an animal cell with the exogenous DNA fragment desired to be introduced into the nucleus and integrated into the animal cell genome, bounded by T-DNA borders or functional parts thereof, along with at least one Agrobacterium-derived protein that targets said fragment to the nucleus and promotes the integration of the exogenous DNA into the host cell genome. The Agrobacterium-derived protein used according to the invention is selected from the group consisting of VirD1, VirD2, VirE2, and VirC. Preferably, a combination of VirD2 and either VirD1, VirC, VirE2, or a subcombination thereof, is used. Most preferably, use is made of the Agrobacterium-derived proteins VirD2 and VirE2 in combination, although in certain cases sole use of VirD2 may be sufficient.
According to the invention, the nucleic acid/protein complex comprising the exogenous nucleic acid, such as e.g. a DNA fragment bounded by T-DNA border sequences or functional parts thereof, may be delivered to the animal cell by non-biological means such as, but not restricted to, electroporation, microinjection, induced uptake, microprojectile bombardment, or other means as are known in the art.
In another aspect of the invention, animal cells or tissues stably transformed with a discrete DNA fragment are regenerated to produce transgenic animal organs or whole animals that stably express a desired homologous or heterologous nucleic acid and, in the latter case, pass it on to progeny in which stable expression of the transgene is inherited as a Mendelian trait.
Furthermore, the present invention provides novel means for the in vivo and ex vivo/in vitro transformation and integration or transient transfection/transformation of exogenous nucleic acids desired to be ex
Hohn Barbara
Relic Biserka
Rossi Luca
Ziemienowicz Alicja
Hess Susan
Novartis AG
Wang Andrew
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