Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-02-11
2003-06-03
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C424S450000
Reexamination Certificate
active
06573101
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the delivery of nucleic acid molecules into cells and, more specifically, to compositions and methods for the high efficiency delivery of nucleic acid molecules into cells.
2. Background Information
Methods that allow the introduction and expression of foreign or exogenous nucleic acid molecules into cells in culture are useful for manipulating the function and expression of various genes, as well as for efficiently expressing a desired protein. In addition, the ability to transfer genes to human cells provides the means to treat a wide range of genetic and acquired diseases, including cystic fibrosis, sickle cell anemia, and AIDS. Unfortunately, methods for introducing a gene of interest into a cell have provided relatively low transfection efficiencies or have been successful only in cultured cell lines.
Transfection methods can be used to replace a defective gene or to correct an error in an existing one. Replacement therapy entails inserting a gene into a cell in order to synthesize a gene product that is not being produced or is being synthesized in inadequate amounts. Corrective therapy, on the other hand, attempts to correct an error in a gene by providing conditions for a recombinational event that replaces all or part of the defective gene with the correct DNA sequence.
Various transfection methods have been developed for eukaryotic cells, particularly mammalian cells. Some transfection methods use calcium phosphate or DEAE-dextran as a carrier to promote the uptake of an exogenous nucleic acid molecule. Other methods use “lipofection” techniques, which incorporate the use of synthetic anionic or cationic lipids to effect the transfection. Osmotic shock, treatment of the cells with liposomal inhibitors, and high voltage electric pulses, which create pores in cell membranes, also have been used in attempts to enhance transfection efficiencies. However, the efficiencies obtained by these methods are relatively low, ranging from 0.001% to 1%, depending on the recipient cell line.
In further efforts to increase the efficiency of introducing nucleic acid molecules into cells, viral vectors have been used. For example, when retroviral vectors are used, the introduced DNA replaces some of the retroviral genes required for the production of viral structural proteins while the viral sequences directing integration into the cellular DNA remain intact. However, the use of retroviral vectors is limited to dividing cells. Adenovirus vectors also have been used with fairly high efficiency, but they only infect specific cell types. In addition, the use of viral vectors is limited by the induction of an immune response against the viral components of the vectors.
Other methods of introducing a nucleic acid molecule into a cell include complexing the nucleic acid molecule with proteins that bind to specific receptors expressed by the target cells, or by incorporating the nucleic acid molecule into liposomes, which fuse with the target cell membrane. Gene transfection of mammalian cells using cationic liposomes, for example, has achieved an efficiency of up to 15% and transferrin-poly-L-lysine mediated transfection has yielded 7% to 8% transfection efficiency. A transfection efficiency of about 90% has been achieved in a cultured cell line using a combination of transferrin and cationic liposomes.
While these approaches represent an improvement over prior techniques, a need exists for compositions and methods for introducing a nucleic acid molecule into any target cell, particularly primary mammalian cells, with efficiencies of about 50% or greater. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a composition for introducing a nucleic acid molecule into a target cell, comprising a liposome, ligand and polymeric scaffold, wherein the ligand can bind to a receptor on the target cell. The liposome can be cationic. The ligand can be attached to the polymeric scaffold. The polymeric scaffold can be positively charged. Thus, for example, the invention provides a composition comprising a cationic liposome, and transferrin, wherein the transferrin is attached to poly-L-lysine.
The invention also provides a method for introducing a nucleic acid molecule into a target cell by contacting the target cell with the nucleic acid molecule, a liposome, a ligand and a polymeric scaffold, wherein the ligand binds to a cell surface receptor on the target cell. The ligand can be attached to the polymeric scaffold. The polymeric scaffold can be positively charged. Thus, for example, the invention provides a method for introducing a nucleic acid molecule into primary mammalian cells, wherein at least about 50% of the transfected cells contain the introduced nucleic acid molecule. A method of the invention further comprises permeabilizing the target cells to facilitate uptake of the nucleic acid molecule.
REFERENCES:
patent: 5128257 (1992-07-01), Baer
patent: 5605890 (1997-02-01), Agarwal et al.
patent: 5736392 (1998-04-01), Hawley-Nelson et al.
patent: 5776746 (1998-07-01), Denney, Jr.
patent: 5830730 (1998-11-01), German et al.
patent: 5842477 (1998-12-01), Naughton et al.
patent: 6074667 (2000-06-01), Kinnunen et al.
Lee et al (1996). J. Biol. Chem 271(14): 8481-8487. See entire document, especially the abstract; Materials and methods: p. 8482, column 1, lines 3 and 4 of first paragraph, and lines 1-6 of second paragraph, and lines 1-6 of fifth paragraph. See also Apr. 1996.*
Kreuzner et al (1996). Atherosclerosis 124(1): 49-60, entire document, see especially abstract, 1996.*
Raja-Walia et al (1995). Gene Therapy 2(8): 521-530. Abstract only, Oct. 1995.*
Chernomordik (Biochim. Biophys. Acta. 1070: 193-197, 1991. See entire document, especially abstract, and p. 194, column 1, lines 5 and 6, 1991.*
Cheng (Human Gene Therapy 7: 275-282, 1996).See entire document, especially lines 2 and 3 of abstract, Feb. 1996.*
Weissmann et al. Trans. Assoc. Am. Phys. 89: 171-183, abstract only, 1976.*
Bolin et al. Scand. J. Gastroent. 16(7): 897-901, abstract only, 1981.*
Hapala (Critical Reviews in Biotechnology 17(2): 105-122, see entire document, especially pp. 108-111, particularly the paragraph bridging pp. 108-109, the first full paragraph on p. 110, and the first full paragraph on p. 111, 1997.*
Voet et al., Biochemistry, Second Edition, New York, John Wiley & Sons, publishers, 1995. See Fig. 23-2 on p. 664.*
Verma et al., Gene therapy—promises, problems and prospects; 1997, Nature vol. 389, 239-242.*
W. Anderson, Human gene therapy; 1998, Nature vol. 392, 25-30.*
S. Orkin; Report and recommendations of the panel to assess the NIH investment in research on gene therapy, Dec. 1995; 1-38.*
You et al.; Surfactant-medicated gene transfer for animals cells, 1997, Cytotechnology 25; 45-52.*
Staedal et al.; High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine: DNA complexes, 1994, The Society for Investigative Dermatology, vol. 102, No. 5: 768-772.*
Ammitzboli et al. (Acta Neurol. Scand. 53(2): 137-1511976.*
Ohno-Shosaku et. al.(J. Membr. Biol.85(3): 269-280,1985.*
Machy et. al.; Gene transfer from targeted liposomes to specific lymphoid cells by electroporation, 1988, Proc. Natl. Acad. Sci. vol. 85: 8027-8031.*
Boucher; Current status of CF gene therapy, 1996, TIG, vol. 12, NO.3:1-4.*
Boucher, Status of gene therapy for cystic fibrosis lung disease, 1999, Journal of Clinical Investigation, vol. 103, No. 4: 441-445.*
Resenfeld et.al.; Impact of basic research on tomorrow's medicine, 1996, Chest 109: 241-252.*
Klareskog et.al.; Expression of HLA-DR and HLA-DQ antigens on cells within the cartilage-pannus junction in rheumatoid arthritis, 1984, Rheumatol Int. 4: 11-15.*
Zhao et.al.; Amplified Gene Expression in CD59-transfected Chinese Hamster Ovary Cells . . . of Human Complement, 1991, Journal of Biological Chemistry, vol. 266: 13418-13422.*
Trubetsky et.al.; Cationic Liposomes enhance
Ketter James
Medlen & Carroll LLP
Schnizer Richard
The Regents of the University of California
LandOfFree
Compositions for receptor/liposome mediated transfection and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Compositions for receptor/liposome mediated transfection and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Compositions for receptor/liposome mediated transfection and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3124615