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FIELD OF THE INVENTION
This invention relates to methods of preparing lipid-nucleic acid particles
which are useful for the introduction of nucleic acids into cells. The
lipid-nucleic acid particles prepared by this method are stable in vivo
and are suitable as nucleic acid or antisense transfer delivery vehicles,
practical for clinical use.
BACKGROUND OF THE INVENTION
Developments in recombinant deoxyribonucleic acid ("DNA") technology have
opened up new avenues for medical treatment. The location and sequences of
an increasing number of disease-related genes are being identified, and
clinical testing of nucleic acid-based therapeutics for a variety of
diseases is now underway.
Gene therapy involves the introduction of genetic material into a cell to
facilitate expression of a deficient or defective protein. Missing or
defective genes (sequences of DNA encoding messenger RNA which are used as
templates for protein construction) which are responsible for the
production of these proteins result in a class of genetic disease often
referred to as `inborn errors of metabolism`. In some cases the disease
can be treated by controlling the diet, as in the case of phenylketonuria,
in which the liver enzyme responsible for the conversion of phenylalanine
to tyrosine is defective. Untreated, this disease can result in mental
retardation.
Treatments available for most genetic diseases are not as straightforward
as merely altering the diet. For example, adenosine deaminase (ADA)
deficiency results from a missing or defective gene that makes the
adenosine deaminase enzyme. This enzyme is essential for a healthy immune
system. ADA deficiency, however, is the disease successfully treated by
the first human "gene transfer" experiment conducted by Kenneth Culver in
1990 (see, Culver, GENE THERAPY: A HANDBOOK FOR PHYSICIANS, MaryAnn
Liebert, Inc. publishers, p. 33-40 (1994)).
One method of introducing nucleic acids into a cell is mechanically, using
direct microinjection. However this method is only practical for
transfecting eukaryotic germline cells for the production of transgenic
systems. To be effective in treating a disease, a nucleic acid-based
therapy must enter many cells.
Systemic gene transfer entails distributing nucleic acids to target cells
and then transferring the nucleic acid across a target cell membrane
intact and in a form that can function in a therapeutic manner. In vivo
gene transfer is complicated by serum interactions, immune clearance,
toxicity and biodistribution.
The in vivo gene transfer methods under study in the clinic consist almost
entirely of vital vectors. Although vital vectors have the inherent
ability to transport nucleic acids across cell membranes and some can
integrate exogenous DNA into the chromosomes, they can carry only limited
amounts of DNA and also pose risks. One such risk involves the random
integration of viral genetic sequences into patient chromosomes,
potentially damaging the genome and possibly inducing a malignant
transformation. Another risk is that the vital vector may revert to a
pathogenic genotype either through mutation or genetic exchange with a
wild type virus.
Lipid-based vectors have also been used in gene transfer and have been
formulated in one of two ways. In one method, the nucleic acid is
introduced into preformed liposomes made of mixture of cationic lipids and
neutral lipids. The complexes thus formed have undefined and complicated
structures and the transfection efficiency is severely reduced by the
presence of serum. Preformed liposomes are commercially available as
LIPOFECTIN.RTM. and LIPOFECTAMINE.RTM.. The second method involves the
formation of DNA complexes with mono- or poly-cationic lipids without the
presence of a neutral lipid. These complexes are prepared in the presence
of ethanol and are not stable in water. Additionally, these complexes are
adversely affected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)).
An example of a commercially available poly-cationic lipid is
TRANSFECTAM.RTM..
Other efforts to encapsulate DNA in lipid-based formulations have not
overcome these problems (see, Szoka, et al., Ann. Rev. Biophys. Bioeng.
9:467 (1980); and Deamer, U.S. Pat. No. 4,515,736).
Ideally, a delivery vehicle for nucleic acid will be small enough (<200 nm)
and stable enough in circulation to distribute from local injection sites
or following intravenous injection. The composition will have the maximum
amount of nucleic acid per particle and will be homogenous and
reproducible. The composition should also maintain the nucleic acid in a
configuration which is protected from degradation prior to nuclear
delivery and should efficiently transfect the target cells.
Surprisingly, the present invention provides such compositions and methods
for their preparation.
SUMMARY OF THE INVENTION
The present invention provides novel, lipid-nucleic acid particles via
formation of hydrophobic lipid-nucleic acid complexes. The complexes are
charge-neutralized. Formation of these complexes in either detergent-based
or organic solvent-based systems, followed by removal of the detergent or
organic solvent, leads to particle formation.
Thus, the present invention also provides methods of preparing
lipid-nucleic acid particles which are useful for the therapeutic delivery
of nucleic acids. The particles are constructed via a hydrophobic
lipid-nucleic acid intermediate (or complex). Upon removal of a
solubilizing component (i.e., detergent or an organic solvent) the nucleic
acid becomes protected from degradation. The particles thus formed are
suitable for use in intravenous nucleic acid transfer as they are stable
in circulation, of a size required for pharmacodynamic behavior resulting
in access to extravascular sites and target cell populations.
Briefly, one method of forming lipid-nucleic acid particles, involves:
(a) contacting nucleic acids with a solution of non-cationic lipids and a
detergent to form a nucleic acid-lipid mixture;
(b) contacting cationic lipids with the nucleic acid-lipid mixture to
neutralize the negative charge of said nucleic acids and form a
charge-neutralized mixture of nucleic acids and lipids: and
(c) removing the detergent from the charge-neutralized mixture to provide
the lipid-nucleic acid particles in which the nucleic acids are protected
from degradation.
Another method of forming lipid-nucleic add particles, involves:
(a) contacting an amount of cationic lipids with nucleic acids in a
solution; the solution comprising of from about 15-35% water and about
65-85% organic solvent and the amount of cationic lipids being sufficient
to produce a +/- charge ratio of from about 0.85 to about 2.0, to provide
a hydrophobic, charge-neutralized lipid-nucleic acid complex;
(b) contacting the hydrophobic, charge-neutralized lipid-nucleic acid
complex in solution with non-cationic lipids, to provide a lipid-nucleic
acid mixture; and
(c) removing the organic solvents from the lipid-nucleic acid mixture to
provide lipid-nucleic acid particles in which the nucleic acids are
protected from degradation.
It is a further aspect of the invention to provide in vitro and in vivo
methods for treatment of diseases which involve the overproduction or
underproduction of particular proteins. In these methods, a nucleic acid
encoding a desired protein or blocking the production of an undesired
protein, is formulated through a hydrophobic intermediate into a
lipid-nucleic acid particle, and the particles are administered to
patients requiring such treatment. Alternatively, cells are removed from a
patient, transfected with the lipid-nucleic acid particles described
herein, and reinjected into the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a model for the binding of monocationic lipids to nucleic acids
resulting in the formation of charge-neutralized, lipid-nucleic acid
complexes which are hydrophobic and in which the nucleic acid is present
in an uncondensed form.
FIG. 2 illustrates a protocol for preparing lipid-nucleic acid particles
using detergent dialysis.
FIG. 3 shows the effect of increasing amounts of LIPOFECTIN.RTM.
(DOTMA/DOPE; 50:50 mol ratio) on the recovery of .beta. gal plasmid DNA in
the aqueous phase following Bligh and Dyer extraction of the lipid-nucleic
acid complexes.
FIGS. 4A and 4B show the effect of increasing amounts of cationic lipid on
the recovery of plasmid DNA in the aqueous (A) and organic (B) phase
following Bligh and Dyer extraction of the lipid-nucleic acid complexes.
FIGS. 5A, 5B, 5C and 5D show the recovery of plasmid DNA from aqueous (A
and C) and organic (B and D) fractions following Bligh and Dyer extraction
and expressed as a function of charge ratio (+/-).
FIGS. 6A and 6B illustrate the DNA condensation by poly-L-lysine and DODAC
assayed by TO-PRO-1 dye intercalation. Condensation state was assessed in
a Bligh and Dyer monophase (A) and in 100 mM OGP (B).
FIG. 7 illustrates the effects of increasing amounts of OGP on the recovery
of plasmid DNA from the aqueous and organic phases following Bligh and
Dyer extraction of lipid-nucleic acid complexes (plasmid/DODAC).
FIG. 8 shows the effects of increasing amounts of NaCl on the recovery of
plasmid DNA from the aqueous phase following Bligh and Dyer extraction of
lipid-nucleic acid complexes.
FIGS. 9A and 9B show the effect of poly-L-lysine and DODAC on the
electrophoretic mobility of plasmid DNA.
FIG. 10 is a bar graph which illustrates the QELS results of a typical
lipid-nucleic acid complex mixture prepared from .beta.-gal
plasmid/DODAC/ESM.
FIG. 11 is a bar graph which illustrates the fluorescence spectroscopic
evaluation of DNA condensation in the lipid-nucleic acid complexes using
TO-PRO-1 dye intercalation. The results show that .beta.-gal plasmid in
DODAC/ESM is condensed and protected against dye intercalation by the
lipid, and that OGP can uncondense the particle.
FIG. 12 shows the results of electrophoresis of DNA extracted from
lipid-nucleic acid complexes following digestion with DNase I. DNA within
the complex is protected from DNase I degradation whereas uncomplexed DNA
is not protected.
FIG. 13 provides the results of CHO cell transfection using .beta.-gal
plasmid/DODAC/ESM as assayed by .beta.-gal enzyme activity.
DETAILED DESCRIPTION OF THE INVENTION
CONTENTS
I. Glossary
II. General
III. Methods of Formulating Lipid-Nucleic Acid Complexes and Particles
IV. Pharmaceutical Preparations
V. Administration of Lipid-Nucleic Acid Particle Formulations
VI. Examples
VII. Conclusion
I. Glossary
Abbreviations and Definitions
The following abbreviations are used herein: CHO, Chinese hamster ovary
cell line; B16, murine melanoma cell line; 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, incorporated herein by
reference); DOGS, diheptadecylamidoglycyl spermidine; DOPE,
1,2-sn-dioleoylphoshatidylethanolamine; 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-trimethylammonium chloride; DOTMA,
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; ESM, egg
sphingomyelin; RT, room temperature; TBE, Tris-Borate-EDTA (89 mM in
Tris-borate and 2 mM in EDTA); HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PBS,
phosphate-buffered saline; EGTA,
ethylenebis(oxyethylenenitrilo)-tetraacetic acid.
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.
As used herein, the term "pharmaceutically acceptable anion" refers to
anions of organic and inorganic acids which provide non-toxic salts in
pharmaceutical preparations. Examples of such anions include chloride,
bromide, sulfate, phosphate, acetate, benzoate, citrate, glutamate, and
lactate. The preparation of pharmaceutically acceptable salts is described
in Berge, et al., J. Pharm. Sci. 66:1-19 (1977), incorporated herein by
reference.
The term "lipid" refers to any suitable material resulting in a bilayer
such that a hydrophobic portion of the lipid material orients toward the
bilayer while a hydrophilic portion orients toward the aqueous phase.
Amphipathic lipids are necessary as the primary lipid vesicle structural
element. 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). The preferred
amphipathic compounds 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 "neutral lipid" refers to any of a number of lipid species which
exist either in an uncharged or neutral zwitterionic form at physiological
pH. Such lipids include, for example diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, and
cerebrosides.
The term "non-cationic lipid" refers to any neutral lipid as described
above as well as anionic lipids. Examples of anionic lipids include
cardiolipin, diacylphosphatidylserine and diacylphosphatidic acid.
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 lipids comprising DOGS in ethanol from Promega Corp.,
Madison, Wis., USA).
The term "transfection" as used herein, refers to the introduction of
polyanionic materials, particularly nucleic acids, into cells. The term
"lipofection" refers to the introduction of such materials using liposome
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 vector sequences. 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.
The term "hydrophobic" as applied to DNA and DNA complexes, refers to
complexes which are substantially more soluble in organic solvents than in
aqueous solutions. More particularly, hydrophobic DNA and DNA complexes
are those which are at least 50% soluble in organic solvents such as
chloroform/methanol mixtures, and preferably more than 70% soluble, more
preferably more than 90% soluble in such organic solvents.
II. General
The present invention provides lipid-nucleic acid particles produced via
novel, hydrophobic nucleic acid-lipid intermediate complexes. The
complexes are charge-neutralized. Manipulation of these complexes in
either detergent-based or organic solvent-based systems can lead to
particle formation in which the nucleic acid is protected and in which
particle components can be altered to improve transfection efficiencies in
vitro and in vivo. Gene delivery in vitro can be improved, for example,
through incorporation of a lipid, such as biotinylated phospholipids, that
can facilitate targeting via avidin linked monoclonal antibodies. In vivo
pharmacokinetic properties can be improved for example, by i)
incorporation of cholesterol, ii) control of particle size, iii)
elimination of surface charge and/or iv) incorporation of lipids (e.g.,
PEG-modified lipids) that reduce protein binding and reticuloendothelial
cell uptake.
Although directed to the transfer of nucleic acid, the particles and method
of formulating the particles can be used for delivering essentially any
polyanionic molecule including nucleic acid. 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, thereby forming a heterogeneous aggregate. This aggregation is
typically associated with the charge neutralization which occurs upon
mixing polyanionic nucleic acids with polyvalent cations.
The present invention also provides methods of forming lipid-nucleic acid
particles, however, the nucleic acid is not condensed during the
intermediate stages of particle formation. Additionally, the particles
formed in the present invention are preferably neutral or
negatively-charged at physiological pH. For in vivo applications, neutral
particles are particularly preferred, while for in vitro applications the
particles are more preferably negatively charged. This provides the
further advantage of reduced aggregation over the positively-charged
liposome formulations in which a nucleic acid can be encapsulated in
cationic lipids. Still further, the particles formed in the present
invention provide significantly enhanced protection of the nucleic acid
against degradation by DNases, compared to earlier methods.
III. Methods of Formulating Lipid-Nucleic Acid Complexes and Particles
In one aspect, the present invention provides novel, lipid-nucleic acid
complexes consisting essentially of cationic lipids and nucleic acids.
These complexes can be distinguished from other complexes by several
features. In particular, these complexes have a hydrophobic character
(being soluble in organic solvents) and are charge-neutralized.
Additionally, the nucleic acid portion of the complexes exists in an
uncondensed form. These complexes can be used in the preparation of the
lipid-nucleic acid particles which are described below and which are
themselves useful for transfecting cells in vitro or in vivo.
The complexes consist essentially of cationic lipids and nucleic acids. The
cationic lipids can be any of a number of lipid species which carry a net
positive charge at physiological pH, including, for example DODAC, DOTMA,
DDAB, DOTAP, DOSPA, DC-Chol, DOGS 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.;
LIPOFECTAMINE.RTM. and TRANSFECTAM.RTM..
The nucleic acids which are useful in the present invention (including both
the complexes and particles) are typically nucleotide polymers having from
10 to 100,000 nucleotide residues. Typically, the nucleic acids are to be
administered to a subject for the purpose of repairing or enhancing the
expression of a cellular protein. Additionally, the nucleic acid can carry
a label (e.g., radioactive label, fluorescent label or colorimetric label)
for the purpose of providing clinical diagnosis relating to the presence
or absence of complementary nucleic acids. Accordingly, the nucleic acids,
or nucleotide polymers, can be polymers of nucleic acids including genomic
DNA, cDNA, mRNA or oligonucleotides containing 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.
Still further, the nucleic acids may encode transcriptional and
translational regulatory sequences including promoter sequences and
enhancer sequences.
The nucleotide polymers can be single-stranded DNA or RNA, or
double-stranded DNA or DNA-RNA hybrids. Examples of double-stranded DNA
include structural genes, genes including 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 increase stability, some 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, phosphoroselenate, 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 that 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.
The formation of the lipid-nucleic acid complexes can be carried out either
in a monophase system (e.g., a Bligh and Dyer monophase or similar mixture
of aqueous and organic solvents) or in a two phase system with suitable
mixing.
When formation of the complexes is carried out in a monophase system, the
cationic lipids and nucleic acids are each dissolved in a volume of the
monophase mixture. Combination of the two solutions provides a single
mixture in which the complexes form. Alternatively, the complexes can form
in two-phase mixtures in which the cationic lipids bind to the nucleic
acid (which is present in the aqueous phase), and "pull" it in to the
organic phase. Without intending to be bound by any particular theory of
formation, FIG. 1 provides a model for the binding of monocationic lipids
to DNA which results in the formation of a hydrophobic (organic-soluble)
lipid-nucleic acid complex. In this figure, cationic lipids first bind to
the DNA to form a complex in which the DNA is uncondensed. This complex is
soluble in the organic phase or in a monophase and the DNA remains
uncondensed. Upon the addition of other lipids and removal of solvent, and
hydration, the complexes form particles (described in more detail below).
In another aspect, the present invention provides a method for the
preparation of lipid-nucleic acid particles, comprising:
(a) contacting nucleic acids with a solution comprising non-cationic lipids
and a detergent to form a nucleic acid-lipid mixture;
(b) contacting cationic lipids with the nucleic acid-lipid mixture to
neutralize a portion of the negative charge of the nucleic acids and form
a charge-neutralized mixture of nucleic acids and lipids; and
(c) removing the detergent from the charge-neutralized mixture to provide
the lipid-nucleic acid particles in which the nucleic acids are protected
from degradation.
Without intending to be limited by any particular aspect of the
illustration, FIG. 2 provides a depiction of one method of forming the
particles using detergent dialysis. In this figure, DNA in an aqueous
detergent solution (OGP) is combined with non-cationic lipids (ESM) in an
aqueous detergent solution and allowed to anneal for about 30 min. A
previously sonicated mixture of cationic lipid (DODAC) in detergent is
added and the resulting mixture is dialyzed for 3 days to remove detergent
and thereby form lipid-nucleic acid particles. One of skill in the art
will understand that for the kinetic formation of such particles, the
order of addition of cationic lipids and non-cationic lipids could be
reversed, or the lipids could be added simultaneously.
The nucleic acids used in this aspect of the invention can be any of those
described for the above complexes. In preferred embodiments, the nucleic
acid is a plasmid.
The non-cationic lipids used in the present invention can be any of a
variety of neutral uncharged, zwitterionic or anionic lipids. Examples of
neutral lipids which are useful in the present methods are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,
sphingomyelin, cephalin, and cerebrosides. Other lipids such as
lysophosphatidylcholine and lysophosphatidylethanolamine may be present.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine (e.g., dioleoylphosphatidylcholine,
dipalmitoylphosphatidylcholine and dilinoleoylphosphatidylcholine),
diacylphosphatidylethanolamine (e.g., dioleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylethanolamine), ceramide or sphingomyelin. The
acyl groups in these lipids are preferably acyl groups derived from fatty
acids having C.sub.10 -C.sub.24 carbon chains. More preferably the acyl
groups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In
particularly preferred embodiments, the non-cationic lipid will be
1,2-sn-dioleoylphosphatidylethanolamine, or egg sphingomyelin (ESM).
Additionally, the non-cationic lipids will include polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol
conjugated to phospholipids or to ceramides (referred to as PEG-Cer), as
described in co-pending U.S. Ser. No. 08/316,429, incorporated herein by
reference.
The detergents which are useful in the present invention are typically one
or more neutral detergents or combinations of detergents and organic
solvents. The detergents are preferably,
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; Triton X-405; hexyl-, heptyl-,
octyl- and nonyl-.beta.-D-glucopyranoside; with octyl
.beta.-D-glucopyranoside and Tween 20 being the most preferred. The
organic solvents which are useful in combination with a detergent include
chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane,
benzene, toluene, acetone, benzyl alcohol, methanol, or other aliphatic
alcohols such as propanol, isopropanol, butanol, tert-butanol,
iso-butanol, pentanol and hexanol. The selection of an organic solvent
will typically involve consideration of solvent polarity and the ease with
which the solvent can be removed at the later stages of particle
formation. Accordingly, the preferred organic solvents used in conjunction
with the detergent are ethanol, dichloromethane, chloroform, methanol and
diethyl ether with chloroform and methanol being the most preferred.
In one group of embodiments, the solution of non-cationic lipids and
detergent is an aqueous solution. Contacting the nucleic acids with the
solution of non-cationic lipids and detergent is typically accomplished by
mixing together a first solution of nucleic acids and a second solution of
the lipids and detergent. One of skill in the art will understand that
this mixing can take place by any number of methods, for example by
mechanical means such as by using vortex mixers. Preferably, the nucleic
acid solution is also a detergent solution. The amount of non-cationic
lipid which is used in the present method is typically determined based on
the amount of cationic lipid used, and is typically of from about 0.2 to 5
times the amount of cationic lipid, preferably about 0.5 to 2 times the
amount of cationic lipid used.
The nucleic acid-lipid mixture thus formed is contacted with cationic
lipids to neutralize a portion of the negative charge which is associated
with the nucleic acids (or other polyanionic materials) present. The
amount of cationic lipids used will typically be sufficient to neutralize
at least 50% of the negative charge of the nucleic acid. Preferably, the
negative charge will be at least 70% neutralized, more preferably at least
90% neutralized. 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 have been described in co-pending
U.S. Ser. No. 08/316,399; 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 D | | |
