Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-10-09
2004-05-11
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S450000, C435S320100, C435S455000, C435S458000
Reexamination Certificate
active
06734171
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to lipid-based formulations for nucleic acid delivery to cells, methods for the preparation of such formulations and, in particular, to lipid encapsulated plasmids. The compositions are safe and practical for clinical use.
BACKGROUND OF THE INVENTION
Gene therapy is an area of current interest which involves the introduction of genetic material into a cell to facilitate expression of a deficient protein. Plasmid DNA has been encapsulated or complexed with lipid-based carriers by a number of methods including reverse phase evaporation (Fraley, et al.,
J. Biol. Chem
., 255:10431-10435 (1980); Soriano, et al.,
Proc. Natl. Acad. Sci. USA
, 80:7128-7131 (1983); Nakanishi, et al.,
Exper. Cell Res
., 159:399-409 (1985); Nandi, et al.,
J. Biol. Chem
., 261:16722-16726 (1986); and Alino, et al.,
Biochem. Biophys. Res. Commun
., 192:174-181 (1993)); Ca
2+
EDTA chelation (Szelei, et al.,
Biochem. J
., 259:549-553 (1989)); detergent dialysis (Wang, et al.,
Proc. Natl. Acad. Sci. USA
, 84:7851-7855 (1987)); lipid hydration (Lurquin,
Nucleic Acids Res
., 6:3773-3784 (1979); Yagi, et al.,
Biochem. Mol. Biol. International
, 32:167-171 (1994)); ether injection (Fraley, et al.,
Proc. Natl. Acad. Sci
., 76:3348-3352 (1979); Nicolau, et al.,
Biochem. Biophys. Res. Comm
., 108:982-986 (1982)); and sonication (Jay, et al.,
Bioconj. Chem
., 6:187-194 (1987) and Puyal, et al.,
Eur. J. Biochem
., 228:697-703 (1993)).
Reverse phase techniques typically encapsulate only about 10 to 20% of DNA in solution and the final DNA to lipid ratio is quite low. For example, Nakanishi, et al. (
Exper. Cell Res
., 159:399-409 (1985)) reported a final DNA to lipid ratio of 1.5 &mgr;g DNA to 2.5 mg lipid, while Soriano, et al. (
Proc. Natl. Acad. Sci. USA
, 80:7128-7131 (1983)) reported a DNA to lipid ratio of about 14 &mgr;g DNA to 60 &mgr;mol of lipids. The maximum theoretical encapsulation efficiency expected by reverse phase is only about 40%. Other methods, such as rehydration of freeze dried vesicles with DNA, have been shown to yield trapping efficiencies between 30 and 40% (Baru, et al.,
Gene
, 161:143-150 (1995)). Others have sought to increase the entrapment of DNA by the inclusion of cationic lipids in the lipid suspension (Stavridis, et al., 1986; Puyal, et al.,
Eur. J. Biochem
., 228:697-703 (1995)), or by rendering the DNA positively charged by 10 coating it with basic proteins such as lysozymes (Jay, et al.,
Proc. Natl. Acad. Sci. USA
, 84:1978-1980 (1987)). Although trapping efficiencies as high as 50% were achieved by the lysozyme method, the amount of DNA loaded per mg of lipid was low (5 &mgr;g/mg lipid) and the largest DNA molecule tested was only 1 kb. Trapping efficiencies as high as 60-90% were achieved by Puyal, et al. (
Eur. J. Biochem
., 228:697-703 (1995)) with a higher DNA to lipid ratio (13 &mgr;g/&mgr;mole lipid) using a 6.3 kb ssDNA (M13 phage). The major drawback of this technique and the one described by Jay, et al.,
Bioconj. Chem
., 6:187-194 (1987)) is that sonication was used. Sonication of DNA typically leads to some degradation of the lipid vesicle.
Detergent dialysis is a method of encapsulation which has no deleterious effects on the DNA. Wang, et al.,
Proc. Natl. Acad. Sci. USA
, 84:7851-7855 (1987) applied a detergent dialysis technique followed by extrusion through a 0.2 &mgr;m polycarbonate filter. A 4.6 kb plasmid was entrapped in vesicles approximately 200 nm in diameter with a trapping efficiency of about 14-17%, giving a DNA to lipid ratio of about 26 &mgr;g DNA to 10 &mgr;mole lipid.
Ideally, a delivery vehicle for a nucleic acid or plasmid will have the following characteristics: a) small enough and long lived enough to distribute from local injection sites when given intravenously, b) capable of carrying a large amount of DNA per particle to enable transfection of all sizes of genes and to reduce the volume of injection, c) homogeneous, d) reproducible, e) protective of DNA from extracellular degradation and f) capable of transfecting target cells in such a way that the DNA is not digested intracellularly.
The present invention provides such compositions and methods for their preparation and use.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides compositions which are nucleic acid (e.g., plasmid)-lipid compositions. In these compositions, a nucleic acid (e.g., plasmid or an antisense molecule) is encapsulated in a self-assembling lipid vesicle in an amount of from about 20 &mgr;g nucleic acid/mg lipid to about 400 &mgr;g nucleic acid/mg lipid. The lipid vesicle will typically be a liposome or lipid particle (a bilayer vehicle coating the plasmid and having little or no aqueous interior). The lipid vesicle can be prepared from a wide variety of lipids or combinations of lipids. The compositions can also include targeting groups and modified lipids (e.g., ATTA-lipids, gangliosides, such as ganglioside G
M1
), PEG-lipids, such as PEG-ceramides, and lipids having reactive functional groups for the attachment of targeting groups or circulation stabilizers). Preferably, the lipid vesicles will comprise cationic lipids and fusogenic lipids. Additionally, the nucleic acid (e.g., plasmid)-lipid compositions described herein can be prepared having a narrow size distribution (typically 50 nm to about 150 nm) without the use of sizing methods, such as extrusion and sonication methods.
In another aspect, the present invention provides methods for the encapsulation of nucleic acids, antisense, ribozymes and, particularly, plasmids in a lipid bilayer carrier. Such methods are related to a detergent dialysis method using cationic lipids of any desired concentration in combination with a dialysis buffer of an ionic strength (salt concentration, type of ions) specific for the given cationic lipid concentration. With the dialysis buffer of appropriate ionic strength, the methods provide encapsulation of 40-80% of the nucleic acid solution. The compositions above, and those formed by the methods described below, exhibit preferably less than about 30% degradation, more preferably, less than about 15% degradation and, even more preferably, less than about 5% degradation when digested with 0.1 to 10 U and, more preferably, 1 U of a nuclease after 30 minutes at 37° C.
In particular, the invention provides a method for encapsulating a nucleic acid in a lipid bilayer carrier, comprising:
(a) combining a nucleic acid with a lipid-detergent mixture comprising an aggregation-preventing agent (e.g., an ATTA-lipid, a PEG-lipid, such as a PEG-ceramide, a ganglioside, etc.) in an amount of about 5 mol % to about 20 mol %, cationic lipids in an amount of about 0.5 mol % to about 50 mol % by weight, neutral or fusogenic lipids in an amount of from about 30 mol % to about 70 mol % and a detergent, to provide a nucleic acid-lipid-detergent mixture; and
(b) dialyzing the nucleic acid-lipid-detergent mixture against a buffered salt solution and to encapsulate the nucleic acid in a lipid bilayer carrier. In these methods, the ionic strength (salt concentration) is adjusted for the cationic lipid concentration used in the lipid mixture and when necessary for the polynucleotide selected for encapsulation to entrap from about 40% to about 80% of the nucleic acid for any given concentration of cationic lipid.
In another aspect, the present invention provides methods for introducing nucleic acids into cells and for inhibiting tumor growth in cells using the lipid-nucleic acid formulations described above.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.
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Cullis Pieter R.
Kojic Ljiljana D.
Ludkovski Olga
Saravolac Edward George
Scherrer Peter
Inex Pharmaceuticals Corp.
Nguyen Dave T.
Townsend & Townsend & Crew LLP
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