Cationic phospholipids for transfection

Details Cationic phospholipids for transfection Cationic phospholipids for transfection

1999-02-05

2001-02-13

Riley, Jezia (Department: 1656)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S001000, C435S006120, C424S450000

Reexamination Certificate

active

06187760

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel cationic phospholipids and methods for making them. This invention also relates to novel liposomes and aggregates comprising the phospholipids of the present invention that are useful for the delivery of nucleic acids and drugs to cells, both in vitro and in vivo. This invention also relates to the treatment of diseases by gene therapeutics involving transfection with DNA and introduction into cells of antisense nucleotides, as well as stable transfection with DNA engineered to become incorporated into the genome of living cells.
2. Description of the Related Art
The introduction of foreign nucleic acids and other molecules is a valuable method for manipulating cells and has great potential both in molecular biology and in clinical medicine. Many methods have been used for insertion of endogenous nucleic acids into eukaryotic cells. E.g., Graham and Van der Eb,
Virology
52, 456 (1973) (co-precipitation of DNA with calcium phosphate); Kawai and Nishizawa,
Mol. Cell. Biol.
4, 1172 (1984) (polycation and DMSO); Neumann et al.,
EMBO Journal
1, 841 (1982) (electroporation); Graessmann and Graessmann in
Microinjection and Organelle Transplantation Techniques,
pp. 3-13 (Cells et al., Eds., Academic Press 1986) (microinjection); Cudd and Nicolau in
Liposome Technology,
pp. 207-221 (G. Gregoriadis, Ed., CRC Press 1984) (liposomes); Cepko et al.,
Cell
37, 1053 (1984) (retroviruses); and Schaffner,
Proc. Natl. Acad. Sci. USA
77, 2163 (1980) (protoplast fusion). Both transient and stable transfection of genes has been demonstrated.
Some of the first work on liposome delivery of endogenous materials to cells occurred some twenty years ago. Foreign nucleic acids were introduced into cells (Magee et al.,
Biochim. Biophys. Acta
451, 610-618 (1976), Straub et al.,
Infect. Immun.
10, 783-792 (1974)), as were foreign lipids (Martin and MacDonald,
J. Cell Biol.
70, 515-526 (1976)), Proteins (Magee et al.,
J. Cell. Biol.
63, 492 (1974), Steger and Desnick,
Biochim. Biophys. Acta
464, 530 (1977)), fluorescent dyes (Leventis and Silvius), and drugs (Juliano and Stamp,
Biochem. Pharm.
27, 21-27 (1978), Mayhew et al.,
Cancer Res.
36, 4406 (1976), Kimelberg,
Biochim. Biophys. Acta
448, 531 (1976)), all using positively charged lipids.
Of the many methods used to facilitate entry of DNA into eukaryotic cells, cationic liposomes are among the most efficacious and have found extensive use as DNA carriers in transfection experiments. See, generally, Thierry et al. in
Gene Regulation: Biology of Antisense RNA and DNA,
p. 147 (Erickson and Izant, Eds., Raven Press, New York, 1992); Hug and Sleight,
Biochim. Biophys. Acta
1097, 1 (1991); and Nicolau and Cudd,
Crit. Rev. Ther. Drug Carr. Sys.
6, 239 (1989) The process of transfection using liposomes is called lipofection. Senior et al.,
Biochim. Biophys. Acta
1070, 173 (1991), suggested that incorporation of cationic lipids in liposomes is advantageous because it increases the amount of negatively charged molecules that can be associated with the liposome. In their study of the interaction between positively charged liposomes and blood, they concluded that harmful side-effects associated with macroscopic liposome-plasma aggregation can be avoided in humans by limiting the dosage.
Felgner et al.,
Proc. Natl. Acad. Sci. USA
84, 7413 (1987), demonstrated that liposomes of dioleoylphosphatidylethanolamine (DOPE) and the synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTIVIA) are capable of both transiently and stably transfecting DNA. Rose et al.,
BioTechiques
10, 520 (1991), tested lipofection with liposomes consisting of DOPE and one of the cationic lipids cetyldimethylethylammonium bromide (CDAB), cetyltrimethylethylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride (MBC) and stearylamine. All of the liposomes (except that with CTAB) successfully transfected DNA into HeLa cells. At high concentrations, however, CDAB and MBC caused cell lysis. Only DDAB was found to be effective in mediating efficient DNA transfection into a variety of other cell lines. Malone et al.,
Proc. Natl. Acad. Sci. USA
86, 6077 (1989), successfully transfected RNA, in vitro, into a wide variety of cells lines. Zhou and Haung,
J. Controlled Release
19, 269 (1992), disclosed successful lipofection by DOPE liposomes stabilized in the lamellar phase by cationic quaternary ammonium detergents. The authors noted, however, that the relatively high cytotoxicity of these compounds would limit their use in vivo.
Hawley-Nelson et al.,
Focus
15, 73 (1990, BRL publications), disclosed the cationic lipid “LIPOFECTAMINE”, a reagent containing 2,3-dioleyloxy-N-[2(sperminecarboxy-amido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). “LEPOFECTAMINE” was found to have higher transfection activity than several monocationic lipid compounds (“LIPOFECTIN”, “LIPOFECTACE”, and DOTAP) in six of eight cell types tested. They observed toxicity when both lipid and DNA were included in the same mixture.
Both Farhood et al.,
Biochim. Biophys. Acta
1111, 239 (1992), and Gao and Huang,
Biochem. Biophys. Res. Comm.
179, 280 (1991), disclose cationic derivatives of cholesterol as components of liposomes capable of transfecting cells in vitro.
Liposomes comprising cationic lipids may also find use as carriers for gene therapy in in vivo applications. Some of the first in vivo applications of delivery of endogenous materials via liposomes was demonstrated twenty years ago. See, e.g., Straub et al., supra, and Magee et al., supra, (nucleic acids), and Mayhew et al., supra (drugs),
Holt et al.,
Neuron
4, 203 (1990), describe a DOTMA dioleoxylphosphatidylethanolamine liposome that successfully transfected a vector expressing luciferase cDNA into embryonic brain of Xenopus in vivo.
Malone,
Focus
11, 4 (1989, BRL publications), reported a similar study on Xenopus neural tissue as did Ono et al.,
Neurosci. Lett.
117, 259 (1990), in mouse brain.
Brigham et al.,
Am J. Med. Sci.
298, 278 (1989), disclosed intravenous injection of “LIPOFECTIN” and chloramphenicol acetyl transferase (CAT) plasmid into mouse lungs.
Nabel et al.,
Science
249, 1285 (1990), reported the expression of a &bgr;-galactosidase gene in a specific arterial segment in vivo in Yucatan pigs by DNA transfection with cationic liposomes. Lim et al.,
Circulation
83, 2007 (1991), disclosed in vivo gene transfer of reporter genes (&bgr;-galactosidase and luciferase) into arteries of dogs using cationic liposomes.
Hazinski,
Sem. Perinatol.
16, 200 (1992) disclosed cationic liposome-mediated transfer of fusion reporter genes to the epithelial cells and transient protein expression via direct injection of DNA-liposome solution into the trachea.
Yosimura et al.,
Nucleic Acids Res.
20, 3233 (1992) demonstrated successful in vivo lipofection of the cystic fibrosis trans-membrane conductance regulator gene (CFTR) into airway epithelium of mice using the cationic liposome “LIPOFECTIN”. Hyde et al.,
Nature
362, 250 (1993), also disclosed lipofection of CFRR using “LIPCFECTIN”. They demonstrated successful delivery of the gene to epithelia of the airway and to alveoli deep in the lung of transgenic mice.
Several cationic amphiphiles have been reported as transfection agents. Ballas et al.,
Biochim. et Biophys. Acta
939, 8 (1988), reported the successful lipofection of tobacco mosaic virus RNA into tobacco and petunia protoplasts via liposomes composed of phosphatidylcholine (PC), cholesterol, and the hydroxyl form of the quaternary ammonium detergent diisobutylcresoxyethoxyethyldimethylbenzylammonium (DEBDA [OH
−
]). Liposomes lacking the quaternary ammonium detergent practically failed to transfect the RNA. Importantly, Ballas et al. also observed that RNA and DNA complexed to liposomes bearing DEBDA[OH
−
] were highly resistant to added RNAses and DNAses.
Pinnaduwage et a

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