Nucleic acid transfer complexes

Details Nucleic acid transfer complexes Nucleic acid transfer complexes

2000-11-10

2002-10-01

Yucel, Remy (Department: 1636)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C435S455000, C435S458000

Reexamination Certificate

active

06458382

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compositions and methods for transferring nucleic acids into cells in vitro and in vivo. The compositions comprise a transfection reagent and one or more detergents. In preferred embodiments, the compositions comprise delivery systems providing nucleic acid transfer complexes that transfect cells with high efficiency.
BACKGROUND OF THE INVENTION
The efficient delivery of biologically active compounds to the intracellular space of cells has been accomplished by the use of a wide variety of vesicles. One particular type of vesicle, liposomes, is one of the most developed types of vesicles for drug delivery. Liposomes are microscopic vesicles that comprise amphipathic molecules that contain both hydrophobic and hydrophilic regions. Liposomes can contain an aqueous volume that is entirely enclosed by a membrane composed of amphipathic molecules (usually phospholipids).
Liposome drug carriers have been under development since the 1970's. Liposomes are formed from one to several different types of amphipathic molecules. Several methods have also been developed to complex biologically active compounds with liposomes. For example, a biologically active compound can be entrapped within the internal aqueous phase, within the lipid phase, or complexed to the outside of the liposome.
Liposomes can be divided into three groups based upon their overall size and lamellar structure. Small uni-lamellar vesicles (SUV), which are typically prepared by sonication, are 20 to 30 nm in diameter and contain one single lipid bilayer surrounding the aqueous compartment. Multi-lamellar vesicles (MLV) are prepared by simply mixing amphipathic molecules in an aqueous phase and contain multiple aqueous compartments and bilayers. Large uni-lamellar vesicles (LUV) are most commonly prepared by reverse-phase evaporation. After subsequent pore filtration, LUV's are usually 150 to 200 nm in diameter.
Liposomes can also be classified according to mechanisms by which they attach to a target cell. Gangliosides, polysacharrides and polymers such as polyethylene glycol have been attached to liposomes (termed “Stealth Liposomes”) to decrease their non-specific uptake by the reticuloendothelial system in vivo. Antibodies, polysaccharides, sugars, and other ligands have been attached to liposomes to enable the tissue and cell specific delivery of biologically active compounds. Other cellular and viral proteins have also been incorporated into liposomes for targeting purposes and for their fusogenic properties.
Liposomes typically deliver a biologically active compound found within their aqueous space to target cells by fusing with either the plasma membrane or an internal membrane of the cell after endocytosis of the liposome. Fusion of the liposome membrane with the cellular membrane is one of the critical steps in the efficient delivery of substances to the cell. Certain types of liposomes are endocytosed by certain types of cells. If a liposome is endocytosed by a receptor-mediated pathway, then it enters an endosome. In order for the biologically active compound contained within or associated with the liposome to reach its target sites and receptors, it is essential that the compound escape or be released from the endosome and avoid degradation in the lysosomes.
Efficient nucleic acid transfer in vitro has been accomplished with the use of positively-charged liposomes that contain cationic lipids. For example, the cationic lipid, N-1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) was the first cationic lipid used for DNA transfections. DOTMA was combined with dioleoylphosphatidylethanolamine (DOPE) to form liposomes that spontaneously complexed with nucleic acids (DNA and RNA) and provided relatively efficient transfections. Other neutral lipids have been used in conjunction with amphipathic compounds to form liposomes. These have generally been chosen from the group consisting of phosphatidylethanolamines (e.g., DOPE), phosphatidylcholines, or phosphatidylserines, wherein the acyl group chain length is between 16 and 20. Another compound used to form liposomes suitable for transfecting nucleic acids into cells is cholesterol.
These liposomes are simply mixed with the nucleic acid and then applied to the cells in culture. Complete entrapment of the DNA or RNA molecules occurs because the positively-charged liposomes naturally complex with negatively-charged nucleic acids. DNA has been shown to induce fusion of cationic liposomes containing DOTMA/DOPE. The procedure with the cationic lipids is generally as or more efficient than the commonly-used procedure involving the co-precipitation of calcium phosphate and DNA.
DOTMA/DOPE liposomes have, however, substantial cytotoxicity, particularly in vivo. A variety of cationic lipids have been made in which a glycerol or cholesterol hydrophobic moiety is linked to a cationic headgroup by metabolically degradable ester bond. These have included 1,2-bis(oleoyloxy)-3-(4′-trimethylammonio)propane (DOTAP), 1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesteryl (4′-trimethylammonio)butanoate (ChoTB). However, there is no evidence of reduced cytotoxicity in comparison of these ester bond-containing cationic lipids as compared to DOTMA. Stearylamine, a cationic lipid has been used in liposomes but it had great cytotoxicity and was not been reported to mediate DNA transfer. Another detergent, cetyltrimethylammonium bromide (CTAB), when combined with DOPE, was able to mediate DNA transfection, but it had significant cytotoxicity. A series of cationic, non-pH sensitive lipids that included DORI (1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide), DORIE (1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), and DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) have been reported and studied. Other non-pH-sensitive, cationic lipids include: O,O′-didodecyl-N-p-(2-trimethylammonioethyloxy)benzoyl-N,N,N-trimethylam monium chloride, Lipospermine, DC-Chol (3á-N-(N′,N″-dimethylaminoethane)carbonylcholesterol), lipopoly(L-lysine), cationic multilamellar liposomes containing N-(á-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), TRANSFECTACE (1:2.5 (w:w) ratio of DDAB which is dimethyl dioctadecylammonium bromide and DOPE) (Life Technologies) and LIPOFECTAMINE (3:1 (w:w) ratio of DOSPA which is 2,3-dioleyloxy-N-20({2,5-bis (3-aminopropyl)amino-1-oxypentyl}amino)ethyl-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminium trifluoroacetate and DOPE) (Life Technologies). While much development has occurred, the art is still in need of improved transfection compositions and methods for providing efficient, low toxicity delivery of agents in vitro and in vivo.
Furthermore, most transfection reagents have been optimized to transfect mammalian cells. In vitro, these cells are generally maintained at 37° C. Only two commercially available transfection reagents are claimed to be most efficient for the transfection of insect cells, which are maintained at a temperature of 25-27° C. Studies on cell membrane composition demonstrated that there are significant composition differences between mammalian and insect cells. Thus, the art is also in need of efficient, low toxicity delivery systems for the delivery of agents to cells other than mammalian cells or to cells under non-standard growth conditions.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for transferring nucleic acids into cells in vitro and in vivo. The compositions comprise a transfection reagent and one or more detergents. In preferred embodiments, the compositions comprise one or more amphipathic compounds and one or more neutral lipids. In preferred embodiments, the compositions comprise delivery systems providing nucleic acid transfer complexes that transfect cells with high efficiency. The transfection complexes of the present invention find use

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