Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-04-02
2004-10-05
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S029000, C435S320100, C435S325000, C435S455000, C536S023100
Reexamination Certificate
active
06800484
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the use of electric pulses to increase the permeability of a cell and more specifically to the introduction of nucleic acids into a cell using electric pulses at a low electric field strength for a long pulse length.
BACKGROUND OF THE INVENTION
The ability to introduce foreign DNA into host cells on one of the principal tools of molecular biology. There are five general types of methods for transfecting eukaryotic host cells: (1) direct introduction of cloned DNA by microinjection, (2) use of viral vectors, (3) encapsulation in a carrier system such as a liposome, (4) the use of facilitators such as calcium phosphate or diethylaminoetyl (DEAE) dextran, and (5) electroporation. Although all of these methods allow the transfer of DNA into dividing cells, fewer methods are available for the transfer of DNA into non-dividing cells.
In the 1970's it was discovered that electric fields could be used to create pores in cells without causing permanent damage. This discovery made possible the insertion of large molecules into cell cytoplasm. It is known that genes and other molecules such as pharmacological compounds can be incorporated into live cells through a process known as electroporation. The genes or other molecules are mixed with the live cells in a buffer medium and short pulses of high electric fields are applied. The cell membranes are transiently made porous and the genes or molecules enter the cells, where they can modify the genome of the cell.
Studies have shown that large size nucleotide sequences (up to 630 kb) can be introduced into mammalian cells via electroporation (Eanault, et al.,
Gene
(Amsterdam), 144(2):205, 1994
; Nucleic Acids Research
, 15(3): 1311, 1987; Knutson, et al.,
Anal. Biochem
., 164:44, 1987; Gibson, et al.,
EMBO J
., 6(8):2457, 1987; Dower, et al.,
Genetic Engineering
, 12:275, 1990; Mozo, et al.,
Plant Molecular Biology
, 16:917, 1991). However, the efficiency of electroporation, as reflected in the current literature, is low (see U.S. Pat. No. 5,019,034, herein incorporated by reference). A typical result is from 5 to 20 percent transfection depending on conditions, parameters and cell type. Creation of a high efficiency method for the transfer of nucleic acid via electroporation would make the method useful for the introduction of nucleic acid into cells both in vivo and in vitro.
SUMMARY OF THE INVENTION
The present invention provides a method for introducing nucleic acid into a cell by contacting the cell with a nucleic acid and applying a low voltage electrical impulse for a long pulse length. The electrical impulse is of sufficient duration and strength to introduce the nucleic acid into the cell. The method can be utilized in vitro or in vivo. The cells can be dividing or non-dividing.
The invention also provides a method for introducing polypeptides into a cell by contacting the cell with a polypeptide and applying a low electrical field impulse for a long pulse length. The electrical impulse is of sufficient duration and strength to introduce the polypeptide into the cell. The method can be utilized in vitro or in vivo. The cells can be dividing or non-dividing.
REFERENCES:
patent: 5019034 (1991-05-01), Weaver et al.
patent: 5859327 (1999-01-01), Dev et al.
patent: 5944710 (1999-08-01), Dev et al.
patent: 5993801 (1999-11-01), Greenberger et al.
patent: WO 97/07826 (1997-03-01), None
patent: WO 99/01158 (1999-01-01), None
Luo, et al. Nature Biotechnology, Jan. 2000, vol. 18, pp. 33-37.*
Palu, et al. Journal of Biotechnology, 1999, vol. 68, pp. 1-13.*
Verma, et al. Nature, Sep. 1997, vol. 389, pp. 239-242.*
Yukiko Yamazaki et al, In Vivo Gene Transfer to Mouse Spermatogenic cells by Deoxyribonucleic Acid Injection into Seminiferous Tubules and Subsequent Electroporation, Biology Of Reproduction 59, 1439-1444 (1998).*
Andreason and Evans, Optimization of Electroporation . . . , Analytical Biochemistry 180, p269-274, Aug. 1, 1989.*
Andreason and Evans, “Optimization of Electroporation for Transfection of Mammalian Cell Lines,”Analytical Biochemistry180, 269-275 (1989).
Prausnitz et al., “Electroporation of mammalian skin: A mechanism to enhance transdermal drug delivery,” Proc. Natl. Acad. Sci., Nov. 1993, vol. 90, pp. 10504-10508.
Giordano, Frank J. et al., “In vivo Gene Delivery to the Rabbit Carotid by Electroporation,”Supplement to Journal of the American College of Cardiology, Abstract 1 page, Mar. 24-27, 1996.
Muramatsu, Tatsuo et al., “In Vivo Electroporation: A Convenient Method for Gene Transfer to Testicular Cells in Mice,”Anim. Sci. Technol. (Jpn), vol. 57, No. 11, pp. 975-982, 1996.
Nishi, Toru et al., “High-Efficiency in Vivo Gene Transfer Using Intraarterial Plasmid DNA Injection Following in Vivo Electroporation,”Cancer Res., vol. 56, pp. 1050-1055, 1996.
Nishi, Toru et al., “Treatment of Cancer Using Pulsed Electric Field in Combination with Chemotherapeutic Agents or Genes,”Human Cell, vol. 10, No. 1, pp. 81-86, 1997.
Nishi, Toru et al., “High Efficiency Gene Transfer in Solid Tumors by In Vivo Electroporation,”Conference Supplement Cancer Gene Therapy, vol. 4, No. 6, Abstract 1 page, Nov./Dec. 1997.
Nishi, T. et al., “High Efficiency Gene Transfer into Solid Tumors Using In Vivo Electroporation,”Proceedings of the American Association of Cancer Research, vol. 39, Abstract p. 59, Mar. 1998.
Nomura, Masayuki et al., “In Vivo Induction of Cytotoxic T Lymphocytes Specific for a Single Epitope Introduced into an Unrelated Molecule,”Journal of Immunological Methods, vol. 193, pp. 41-49, 1996.
Titomirov, Alexander V. et al., “In Vivo Electroporation and Stable Transformation of Skin Cells of Newborn Mice by Plasmid DNA,”Biochimica et Biophysica Acta., vol. 1088, pp. 131-134, 1991.
Aihara and Mlyazaki, “Gene transfer into muscle by electroporation in vivo,”Nature Biotechnology, 16:867-870 (1998).
Filshie Robin
Nolan Ed
BioTechnology Law Group
Chambers Daniel M.
Genetronics Inc.
Leffers Gerry
Murdock Douglas C.
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