Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-03-31
2001-01-09
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S458000, C530S344000, C530S350000
Reexamination Certificate
active
06171862
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of molecular biology and gene therapy, and more particularly to improved methods for transfecting cells in the presence of culture media containing serum.
BACKGROUND OF THE INVENTION
Cationic lipid-mediated gene transfer (lipofection) is a simple and efficient technique for introducing foreign genetic information into cultured mammalian cells (P. L. Feigner et al.,
Proc Natl Acad Sci USA
(1987) 84:7413-17; P. L. Feigner et al.,
Ann NY Acad Sci
(1995) 772:126-39). Although it is a widely used gene transfer technique, it is hampered by several disadvantages, including low gene transfer efficiency in some cell types, instability and serum-induced inactivation of the DNA-lipid complex, and cell toxicity of the lipofection procedure. In most cell lines, cationic liposome-mediated transfection requires serum depletion.
A number of approaches have been tried to overcome these disadvantages. H. E. Hofland et al.,
Proc Natl Acad Sci USA
(1996) 93:7305-09, reported the formation of stable DNA-lipid complexes that retain their efficiency of gene transfer even in the presence of serum in culture medium, and that remain active for up to three months. Mizuguchi et al. reported that the fusogenic liposomes formed by cationic lipid-DNA complex and Sendai virus retained 70% of their transfection efficiency even in the presence of 40% fetal bovine serum (FBS) in contrast to the virtual inactivity of complexes formed without Sendai virus, even in the presence of as little as 5% FBS.
The polycation polybrene is used routinely to enhance the efficiency of retrovirus vector-mediated gene transfer. S. Andreadis et al.,
Hum Gene Ther
(1997) 8:285-91, have recently reported that the concentrations of polybrene required for optimum retrovirus-mediated gene transfer increase with increasing concentrations of serum. X. Gao et al.,
Biochemistry
(1996) 35:1027-36, have also reported that the efficiency of cationic liposome mediated gene transfer in vitro can be enhanced up to 2 to 28-fold by the use of a polycation. Adding a polycation during DNA-lipid complex formation resulted in complexes having a reduced particle size, leading to higher efficiency.
SUMMARY OF THE INVENTION
We have now invented an improved method for transfecting cells in the presence of serum. The method comprises preparing a complex of cationic lipid and nucleic acid, and adding VSV-G protein and/or polybrene to the preformed complex. Alternatively, the polybrene may be added to the culture medium just prior to adding the preformed complex. The resulting complex is capable of transfecting cells even in the presence of substantial amounts of serum.
One aspect of the invention is a method for introducing nucleic acids into a host cell in the presence of interfering serum components, by providing a nucleic acid-lipid complex and a host cell in a medium comprising an interfering component, and contacting the host cell with the nucleic acid-lipid complex and an effective amount of a transfection aid, wherein the transfection aid is either VSV-G or a polycation.
Another aspect of the invention is a method for preparing VSV-G for use as a transfection aid, by providing a producing cell comprising an expression vector encoding VSV-G operable in the producing cell, culturing the producing cell in medium under conditions which result in expression of VSV-G to provide a conditioned medium, and purifying VSV-G from the conditioned medium by centrifugation under non-denaturing conditions to provide fusogenically active VSV-G.
Another aspect of the invention is a composition for effecting lipid-mediated transfection in the presence of interfering cell culture components, said composition comprising a transfection-effective lipid, and an effective amount of a transfection aid selected from the group consisting of VSV-G and a polycation.
Another aspect of the invention is a composition for effecting lipid-mediated transfection in the presence of interfering cell culture components, said composition comprising an effective amount of non-denatured, fusogenically active VSV-G and a non-toxic carrier.
One object of the invention is to provide a method for transfecting cells by lipofection in the presence of interfering components, for example in the presence of serum-containing medium.
Another object of the invention is to provide a composition suitable as a lipofection aid, capable of overcoming the deleterious effects of serum on lipofection.
Another object of the invention is to provide a lipofection aid that is simple to prepare and use.
Another object of the invention is to provide a method for preparing VSV-G under conditions that preserve its fusogenic activity, rendering it usable as a lipofection aid.
REFERENCES:
patent: 5512421 (1996-04-01), Burns et al.
patent: 5578475 (1996-11-01), Jessee
patent: 5627159 (1997-05-01), Shih et al.
Andreadis, S., and Palsson, B., 1997, “Coupled Effects of Polybrene and Calf Serum on the Efficiency of Retroviral Transduction and the Stability of Retroviral Vectors,” Hum. Gen Ther. 8:285-291.
Brunette, E., et al., 1992, “Lipofection does not require the removal of serum,” Nucl. Acids Res. 20:1151.
Burns, J.C., et al., 1993, “Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: Concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells,” Proc. Natl. Acad. Sci. USA 90:8033-8037.
Chen, S.T., et al., 1996, “Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system,” Proc. Natl. Acad. Sci. USA 93:10057-10062.
Emi, N., et al., 1991, “Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus,” J. Virol 65:1202-1207.
Felgner, P.L. et al., 1987, “Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure,” Proc. Natl. Acad. Sci. USA 84:7413-17.
Felgner, P.L., et al., 1995, “Improved Cationic Lipid Formulations for In Vivo Gene Therapy,”DNA Vaccines, a New Era in Vaccinology, Ann. NY Acad. Sci. 772:126-139.
Gao, X., and Huang, L., 1996, Biochemistry 35:1027-1036.
Hofland, E.E., et al., 1996, “Formation of stable cationic lipid/DNA complexes for gene transfer,” Proc. Natl. Acad. Sci. USA 93:7305-7309.
Hong, K., et al., 1997, “Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery,” FEBS Lett. 400:233-237.
Hug, P., and Sleight, R.G., 1994, “Fusogenic Virosomes Prepared by Partitioning of Vesicular Stomatitis Virus G Protein Into Preformed Vesicles,” J. Biol. Chem 269:4050-4056.
Metsikko, K., et al., 1986, “Reconstitution of the fusogenic activity of fsicular stomatitis virus,” EMBO J. 5:3429-3435.
Mizuguchi, H., et al., 1996, “Efficient Gene Transfer Into Mammalian Cells Using Fusogenic Liposome,” Biochem. Biophys. Res. Com. 218:402-407.
Petri, W.A., and Wagner, R.R., 1979, “Reconstitution Into Liposomes of the Glycoprotein of Vesciular Stomatitis Virus by Detergent Cialysis,” J. Biol. Chem. 254:4313-4316.
Sharma, S., et al., 1997, “Noninfectious virus-like particules produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents,” Proc. Natl. Acad. Sci. USA 94:10803-10808.
Singh, D., and Rigby, P.W.J., 1996, “The use of histone as a facilitator to improve the efficiency of retroviral gene transfer,” Nucl. Acids Res. 24:3113-3114.
Stephan, D.J., et al., 1996, “A New Cationic Liposome DNA Complex Enhances the Efficiency of Arterial Gene Transfer In Vivo,” Hum. Gene Ther. 7:1803-1812.
Yee, J.K., et al., 1994, “A general method for the generation of high-titer, pantropic retroviral vectors: Highly efficient infection of primary hepatocytes,” Proc. Natl. Acad. Sci. USA 914:9564-9568.
Zabner, et al., 1995, “Cellular and Molecular Barriers to Gene Transfer by a Cationic Lipid,” J. Biol. Chem. 270:18997-19007.
Abe Akihiro
Friedmann Theodore
Miyanohara Atsushi
Foley & Lardner
Guzo David
The Regents of the University of California
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