Organic compounds -- part of the class 532-570 series – Organic compounds – Unsubstituted hydrocarbyl chain between the ring and the -c-...
Reexamination Certificate
1998-01-17
2001-01-30
Lambkin, Deborah C. (Department: 1613)
Organic compounds -- part of the class 532-570 series
Organic compounds
Unsubstituted hydrocarbyl chain between the ring and the -c-...
C544S402000, C564S509000, C564S512000
Reexamination Certificate
active
06180784
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to techniques for transferring genes into mammalian cells. The method provides a method for transfecting cells with high efficiency and low cellular toxicity.
BACKGROUND OF THE INVENTION
Despite the great promise of gene therapy, there remains to be solved the challenging problem of efficiently transferring and stably expressing transgenes in appropriate tissues. This problem has recently been termed the “vector void” (Hodgson, C P. Bio/Technol. 1995;13:222-225). After the description of DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) (Felgner, P L, Gadek, T R, Holm, M, et al. Proc. Natl. Acad. Sci. USA. 1987;84:74137417), a plethora of cationic lipids have been synthesized. Basically, all the cationic lipids are amphipathic compounds that contain a hydrophobic domain, a spacer, and positively-charged amine(s). The cationic lipids are mixed with a fusogenic lipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to form liposomes. The cationic liposomes are then mixed with plasmid DNA and the binary complex of the DNA and liposomes are then applied to cells in a tissue culture dish. The ease of mixing the plasmid DNA with the cationic liposome formulation, the ability of the cationic lipids to complex with DNA and the relative high levels of transfection efficiency has led to he increasing use of these formulations. However, these cationic lipid formulations can also be toxic to the cells in culture (Gao, X and Huang, L. Biochem. Biophys. Res. Com. 1991;179:280-285) (Leventis, R and Silvius, J R. Biochim. et Biophys. Acta 1990;1023:124-132). Although, LipofectAMINE can transfect many cell types more efficiently than other types of cationic lipid formulations (Harms, J S and Splitter, G A. Focus 1994;17:34-35), it also has greater toxicity (Hawley-Nelson, P, Ciccarone, V and Jessee, J. Focus 1993;15:73-79) (Ciccarone, V, Hawley-Nelson, P and Jessee, J. Focus 1993;15:80-83). If the transfection method is toxic to cells, then this would reduce its applicability for gene therapy. Cellular toxicity would also reduce its applicability to the study of genes since the cells would be in an altered state and it may be difficult to differentiate an effect of the transfection reagent from expression of the foreign gene.
Accordingly, there is a need for a means of transfecting cells with great efficiency and little toxicity. In addition, given the vector void for gene therapy, there is a need for new types of methods to transfer genes into mammalian cells.
SUMMARY OF THE INVENTION
The present invention provides an enhancement of gene transfer into cells using a ternary complex of DNA, amphipathic compounds, and a DNA-binding protein. Cultured cells exposed to these ternary complexes expressed foreign genes at very high levels and with minimal cellular toxicity. Further, the use of a DNA-binding protein and novel amphipathic compounds together increased gene transfer efficiency by several orders of magnitude.
In one aspect, the present invention provides a process of transfecting a cell with DNA comprising exposing the cell to the DNA mixed with amphipathic compounds and an effective amount of a DNA-binding protein.
A preferred DNA-binding protein is a histone such as H1, H2A, or H2B. Natural DNA binding proteins such as histone also have several advantages over polycationic compounds such as polylysine. Human H1 histone protein is not immunogenic and does not induce anaphylaxis. Polylysine induces anaphylactic shock and is very immunogenic.
In one preferred embodiment, the DNA-binding protein is linked to a nuclear localization signal. A recombinant histone (NLS-H1) containing both the SV40 large T antigen nuclear localization signal and the C-terminal domain of human histone H1 was produced in bacteria. NIH3T3 or COS-7 cells transfected with NLS-H1 plasmid DNA-Lipofectin complexes expressed at least 20 times more liuciferase or had at least 2.5 times more B-galactosidase positive cells than those transfected with plasmid DNA-lipofectin complexes alone. Foreign gene expression was also improved by other DNA-binding proteins and cationic lipid formulations, but the greatest enhancement in foreign gene expression was obtained with complexes containing either NLS-H1 or calf-thymus histone H1.
A variety of amphipathic compounds can be used in conjunction with a DNA binding protein such as histone protein to mediate the transfer of the plasmid DNA into the cell. A preferred embodiment is amphipathic compounds that are cationic. The cationic amphipathic compound can be a non-natural polyamine wherein one or more of the amines is bound to at least one hydrophobic moiety wherein the hyudrophobic moiety comprises a C6-C24 alkane, C6-C24 alkene, sterol, steroid, lipid, fatty acid or hydrophobic hormone. The amphipathic compounds may or may not form liposomes. Several novel amphipathic cationic compounds are described. These include compounds with the following structures:
In contrast to the use of previously described cationic liposomes, most of the novel amphipathic cationic compounds described above do not efficiently mediate the transfer of genes into cells when used alone. However, the use of histone proteins with these novel amphipathic cationic compounds enable the efficient gene transfer into a variety of mammalian cells with minimal cellular toxicity. Therefore, the use of histone proteins expands the range and types of cationic lipids that can be used for gene transfer.
Histone H1-plasmid DNA-cationic lipid complexes showed less cytotoxicity and were internalized by a greater number of cells than plasmid DNA-cationic lipid complexes. Thus, histone binding to plasmid DNA prior to the addition of cationic amphipathic compounds improved cellular uptake and transient foreign gene expression while reducing the cellular toxicity associated with cationic-lipid mediated gene transfer.
DETAILED DESCRIPTION
The present invention includes a process of transfecting a polynucleotide into a cell for expression by associating a selected cell with an amphipathic compound, an effective amount of a polynucleotide-binding protein and a selected polynucleotide, in solution. The term “transfecting” means that a polynucleotide becomes associated with a selected cell. The polynucleotide can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organeile of the cell. Other terms sometimes used interchangeably with transfecting include “delivering” or “transferring” to a cell and “transforming” a cell.
The polynucleotide is delivered to the cell by associating the cell with one or more amphipathic compounds along with a polynucleotide-binding protein in a solution. The term “associating” means to put in communication with or to place in close proximity. The term “polynucleotide” is a term of art that refers to a string of at least two base-sugar-phosphate combinations. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of an oligonucleotide, messenger RNA, anti-sense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. The selected cell can be a mammalian cell that is within the tissue in situ; the cell could also be removed and maintained in tissue culture in a primary, secondary, immortalized or transformed state.
The amphipathic compound is a linear molecule with a polar first end causing that end to be hydrophilic (water-soluble); the second end is nonpolar and therefore hydrophobic (water-insoluble). The amphipathic compound can be cationic or incoporated into a liposome that is cationic or anionic. In one embodiment, the amphipathic compound is a non-natural polyamine. “Non-natural”, in this application, means that, for example, the compound is not found in living matter. As contrasted with “natural” which in this application refers to a compound that is derived from animal or plant tissue directly or by recombinant means.
The polynucleotide-binding protein is a protein that can be
Budker Vladimir G.
Fritz Jeffery
Hagstrom James E.
Wolff Jon A.
Johnson Mark K.
Lambkin Deborah C.
Mirus Corporation
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