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Description  |
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FIELD OF THE INVENTION
This invention relates to formulations for therapeutic nucleic acid
delivery and methods for their preparation, and in particular to lipid
encapsulated plasmids or antisense constructs. The invention provides a
circulation-stable, characterizable delivery vehicle for the introduction
of plasmids or antisense compounds into cells. These vehicles are safe,
stable, 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. There are currently five major methods by which this is
accomplished, namely: (i) calcium phosphate precipitation, (ii)
DEAE-dextran complexes, (iii) electroporation, (iv) cationic lipid
complexes and (v) reconstituted viruses or virosomes (see Chang, et al.,
Focus 10:88 (1988)). Cationic lipid complexes are presently the most
effective generally used means of effecting transfection.
A number of different formulations incorporating cationic lipids are
commercially available, namely (i) LIPOFECT.RTM. (which uses
1,2-dioleyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTMA, see
Eppstein, et al., U.S. Pat. No. 4,897,355); LIPOFECTAMINE.RTM. (which uses
DOSPA, see Hawley-Nelson, et al., Focus 15(3):73 (1993)); and
LIPOFECTACE.RTM. (which uses N,N-distearyl-N,N-dimethyl-ammonium bromide,
or DDAB, see Rose, U.S. Pat. No. 5,279,833). Others have reported
alternative cationic lipids that work in essentially the same manner but
with different efficiencies, for example
1,2-dioleoyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTAP, see
Stomatatos, et al., Biochemistry 27:3917-3925 (1988)); glycerol based
lipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990);
lipopolyamines (see, Behr, et al., U.S. Pat. No. 5,171,678) and
cholesterol based lipids (see Epand, et al., WO 93/105162, and U.S. Pat.
No. 5,283,185).
Others have noted that DOTMA and related compounds are significantly more
active in transfection assays than their saturated analogues (see,
Felgner, et al., W091/16024). However, both DOTMA and DOSPA based
formulations, despite being the most efficient of the cationic lipids in
effecting transfection, are prohibitively expensive. DDAB on the other
hand is inexpensive and readily available from chemical suppliers but is
less effective than DOTMA in most cell lines. Another disadvantage of the
current lipid systems is that they are not appropriate for intravenous
injection.
An examination of the relationship between the chemical structure of the
carrier vehicle and its efficiency of transfection has indicated that the
characteristics which provide for effective transfection would make a
carrier unstable in circulation (see, Ballas, et al., Biochim. Biophys.
Acta 939:8-18 (1988)). Additionally, degradation either outside or inside
the target cell remains a problem (see, Duzghines, Subcellular
Biochemistry 11:195-286 (1985)). Others who have attempted to encapsulate
DNA (Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer,
U.S. Pat. No. 4,515,736) made no efforts to ensure a safe, injectable
formulation, or arrived at inefficient loading (Legendre, Pharm. Res.
9:1235-1242 (1992)).
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 reduce the volume of injection, c) homogenous, 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 methods for the preparation
of serum-stable plasmid-lipid particles. In one group of these methods, a
plasmid is combined with cationic lipids in a detergent solution to
provide a coated plasmid-lipid complex. The complex is then contacted with
non-cationic lipids to provide a solution of detergent, a plasmid-lipid
complex and non-cationic lipids, and the detergent is then removed to
provide a solution of serum-stable plasmid-lipid particles, in which the
plasmid is encapsulated in a lipid bilayer. The particles, thus formed,
have a size of about 50-150 nm.
In a related group of methods the serum-stable plasmid-lipid particles are
formed by preparing a mixture of cationic lipids and non-cationic lipids
in an organic solvent; contacting an aqueous solution of plasmid with the
mixture of cationic and non-cationic lipids to provide a clear single
phase; and removing the organic solvent to provide a suspension of
plasmid-lipid particles, in which the plasmid is encapsulated in a lipid
bilayer, and the particles are stable in serum and have a size of about
50-150 nm.
In another aspect, the present invention provides plasmid-lipid particles
prepared by the above methods.
In yet another aspect, the present invention provides methods of
transfecting cells using these plasmid-lipid particles.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a liposome-mediated transfection using "sandwich-type"
complexes of DNA.
FIG. 2 illustrates an aggregation and precipitation which commonly occurs
during the entrapment of large nucleic acids in lipid complexes.
FIG. 3 provides a schematic representation of the preparation of
plasmid-lipid particles using the methods of the present invention.
FIG. 4 illustrates the recovery of .sup.3 H-DNA from encapsulated particles
following the reverse-phase preparation of the particles and extrusion
through a 400 nm filter and a 200 nm filter. Lipid composition is
POPC:DODAC:PEG-Cer(C.sub.20) in proportions as shown in Table 1.
FIG. 5 illustrates the recovery of .sup.3 H-DNA from particles prepared
using a reverse-phase procedure. The particles were extruded through a 200
nm filter and eluted on a DEAE Sepharose CL-6B anion exchange column. The
percent recovery reported is based on the amount recovered after
filtration. Lipid composition is as in FIG. 4.
FIG. 6 illustrates the recovery of .sup.14 C-lipid from encapsulated
particles following the reverse-phase preparation of the particles and
extrusion through a 400 nm filter and a 200 nm filter. Lipid composition
is as in FIG. 4.
FIG. 7 illustrates the recovery of .sup.14 C-lipid from particles prepared
using a reverse-phase procedure. The particles were extruded through a 200
nm filter and eluted on a DEAE Sepharose CL-6B anion exchange column. The
percent recovery reported is based on the amount recovered after
filtration. Lipid composition is as in FIG. 4.
FIG. 8 illustrates recovery of .sup.3 H-DNA and .sup.14 C-lipids from
particles prepared by detergent dialysis after elution on a DEAE Sepharose
CL-6B anion exchange column in HBS, pH 7.4. Lipid composition is
POPC:DODAC:PEG-Cer(C.sub.20) in proportions as shown in Table 2.
FIG. 9 illustrates recovery of .sup.3 H-DNA and .sup.14 C-lipids from
particles prepared by detergent dialysis after elution on a DEAE Sepharose
CL-6B anion exchange column in HBS, pH 7.4. Lipid composition is
DOPE:DODAC:PEG-Cer(C.sub.20) in proportions as shown in Table 3.
FIG. 10 provides an elution profile of free .sup.3 H-DNA (pCMV4-CAT) on a
Sepharose CL-4B column in HBS, pH 7.4.
FIG. 11 provides an elution profile of free .sup.3 H-DNA (pCMV4-CAT) on a
Sepharose CL-4B column in HBS, pH 7.4, after incubation in 80% mouse serum
for 30 min at 37.degree. C.
FIG. 12 shows the recovery of .sup.3 H-DNA and .sup.14 C-lipids from
particles (prepared by reverse-phase methods) after incubation in 80%
mouse serum for 15 min at 37.degree. C. Lipid composition is
POPC:DODAC:PEG-Cer(C.sub.20).
FIG. 13 shows the recovery of .sup.3 H-DNA and .sup.14 C-lipids from
particles (prepared by detergent dialysis methods) after incubation in 80%
mouse serum for 30 min at 37.degree. C. Lipid composition is
DOPE:DODAC:PEG-Cer(C.sub.20).
FIG. 14 provides a density gradient profile of .sup.14 C-lipid complexes
prepared in the absence of DNA by reverse phase methods. Lipid composition
is POPC:DODAC:PEG-Cer(C.sub.20). FIG. 15 provides a density gradient
profile of free .sup.3 H-DNA (pCMV4-CAT).
FIG. 16 provides a density gradient profile of .sup.3 H-DNA and .sup.14
C-lipid from particles prepared by reverse-phase methods. Lipid
composition is as in FIG. 14.
FIG. 17 provides a density gradient profile of free .sup.3 H-DNA, .sup.14
C-lipid complexes prepared in the absence of DNA by detergent dialysis
methods and .sup.3 H-DNA and .sup.14 C-lipid from DNA-lipid complexes
prepared by detergent dialysis. Lipid composition is
DOPE:DODAC:PEG-Cer(C.sub.20).
FIG. 18 provide a size distribution of DNA-lipid particles prepared by
detergent dialysis methods. Lipid composition is
DOPE:DODAC:PEG-Cer(C.sub.20).
FIG. 19 shows the clearance of .sup.3 H-DNA and .sup.14 C-lipid from
particles (prepared by reverse-phase methods) after injection into IRC
mice. The figure includes free .sup.3 H-DNA after injection as a
comparison. Lipid composition is POPC:DODAC:PEG-Cer(C.sub.20).
FIG. 20 shows the clearance of .sup.3 H-DNA and .sup.14 C-lipid from
particles (prepared by detergent dialysis methods) after injection into
IRC mice. Lipid composition is DOPE:DODAC:PEG-Cer(C.sub.20) (83.5:6.5:10
mole %).
FIG. 21 shows the clearance of .sup.3 H-DNA and .sup.14 C-lipid from
particles (prepared by detergent dialysis methods) after injection into
IRC mice. Lipid composition is as in FIG. 20 except that PEG-Cer(C.sub.20)
is replaced with PEG-Cer(C.sub.14).
FIG. 22 shows the results of in vivo transfection which occurs in the lungs
of mice. Lipid composition is DOPE:DODAC:PEG-Cer(C.sub.20 or C.sub.14)
(83.5:6.5:10 mole %).
FIG. 23 shows the results of in vivo transfection which occurs in the liver
of mice. Lipid composition is as in FIG. 22.
FIG. 24 shows the results of in vivo transfection which occurs in the
spleen of mice. Lipid composition is as in FIG. 22.
DETAILED DESCRIPTION OF THE INVENTION
CONTENTS
I. Glossary
II. General
III. Methods of Forming Plasmid-Lipid Particles
IV. Pharmaceutical Preparations
V. Administration of Plasmid-Lipid Particle Formulations
VI. Examples
VII. Conclusion
Glossary
The following abbreviations are used herein: DC-Chol, 3.beta.-(N-(N',
N'-dimethylaminoethane)carbamoyl)cholesterol (see, Gao, et al., Biochem.
Biophys. Res. Comm. 179:280-285 (1991)); DDAB,
N,N-distearyl-N,N-dimethylammonium bromide; DMRIE,
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride (see commonly
owned patent application U.S. Ser. No. 08/316,399, now abandoned
incorporated herein by reference); DOGS, diheptadecylamidoglycyl
spermidine; DOPE, 1,2-sn-dioleylphatidylethanolamine DOSPA,
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl
ammonium trifluoroacetate; DOTAP,
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumchloride; DOTMA,
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride; EPC, egg
phosphatidylcholine; RT, room temperature; HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBS, HEPES buffered
saline (150 mM NaCl and 20 mM HEPES); PEG-Cer-C.sub.20,
1-0-(2-(.omega.-methoxypolyethyleneglycol)succinoyl)-2-N-arachidoyl-sphing
osine; PEG-Cer-C.sub.14,
1-0-(2'-(co-methoxypolyethyleneglycol)succinoyl)-2-N-myristoyl-sphingosine
; PBS, phosphate-buffered saline; EGTA,
ethylenebis(oxyethylenenitrilo)-tetraacetic acid; OGP, n-octyl
.beta.-D-glycopyranoside (Sigma Chemical Co., St. Louis, Mo.); POPC,
palmitoyl oleoyl phosphatidylcholine (Northern Lipids, Vancouver, BC);
QELS, quasielastic light scattering; TBE, 89 mM Tris-borate with 2 mM
EDTA; and EDTA, Ethylenediaminetetraacetic acid (Fisher Scientific, Fair
Lawn, N.J.);
The term "acyl" refers to a radical produced from an organic acid by
removal of the hydroxyl group. Examples of acyl radicals include acetyl,
pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl and oleoyl.
The term "lipid" refers to any fatty acid derivative which is capable of
forming a bilayer such that a hydrophobic portion of the lipid material
orients toward the bilayer while a hydrophilic portion orients toward the
aqueous phase. Hydrophilic characteristics derive from the presence of
phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like
groups. Hydrophobicity could be conferred by the inclusion of groups that
include, but are not limited to, long chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or more
aromatic, cycloaliphatic or heterocyclic group(s). Preferred lipids are
phosphoglycerides and sphingolipids, representative examples of which
include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine or
dilinoleoylphosphatidylcholine could be used. Other compounds lacking in
phosphorus, such as sphingolipid and glycosphingolipid families are also
within the group designated as lipid. Additionally, the amphipathic lipids
described above may be mixed with other lipids including triglycerides and
sterols.
The term "non-cationic lipid" refers to any of a number of lipid species
which exist either in an uncharged form, a neutral zwitterionic form, or
an anionic form at physiological pH. Such lipids include, for example
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,
sphingomyelin, cephalin, cardiolipin, and cerebrosides.
The term "cationic lipid" refers to any of a number of lipid species which
carry a net positive charge at physiological pH. Such lipids include, but
are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE.
Additionally, a number of commercial preparations of cationic lipids are
available which can be used in the present invention. These include, for
example, LIPOFECTIN.RTM. (commercially available cationic liposomes
comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y., USA);
LIPOFECTAMINE.RTM. (commercially available cationic liposomes comprising
DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM.RTM. (commercially
available cationic liposomes comprising DOGS from Promega Corp., Madison,
Wis., USA).
The terms "transfection" and "transformation" are used herein
interchangeably, and refer to the introduction of polyanionic materials,
particularly nucleic acids, into cells. The term "lipofection" refers to
the introduction of such materials using liposome or lipid-based
complexes. The polyanionic materials can be in the form of DNA or RNA
which is linked to expression vectors to facilitate gene expression after
entry into the cell. Thus the polyanionic material used in the present
invention is meant to include DNA having coding sequences for structural
proteins, receptors and hormones, as well as transcriptional and
translational regulatory elements (i.e., promoters, enhancers, terminators
and signal sequences) and vectors. Methods of incorporating particular
nucleic acids into expression vectors are well known to those of skill in
the art, but are described in detail in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring
Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F.
Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York
(1987), both of which are incorporated herein by reference.
"Expression vectors", "cloning vectors", or "vectors" are often plasmids or
other nucleic acid molecules that are able to replicate in a chosen host
cell. Expression vectors may replicate autonomously, or they may replicate
by being inserted into the genome of the host cell, by methods well known
in the art. Vectors that replicate autonomously will have an origin of
replication or autonomous replicating sequence (ARS) that is functional in
the chosen host cell(s). Often, it is desirable for a vector to be usable
in more than one host cell, e.g., in E. coli for cloning and construction,
and in a mammalian cell for expression.
II. General
Although directed to the transfer of nucleic acids, and in particular to
the transfer of plasmids to cells, the particles of the present invention
can be used for delivering essentially any polyanionic molecule. As noted
in the Background of the Invention, typical lipid-nucleic acid
formulations are formed by combining the nucleic acid with a preformed
cationic liposome (see, U.S. Pat. Nos. 4,897,355, 5,264,618, 5,279,833 and
5,283,185. In such methods, the nucleic acid is attracted to the cationic
surface charge of the liposome and the resulting complexes are thought to
be of the "sandwich-type" depicted in FIG. 1. As a result, a portion of
the nucleic acid or plasmid remains exposed in serum and can be degraded
by enzymes such as DNAse I. Others have attempted to incorporate the
nucleic acid or plasmid into the interior of a liposome during formation.
These methods typically result in the aggregation in solution of the
cationic lipid-nucleic acid complexes (see FIG. 2). Passive loading of a
plasmid into a preformed liposome has also not proven successful. Finally,
the liposome-plasmid complexes which have been formed are typically 200 to
400 nm in size and are therefore cleared more rapidly from circulation
than smaller sized complexes or particles. The present invention provides
a method of preparing serum-stable plasmid-lipid particles in which the
plasmid is encapsulated in a lipid-bilayer and is protected from
degradation. Additionally, the particles formed have a size of about 50 to
about 150 nm, with a majority of the particles being about 65 to 85 nm.
The particles can be formed by either a detergent dialysis method or by a
modification of a reverse-phase method which utilizes organic solvents to
provide a single phase during mixing of the components. Without intending
to be bound by any particular mechanism of formation, FIG. 3 depicts a
detergent dialysis approach to the formation of the plasmid-lipid
particles. With reference to FIG. 3, a plasmid or other large nucleic acid
is contacted with a detergent solution of cationic lipids to form a coated
plasmid complex. These coated plasmids can aggregate and precipitate.
However, the presence of a detergent reduces this aggregation and allows
the coated plasmids to react with excess lipids (typically, non-cationic
lipids) to form particles in which the plasmid is encapsulated in a lipid
bilayer. As noted above, these particles differ from the more classical
liposomes both in size (liposomes being typically 200-400 nm) in that
there is little or no aqueous medium encapsulated by the particle's lipid
bilayer. The methods described below for the formation of plasmid-lipid
particles using organic solvents follow a similar scheme.
III. Methods of Forming Plasmid-Lipid Particles
The present invention provides methods for the formation of serum-stable
plasmid-lipid particles. While the invention is described with reference
to the use of plasmids, one of skill in the art will understand that the
methods described herein are equally applicable to other larger nucleic
acids or oligonucleotides. In one group of embodiments, the particles are
formed using detergent dialysis. Thus, the present invention provides a
method for the preparation of serum-stable plasmid-lipid particles,
comprising:
(a) combining a plasmid with cationic lipids in a detergent solution to
form a coated plasmid-lipid complex;
(b) contacting non-cationic lipids with the coated plasmid-lipid complex to
form a detergent solution comprising a plasmid-lipid complex and
non-cationic lipids; and
(c) dialyzing the detergent solution of step (b) to provide a solution of
serum-stable plasmid-lipid particles, wherein the plasmid is encapsulated
in a lipid bilayer and the particles are serum-stable and have a size of
from about 50 to about 150 nm.
The plasmids which are useful in the present invention are typically
nucleotide polymers which are to be administered to a subject for the
purpose of repairing or enhancing the expression of a cellular protein.
Accordingly, the nucleotide polymers can be polymers of nucleic acids
including genomic DNA, cDNA, or mRNA. Still further, the plasmids may
encode promoter regions, operator regions, structural regions. When
nucleic acids other than plasmids are used the nucleic acids can contain
nucleic acid analogs, for example, the antisense derivatives described in
a review by Stein, et al., Science 261:1004-1011 (1993) and in U.S. Pat.
Nos. 5,264,423 and 5,276,019, the disclosures of which are incorporated
herein by reference.
The plasmids, or nucleic acids can be single-stranded DNA or RNA, or
double-stranded DNA or DNA-RNA hybrid. Examples of double-stranded DNA
include structural genes, genes including operator control and termination
regions, and self-replicating systems such as plasmid DNA.
Single-stranded nucleic acids include antisense oligonucleotides
(complementary to DNA and RNA), ribozymes and triplex-forming
oligonucleotides. In order to have prolonged activity, the single-stranded
nucleic acids will preferably have some or all of the nucleotide linkages
substituted with stable, non-phosphodiester linkages, including, for
example, phosphorothioate, phosphorodithioate, phophoroselenate, or
O-alkyl phosphotriester linkages.
The nucleic acids used in the present invention will also include those
nucleic acids in which modifications have been made in one or more sugar
moieties and/or in one or more of the pyrimidine or purine bases. Examples
of sugar modifications include replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, azido groups or functionalized as
ethers or esters. Additionally, the entire sugar may be replaced with
sterically and electronically similar structures, including aza-sugars and
carbocyclic sugar analogs. Modifications in the purine or pyrimidine base
moiety include, for example, alkylated purines and pyrimidines, acylated
purines or pyrimidines, or other heterocyclic substitutes known to those
of skill in the art.
Multiple genetic sequences can be also be used in the present methods.
Thus, the sequences for different proteins may be located on one strand or
plasmid. Promoter, enhancer, stress or chemically-regulated promoters,
antibiotic-sensitive or nutrient-sensitive regions, as well as therapeutic
protein encoding sequences, may be included as required. Non-encoding
sequences may be also be present, to the extent they are necessary to
achieve appropriate expression.
The nucleic acids used in the present method can be isolated from natural
sources, obtained from such sources as ATCC or GenBank libraries or
prepared by synthetic methods. Synthetic nucleic acids can be prepared by
a variety of solution or solid phase methods. Generally, solid phase
synthesis is preferred. Detailed descriptions of the procedures for solid
phase synthesis of nucleic acids by phosphite-triester, phosphotriester,
and H-phosphonate chemistries are widely available. See, for example,
Itakura, U.S. Pat. No. 4,401,796; Caruthers, et al., U.S. Pat. Nos.
4,458,066 and 4,500,707; Beaucage, et al., Tetrahedron Lett., 22:1859-1862
(1981); Matteucci; et al., J. Am. Chem. Soc., 103:3185-3191 (1981);
Caruthers, et al., Genetic Engineering, 4:1-17 (1982); Jones, chapter 2,
Atkinson, et al., chapter 3, and Sproat, et al., chapter 4, in
Oligonucleotide Synthesis: A Practical Approach, Gait (ed.), IRL Press,
Washington D.C. (1984); Froehler, et al., Tetrahedron Lett., 27:469-472
(1986); Froehler, et al., Nucleic Acids Res., 14:5399-5407 (1986); Sinha,
et al. Tetrahedron Lett., 24:5843-5846 (1983); and Sinha, et al., Nucl.
Acids Res., 12:4539-4557 (1984) which are incorporated herein by
reference.
Cationic lipids which are useful in the present invention, include, for
example, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. These lipids and
related analogs, which are also useful in the present invention, have been
described in co-pending U.S. Ser. No. 08/316,399; now abandoned U.S. Pat.
Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185, the disclosures of
which are incorporated herein by reference. Additionally, a number of
commercial preparations of cationic lipids are available and can be used
in the present invention. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and DOPE, from
GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM. (commercially
available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL);
and TRANSFECTAM.RTM. (commercially available cationic liposomes comprising
DOGS from Promega Corp., Madison, Wis., USA).
An initial solution of coated plasmid-lipid complexes is formed by
combining the plasmid with the cationic lipids in a detergent solution.
The detergent solution is preferably an aqueous solution of a neutral
detergent having a critical micelle concentration of 15-300 mM, more
preferably 20-50 mM. Examples of suitable detergents include, for example,
N,N'-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide) (BIGCHAP);
BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20;
Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent.RTM.
3-08; Zwittergent.RTM. 3-10; Triton X-405; hexyl-, heptyl-, octyl- and
nonyl-.beta.-D-glucopyranoside; and heptylthioglucopyranoside; with octyl
.beta.-D-glucopyranoside being the most preferred. The concentration of
detergent in the detergent solution is typically about 100 mM to about 2
M, preferably from about 200 mM to about 1.5 M.
The cationic lipids and plasmid will typically be combined to produce a
charge ratio (+/-) of about 1: 1 to about 20: 1, preferably in a ratio of
about 1: 1 to about 12:1, and more preferably in a ratio of about 2:1 to
about 6:1. Additionally, the overall concentration of plasmid in solution
will typically be from about 25 .mu.g/mL to about 1 mg/mL, preferably from
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