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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns a reagent and a method for introducing
nucleic acid into animal cells.
2. Background Information
There are currently four major reagents or methods used to introduce DNA
into animal cells. These are (1) CaPO.sub.4 -DNA precipitates, (2) DEAE
dextran-DNA complexes, (3) electroporation and (4) "LIPOFECTIN".TM.
reagent, a transfection reagent marketed by BRL (Life Technologies, Inc.,
Gaithersburg, Md).
Recently, a liposome-mediated transfection protocol ("LIPOFECTION".TM.) has
been reported for the introduction of DNA into animal cells (Philip L.
Felgner, Thomas R. Gadek, Marilyn Holm, Richard Roman, Hardy W. Chan,
Michael Wenz, Jeffrey P. Northrop, Gordon M. Ringold and Mark Danielsen,
"Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection
Procedure," Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)). This
protocol uses the synthetic cationic lipid DOTMA
(N-[1-(2,3-dioleyloxy)propyl] -N,N,N-trimethylammonium chloride).
Liposomes composed of DOTMA and a neutral lipid PtdEtn
(dioleoylphosphatidylethanolamine) form stable complexes with DNA, and
deliver DNA into several eukaryotic cells with higher efficiency and
reproducibility than other methods.
A transient expression system that requires transfected DNA to be present
in the cytoplasm has recently been described by T.R. Fuerst, E.G. Niles,
W. Studier and B. Moss, Proc. Natl. Acad. Sci. USA, 83, 8122-8126 (1986).
This system is based on use of a recombinant vaccinia virus that
synthesizes bacteriophage T7 RNA polymerase. The plasmid DNA containing
the gene of interest under control of the T7 promoter is transfected as a
CaPO.sub.4 precipitate into the cytoplasm of the vaccinia infected cells
where it is transcribed efficiently by the T7 RNA polymerase. The mRNA
derived from the transfected gene can be as much as 10% of the total
cytoplasmic RNA. This system has facilitated studies of viral glycoprotein
translocation (A.S. Shaw, P.J.M. Rottier and J.K. Rose, Proc. Natl. Acad.
Sci. USA, 85, 7592-7596, (1988)) and virus assembly (M. Whitt, L. Chong
and J.K. Rose, J. Virol., 63, 3569-3578 (1989)), and has allowed for the
definition of interacting domains of the lymphocyte glycoprotein CD4 and
an intracellular tyrosine protein kinase (A. Shaw, K. Amrein, C. Hammond,
D.F. Stern, B.M. Sefton and J.K. Rose, Cell, 59, 627-636 (1989)). A major
difficulty with this system was the lack of reproducibility of the
transfection step using CaPO.sub.4 precipitates of DNA (Fuerst et al,
Proc. Natl. Acad. Sci. USA, 83, 8122-8126, (1986) and F.L. Graham and A.J.
Van Der Eb, Virology, 52, 456-467, (1973)). The percentage of cells
expressing protein showed a high degree of day-to-day variability, some
plasmid DNA preparations were inactive for unknown reasons, and it was not
possible to use impure plasmid DNA preparations containing large amounts
of RNA (DNA from minipreps).
The variability in the transfection was overcome by using lipofection
instead of the CaPO.sub.4 procedure (M. Whitt et al, J. Virol., 63,
3569-3578, (1989)) and a threefold increase over the best expression
levels obtained with CaPO.sub.4 was found.
A major drawback to the DOTMA transfection procedure is that the compound
itself is not commercially available, and the preformed liposomes
containing DOTMA ("LIPOFECTIN".TM. reagent, Life Technologies, Inc.,
Gaithersburg, Md.) are too expensive for large scale use in transient
assays. The cost of lipofection is prohibitive ($145/ml or about $10 per
transfection) to laboratories, especially to laboratories which perform
thousands of transfections per year.
P.L. Felgner and G.M. Ringold, "Cationic Liposome-Medicated Transfection,"
Nature, 337, 387-388, (1989) at page 387 report that liposomes comprised
of stearylamine or dioctaldecyl-dimethylammonium bromide were inactive in
transfection assays.
J.-P. Behr, B. Demeneix, J-P. Loeffler and J.P. Mutul, Proc. Natl. Acad.
Sci., USA, 86, 6982-6986, (1989) described a transfection procedure using
compacted lipopolyamine-coated plasmids.
It would be advantageous to have a reagent and method for introducing
nucleic acids into animal cells using readily available and relatively
inexpensive compounds.
SUMMARY OF THE INVENTION
It is an object of the present invention to introduce nucleic acids into
animal cells using relatively available and relatively inexpensive
reagents. This object, as well as other objects, aims and advantages are
achieved by the present invention.
The present invention concerns a reagent for introducing nucleic acids into
an animal cell comprising
a. a neutral lipid, for example, dioleyl phosphatidylethanolamine, and
b. a cationic lipid selected from the group consisting of
(1) an ammonium salt of the formula
##STR1##
wherein R.sub.1 is a straight hydrocarbon chain of C.sub.14 to C.sub.18
that is saturated or unsaturated,
R.sub.2, R.sub.3 and R.sub.4 are, independently of each other, hydrogen, a
straight hydrocarbon chain of C.sub.1 to C.sub.18 that is saturated or
unsaturated or an aryl, e.g., benzyl or phenyl, and A is an anion,
(2) an amine of the formula
##STR2##
wherein R.sub.5 is a straight chain of C.sub.14 to C.sub.18 that is
saturated or unsaturated and wherein R.sub.6 and R.sub.7, independently of
each other, are hydrogen or C.sub.1 to C.sub.5 alkyl and
(3) a benzethonium salt of the formula
##STR3##
wherein R.sub.8 is a straight chain or branched C.sub.1 -C.sub.10 -alkyl,
R.sub.9 is a chain C.sub.1 -C.sub.10 -alkyl,
R.sub.10 is a chain C.sub.1 -C.sub.10 -alkyl,
R.sub.11 is a chain C.sub.1 -C.sub.10 -alkyl,
R.sub.12 is a chain C.sub.1 -C.sub.10 -alkyl,
R.sub.13 is a chain C.sub.1 -C.sub.10 -alkyl,
R.sub.14 is a chain C.sub.1 -C.sub.10 -alkyl and
A.sub.1.sup..crclbar. is an anion.
The present invention also concerns a method for introducing a nucleic acid
into an animal cell comprising
a. mixing nucleic acid with a reagent as described above to form a liposome
or lipid micelles and
b. contacting the resultant liposome or lipid micelles with an animal cell.
The present invention further relates to a complex between a nucleic acid
and the reagent as defined above. Still further, the present invention is
directed to such complex further comprising a liposome or lipid micelles.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purpose of illustrating the invention there is shown in the
drawings forms which are presently preferred. It is to be understood,
however, that the present invention is not limited to the precise
arrangements and instrumentalities depicted in the drawings.
FIG. 1A and 1B depict flow cytometry profiles showing a high frequency of
VSV glycoprotein expression on the surface of HeLa cells. FIG. 1A is a
profile for a control (mock transfection). FIG. 1B is a profile for pARG
transfected (pARG is a plasmid that encodes a viral glycoprotein).
FIG. 2 is a series of bar graphs depicting the effect of DDAB concentration
on expression frequency for pARG transfected for BHK cells and HeLa cells.
FIG. 3 is a series of bar graphs depicting the effect of DNA concentration
on expression frequency for pARG transfected in BHK cells.
FIG. 4 is a graph showing the effect of DNA concentration on the rate of
VSV G protein synthesis.
FIG. 5A, 5B, 5C, and 5D depict flow cytometry profiles for a control (mock
transfected) (FIG. 5A), DDAB (FIG. 5B), "LIPOFECTIN".TM. reagent that
contains DOTMA (FIG. 5C) and CaPO.sub.4 (FIG. 5D).
DETAILED DESCRIPTION OF THE INVENTION
The invention concerns a reagent and a method for introducing nucleic acid
into animal cells with very high efficiency. The reagent is composed of a
cationic lipid (detergent), for example, dimethyldioctadecylammonium
bromide, and a neutral lipid, for example, dioleoyl
phosphatidylethanolamine. Liposomes are formed from these two compounds.
Nucleic acid, e.g., DNA or RNA, is mixed with the preformed liposomes in
medium and added to cells, e.g., on a tissue culture dish. This results in
nucleic acid, e.g., DNA, delivery into the cells with high efficiency.
Non-limiting examples of other neutral lipids for use in the present
invention include the following: phospholipid-related materials such as
lecithin, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphinogomyelin, cephalin, cardiolipin, phosphatidic
acid, cerebrosides, acetylphosphate, dioleoylphosphatidylcholine (DOPE),
dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol (DOPC),
dipalmitoylphosphatidylglycerol, dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (DOPE-MAL), diheptadecanoyl
phosphatidylethanolamine, dilauroylphosphatilylethanolamine,
dimyristoylphosphatidylethanolamine, distearoyl phosphatidylethanolamine,
beta-linoleoyl-gammapalmitoyl phosphatidylethanolamine and
beta-oleoyl-gammapalmitoyl phosphatidylethanolamine.
The cationic lipid for use in the present invention is
(1) an ammonium salt of the formula
##STR4##
wherein R.sub.1 is a straight hydrocarbon chain of C.sub.14 to C.sub.18
that is saturated or unsaturated,
R.sub.2, R.sub.3 and R.sub.4 are, independently of each other, hydrogen, a
straight hydrocarbon chain of C.sub.1 -C.sub.18 that is saturated or
unsaturated, preferably C.sub.1 -C.sub.5 -alkyl or C.sub.18 a-alkyl or an
aryl, e.g., a benzyl or phenyl and A is an anion, for example, a halogen,
for example, Br, Cl, I or F, preferably Br, or a sulfate, nitrite or
nitrate, wherein preferred embodiments of such salt are
cetyldimethyethylammonium bromide of the formula CH.sub.3
(CH.sub.2).sub.15 N(CH.sub.3).sub.2 (C.sub.2 H.sub.5)Br and
dimethyldioctadecylammonium bromide (DDAB) of the formula
((CH.sub.3)(CH.sub.2).sub.17).sub.2 N(CH.sub.3).sub.2 Br,
(2) an amine of the formula
##STR5##
wherein R.sub.5 is a straight chain of C.sub.14 to C.sub.18 that is
saturated or unsaturated and wherein R.sub.6 and R.sub.7, independently of
each other, are hydrogen or C.sub.1 to C.sub.5 alkyl, preferably the amine
is stearylamine of the formula CH.sub.3 (CH.sub.2).sub.17 NH.sub.2, or
(3) a benzethonium salt of the formula
##STR6##
wherein R.sub.8 is a straight chain or branched C.sub.1 -C.sub.10 -alkyl,
R.sub.9 is a C.sub.1 -C.sub.10 -alkyl,
R.sub.10 is a C.sub.1 -C.sub.10 -alkyl,
R.sub.11 is a C.sub.1 -C.sub.10 -alkyl,
R.sub.12 is a C.sub.1 -C.sub.10 -alkyl,
R.sub.13 is a C.sub.1 -C.sub.10 -alkyl,
R.sub.14 is a C.sub.1 -C.sub.10 -alkyl and A.sub.1.sup..crclbar. is an
anion, for example a halogen, for example, Br, Cl, I or F, preferably Cl,
or a sulfate, nitrate or nitrate, preferably the benzethonium salt is
methylbenzethonium chloride of the formula
##STR7##
(N,N-dimethyl-N-[2-[2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy]e
t
hyl]benzenemethanaminium chloride).
The present invention also encompasses substituted forms of the ammonium
salts, amines and benzethonium salts as described above, wherein the use
of such substituted forms in the reagent functions to allow the
introduction of nucleic acid into an animal cell.
The ratio of cationic lipid to neutral lipid can be widely varied,
depending on the particular cationic lipid employed. Thus for
cetyldimethylethylammonium bromide and methylbenzethonium chloride the
ratio can be 3/30 to 12/30; for dimethyldioctadecylammonium bromide the
ratio can be 6/30 to 12/30; and for stearylamine, the ratio can be 0.3/30
to 30/30.
The ratio of nucleic acid, e.g., DNA, to cationic lipid should not be so
high as to saturate the positive charges on the liposomes, which could
result in that the liposomes would not bind the cell surface.
Animal cells for use in the present invention include cells from humans and
non-human warm blooded animals. Erythrocytes, for example, can be
employed. Also baby hamster kidney (BHK) cells, AtI 20 cells, RK-13 cells
and Madin-Darby canine kidney (MDCK) cells can be used in the present
invention.
Although cytoplasmic gene expression is preferred, gene expression in the
cell nucleus can be conducted according to the invention.
Nucleic acid as used in this application includes DNA, RNA and
oligonucleotides of DNA and RNA.
The nucleic acid, e.g., DNA or RNA, coding for specific genes of interest
may be retrieved, without undue experimentation, from the GenBank of EMBL
DNA libraries. Such sequences may include coding sequences, for example,
the coding sequences for structural proteins, hormones, receptors and the
like, and the DNA sequences for other DNAs of interest, for example,
transcriptional and translational regulatory elements (promoters,
enhancers, terminators, signal sequences and the like), vectors
(integrating or autonomous) and the like. Non-limiting examples of DNA
sequences which may be introduced into cells with the reagent of the
invention include those sequences coding for fibroblast growth factor (WO
87/01728); ciliary neurotrophic factor (Lin et al, Science, 246:1023-1025
(1989); human interferon-.alpha. receptor (Uze, G. et al, Cell, 60:225-235
(1990); the interleukins and their receptors (reviewed in Mizal, S.B.,
FASEB J., 3:2379-2388 (1989); hybrid interferons (see European Patent
Application No. 051,873); the RNA genome of human rhinovirus (see
Callahan, P.L., Proc. Natl. Acad. Sci. (USA), 82:732-736 (1985));
antibodies including chimeric antibodies (see Cabilly et al, U.S. Pat. No.
4,816,567); reverse transcriptase (see Moelling, K., et al. J. Virol.,
32:370-378 (1979)); human CD4 and soluble forms thereof (Maddon et al,
Cell, 47:333-348 (1986); PCT Application Publication Nos. WO 88/01304
(1988) and WO 89/01940 (1989)). See also Seed, B. et al. European Patent
Application Publication No. 330,191, who disclose a rapid immunoselection
cloning method which is useful for the cloning of a large number of
important proteins. The disclosures of the references cited above are
fully incorporated by reference herein.
The present invention can be employed for the introduction of DNA, for
example, into animal cells for research purposes. The most common reason
for doing this is to obtain transient or permanent expression of DNA in
established animal cell lines. This is done on a daily basis in thousands
of laboratories worldwide.
The present invention can also be employed in gene therapy, e.g., to treat
genetic diseases in humans and nonhuman warm blooded animals. For example,
a "missing gene" can be introduced in the bone marrow of a patient by the
present invention.
The invention can further be used to treat infectious diseases, e.g., AIDS,
by blocking infection.
Transformation/Transfection is defined as follows: the introduction of DNA
or RNA into cells in such a way as to allow gene expression.
Liposomes consist of spheres of lipid bilayers (two-molecules thick) that
enclose an aqueous medium.
Liposomes can generally be formed by sonicating a lipid in a aqueous
medium, by resuspension of dried lipid layers in a buffer or by dialysis
of lipids dissolved in an organic solvent against a buffer of choice.
Phospholipids form closed, fluid-filled spheres when they are mixed with
water, in part because the molecules are amphipathic: they have a
hydrophobic (water-insoluble) tail and a hydrophilic (water-soluble), or
"polar," head. Two fatty acid chains containing up to about 24 carbon
atoms generally make up the hydrophobic tail of most naturally occurring
phospholipid molecules. Phosphoric acid bound to any of several
water-soluble molecules composes the hydrophilic head. When a high enough
concentration of phospholipids is mixed with water, the hydrophobic tails
spontaneously herd together to exclude water, whereas the hydrophilic
heads bind to water.
The result is a bilayer in which the fatty acid tails point into the
membrane's interior and the polar head groups point outward. The polar
groups at one surface of the membrane point toward the liposome's interior
and those at the other surface point toward the external environment. As a
liposome forms, any water-soluble molecules that have been added to the
water are incorporated into the aqueous spaces in the interior of the
spheres, whereas any lipid-soluble molecules added to the solvent during
vesicle formation are incorporated into the lipid bilayer.
Liposomes typically range in diameter from 250 angstrom units to several
micrometers (the diameter of a red blood cell is roughly 10 micrometers)
and are usually suspended in a solution. They have two standard forms:
"onion-skinned" multilamellar vesicles (MLV's), made up of several lipid
bilayers separated by fluid, and unilamellar vesicles, consisting of a
single bilayer surrounding an entirely fluid core. The unilamellar
vesicles are typically characterized as being small (SUV's) or large
(LUV's). The unilamellar vesicles are preferred in the present invention.
Under appropriate circumstances liposomes can adsorb to almost any cell
type. Once they have adsorbed the spheres, liposomes may be endocytosed,
or swallowed up, by some cells. Adsorbed liposomes can also exchange
lipids with cell membranes and may at times be able to fuse with cells.
When fusion takes place, the liposomal membrane is integrated into the
cell membrane and the aqueous contents of the liposome merge with the
fluid in the cell.
Endocytosis of liposomes occurs in a limited class of cells; those that are
phagocytic, or able to ingest foreign particles. When phagocytic cells
take up liposomes, the cells move the spheres into subcellular organelles
known as lysosomes, where the liposomal membranes are thought to be
degraded. From the lysosome, the liposomal lipid components probably
migrate outward to become part of the cell's membranes and other liposomal
components that resist lysosomal degradation (such as certain medications)
may enter the cytoplasm.
Lipid exchange involves the transfer of individual lipid molecules from the
liposome into the plasma membrane (and vice versa); the aqueous contents
of the liposome do not enter the cell. For lipid exchange to take place,
the liposomal lipid must have a particular chemistry in relation to the
target cell. Once a liposomal lipid joins the cell membrane it can either
remain in the membrane for a long time or be redistributed to a variety of
intracellular membranes.
In very dilute solutions, lipid micelles may form instead of liposomes.
With respect to screening procedures described herein, alternative
components active in transfection could be screened by substituting them
for PtdEtn in the preparation of liposomes containing cationic lipids.
These liposomes would then be used to transfect cells with plasmid DNA
encoding a protein that is easily assayed (for example the VSV
glycoprotein). The frequency of gene expression in cells would be
determined by flow cytometry as depicted in FIG. 1 and FIG. 5.
The invention will now be described with reference to the following
non-limiting examples.
EXAMPLES
MATERIALS
LIPIDS
L-.alpha.-dioleoylphosphatidylethanolamine (PtdEtn), stearylamine,
cetyldimethlyethyl-ammonium bromide, dimethyldioctadecylammonium bromide
(DDAB), and methylbenzethonium chloride
(N,N-dimethyl-N-[2-(2-[methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy)
ethyl]benzenem chloride), were purchased from Sigma Chemical Co., St.
Louis, Mo. "LIPOFECTIN".TM. reagent was purchased from Life Technologies,
Inc., Gaithersberg, Md.
CELLS AND MEDIA
HeLa cells were grown in Dubecco-Vogt's modified minimal essential medium
(DMEM) containing 10% horse serum or 5% fetal calf serum. Baby Hamster
Kidney (BHK) cells, AtT-20 cells, RK-13 cells, and Madin-Darby canine
kidney cells (MDCK) cells were grown in DME containing 5% fetal calf
serum.
EXAMPLE 1: LIPOSOME PREPARATION
Dioleoylphosphatidylethanolamine (PtdEtn) was supplied as a 10 mg/ml
solution in CHCl.sub.3. The other lipids were dissolved in CHCl.sub.3 at
100 mg/ml. Liposomes were typically prepared as follows. One mg of PtdEtn
(0.1 ml of a 10 mg/ml solution) was combined with varying amounts of each
cationic lipid (e.g., 0.4 mg DDAB) and the solution was evaporated to
dryness overnight in a Speed Vac Concentrator (Savant). Liposomes were
prepared by resuspending the lipids in 1 ml sterile deionized water and
sonicating on ice using an Ultrasonics Sonicator (microprobe, 50V setting)
until the solution was almost clear This typically required 5-10 minutes,
with pausing every 10-15 seconds to prevent overheating. Because
applicants observed variability in the transfection frequencies obtained
with different batches of liposomes sonicated to different extents, a
procedure was adopted of sonicating DDAB/PtdEtn (0.4 mg/ml and 1 mg/ml,
respectively) until a 1:10 dilution in distilled water gave an O.D. of 0.1
at 540 nm. Liposomes were stored at 4.degree. C. and were stable for at
least four months. were stable for at least four months.
EXAMPLE 2: TRANSFECTIONS
Plasmid DNA was normally purified by isopycnic banding in CsCl gradients.
However, it was found that crude DNA preparations which had not been
separated from RNA (minipreps) were also very effective. Cells to be
transfected (typically 5.times.10.sup.5 HeLa or BHK cells) were plated on
3 cm dishes. On the following day they were infected with vaccinia virus
encoding the T7 RNA polymerase (Fuerst et al, Proc. Natl. Acad. Sci. USA,
83, 8122-8126 (1986)) at a multiplicity of 10 and incubated for 30 minutes
at 37.degree. C. in 0.5 ml DME. Details of individual transfections are
given in the FIGURES. A typical transfection giving optimal expression in
BHK or HeLa cells was performed as follows. Five .mu.g of plasmid DNA were
added to 1 ml of DMEM in a polystyrene tube followed by addition 30 .mu.l
of liposomes. This solution was mixed gently and then incubated at room
temperature for 10 minutes to allow binding of DNA to liposomes. The vTF-7
inoculum was replaced with the liposome-DNA solution and incubated with
the cells for 3 hours at 37.degree. C. in a 5% CO.sub.2 incubator. An
additional 1 ml of DME with 10% fetal calf serum was then added without
removing the transfection mixture. It was found that the DNA and liposome
amount can be reduced at least two fold (2.5 .mu.g DNA and 15 .mu.l
liposomes) without reducing the protein expression level significantly.
For transfection using CaPO.sub.4 -DNA precipitates, cells in 6 cm dishes
were infected with VTF-7 as described above. After removing the inoculum,
4 ml of DME with 5% fetal bovine serum were added followed by 0.75 ml of
CaPO4-precipitated DNA containing 15 .mu.g pARG DNA and 15 .mu.g sonicated
calf thymus DNA carrier (added dropwise). The CaPO.sub.4 -DNA suspension
was prepared essentially as described in F.L. Graham and A.J. Van der Eb,
Virology, 52, 456-467 (1973).
EXAMPLE 3: FLUORESCENCE MICROSCOPY
Indirect immunofluorescence was carried out on Hela and BHK cells six hours
after transfection as described in J.K. Rose and J.E. Bergmann, Cell, 34,
513-524 (1983) with the following modifications. Fixed HeLa cells were
incubated with rabbit anti-VSV serum (1:200), followed by FITC conjugated
goat anti-rabbit immunoglobulin G (IgG) (1:50 dilution; Zymed
Laboratories, San Francisco, Calif.) for detection of cell-surface G
protein. For detection of G protein on the surface of BHK cells, a mouse
monoclonal antibody (II, L. Lefrancois and D.S. Lyles, Virology, 121,
157-167 (1982)) was used with an FITC-conjugated goat anti-mouse second
antibody. For determination of the percent of cells expressing, at least
five random fields of cells were counted (250-500 cells) using a Nikon
Microphot-FX fluorescence microscope equipped with a planapochromat 40x
objective.
EXAMPLE 4: FLOW CYTOMETRY
BHK or HeLa cells were infected with vTF7-3 and transfected as described
above with various ratios of DNA and cationic liposome suspensions. The
cells were removed from the dish and fixed in suspension at 6 hours
post-transfection. The cells were processed for flow cytometric analysis
as described in M. Whitt, L. Chong and J.K. Rose, J. Virol., 63, 3569-3578
(1989), except that in some experiments 0.5% bovine serum albumin was
included during the antibody incubations.
EXAMPLE 5: RADIOLABELING AND IMMUNOPRECIPITATION
Four hours post-transfection, cells were metabolically labeled by
incubation in methionine free DMEM containing [.sup.35 S]-methionine
(25.mu.Ci/0.5 ml) for 30 minutes. Cells were lysed, and the VSV G protein
was immunoprecipitated with a rabbit anti-VSV antibody as described in J.
K. Rose and J.E. Bergmann, Cell, 34, 513-524 (1983). Immunoprecipitated
proteins were analyzed by electrophoresis in 10% polyacrylamide gels in
the presence of sodium dodecyl sulfate. Dried gels were subjected to
autoradiography. For quantitation of radioactivity in the bands, films
were scanned with a Hoeffer model GS300 densitometer and the peaks were
integrated.
EXAMPLE 6: USE OF VARIOUS CATIONIC LIPIDS TO FORM LIPOSOMES To Mediate DNA
Transfection
To determine if cationic lipids other than DOTMA could form liposomes that
would mediate DNA transfection into the cytoplasm of animal cells, an
experiment was performed using four commercially available cationic lipids
(see Table 1). These lipids were chosen because they had long alkyl
chains, were relatively insoluble in water, and thus were not expected to
act as strong detergents. Liposomes were formed by sonication with a
constant amount of the neutral lipid, dioleylphosphatidylethanolamine
(PtdEtn), and the amount of each cationic lipid was varied as indicated.
The neutral lipid was added because of the previous report that it
enhanced transfection of liposomes containing DOTMA (Felgner et al, Proc.
Natl. Acad. Sci USA, 84, 7413-7417 (1987)). The vesicular stomatitis virus
(VSV) glycoprotein (G) has been expressed from cloned DNA previously and
is transported to the cell surface (Rose et al, Cell, 34, 513-524 (1983)).
Plasmid DNA designated pARG (M. Whitt, L. Chong and J.K. Rose, J. Virol.,
63, 3569-3578 (1989)) containing a cDNA clone encoding the VSV G protein
under control of the bacteriophage T7 promoter was mixed with the
preformed liposomes and then added to cells. These cells had been infected
with vTF-7 for 30 minutes prior to transfection. vTF-7 is a vaccinia virus
recombinant which expresses the bacteriophage T7 RNA polymerase in the
cytoplasm of infected cells (Fuerst et al, Proc. Natl. Acad. Sci. USA, 83,
8122-8126 (1986)). Six hours after transfection, cells were fixed with
paraformaldehyde, labeled with fluorescent antibodies recognizing the VSV
G protein, and counted using a fluorescence microscope to determine the
percent of cells expressing the protein. Results of these initial
experiments (Table 1) showed that all four of the compounds tested were
effective in HeLa cells when used at an appropriate concentration. Two of
these compounds (cetyldimethlyethylammonium bromide and methylbenzethonium
chloride) also caused cell lysis at higher concentrations. However, only
two of the compounds, namely, stearylamine and dimethyldioctadecylammonium
bromide (DDAB), gave detectable transfection of BHK cells, and DDAB was
far more effective than thearylamine. Because these and other experiments
suggested that DDAB was more effective than the other compounds on a
variety of cells, a more detailed analysis for only DDAB was conducted.
TABLE 1
______________________________________
Effectiveness of several cationic lipids in
transfection of HeLa and BHK cells.
Percent Cells
Expressing
Cationic lipid (.mu.g/ml) HeLa BHK
______________________________________
Cetyldimethylethylammonium
3 70 0
bromide 6 76 0
12 0* 0*
Dimethyldioctadecyl-
6 50 30
ammonium bromide 12 67 64
Methylbenzethonium
3 23 0
chloride 6 63 0
12 0* 0*
Stearylamine 0.3 10 0
3 60 5
30 1 0.5
______________________________________
Cells were transfected with 5 .mu.g pARG DNA bound to liposomes containin
the indicated amounts of cationic lipids and 30 .mu.g PtdEtn as described
herein. Percentage of cells expressing was determined by indirect
immunofluorescence microscopy at 6 hours after transfection. Liposomes
formed with 30 .mu.g/ml dipalmitoylphosphatidylethanolamine and 12
.mu.g/ml of DDAB did not result in transfection of HeLa or BHK cells.
*indicates cell lysis
To determine the transfection efficiency of liposomes containing DDAB on
other cell types, several other cell lines were examined. Transfections
into vTF-7 infected cells were conducted under conditions that were known
to be optimal for BHK and HeLa cells. Determination of the percent of
cells expressing was by indirect immunofluorescence microscopy. For AtT-20
cells (murine anterior pituitary) and RK-13 cells (rabbit kidney
fibroblast), 50-60 percent of the cells expressing was obtained. For MDCK
cells (Madin-Darby canine kidney epithelial), about 10% of the cells
expressing were obtained. For other cells such as mouse L-cells and NIH
3T3 cells, the frequency of cells expressing was usually only about 5
percent. Variability in results obtained with cells that were poorly
transfected suggested that a systematic analysis of DNA and lipid
concentrations might well improve the transfection frequencies in these
lines.
In addition to examining the percentage of cells expressing by direct
counting under a fluorescence microscope, flow cytometry was also used to
assess the accuracy of the direct cell counts and to examine the
distribution of fluorescence intensities for the expressing cells. FIG. 1
shows examples of flow cytometry profiles for control (mock-transfected
with DDAB liposomes) (FIG. 1A) and pARG transfected HeLa cells (FIG. 1B).
Duplicate 6 cm dishes containing 2.times.10.sup.6 HeLa cells were infected
with vTF-7 and then either transfected with 15 .mu.g DNA encoding the VSV
G protein (pARG) and 45 .mu.l liposomes containing 18 .mu.g DDAB or mock
transfected with liposomes only. After six hours, cells were then fixed,
immunostained for cell surface G protein and analyzed by flow cytometry as
described in Whitt et al, J. Virol., 63, 3569-3578, (1989).
The control cells fall in a sharp peak representing background
fluorescence, while the majority of transfected cells (85%) are clearly
positive for cell-surface expression of the VSV G protein. Using
CaPO.sub.4 -DNA transfection, only 10% of the cells expressing (data not
shown) was obtained. The heterogeneity in fluorescence of the positive
cells probably reflects delivery of variable amounts of plasmid DNA into
the cytoplasm of individual cells. The values obtained by direct counting
paralleled those obtained by flow cytometry, but the flow cytometry values
were generally somewhat higher. This difference probably reflects the
greater sensitivity of the flow cytometry measurements.
EXAMPLE 7: EFFECT OF DDAB CONCENTRATION
Liposomes containing a constant amount of PtdEtn and varying amounts of
DDAB were prepared as described herein. Dishes containing 2.times.10.sup.5
HeLa or BHK cells on coverslips were infected with vTF-7 and then
transfected with liposomes containing the indicated amounts of DDAB and 5
.mu.g DNA. The percentage of cells expressing was determined by
immunofluorescence microscopy. The results are depicted in FIG. 2. The
concentration of PtdEtn was held constant at 30 .mu.g/ml. Liposomes formed
with PtdEtn alone gave no transfection. A final concentration of 12
.mu.g/ml DDAB (30 .mu.l of liposomes containing 0.4 mg/ml DDAB and 1 mg/ml
PtdEtn added to 1 ml DMEM) appeared optimal and the percentage of cells
expressing dropped off markedly at higher DDAB concentrations. HeLa cells
with liposomes containing only DDAB were examined and it was found that
these were only about half as effective as those containing PtdEtn (data
not shown).
EXAMPLE 8: EFFECT OF DNA CONCENTRATION
The effect of DNA concentration on the transfection frequency in BHK cells
at the optimal lipid concentration as determined in FIG. 2 was examined.
Dishes containing 2.times.10.sup.5 BHK cells on coverslips were
transfected with the indicated amounts of DNA and 30 .mu.l DDAB liposomes.
The percentage of cells expressing was determined by immunofluorescence
microscopy. The results are depicted in FIG. 3.
With as little as 0.1 .mu.g of DNA, 18% of the cells expressing were
obtained. This increased to a maximum of 62% with 5 .mu.g of DNA in this
experiment and was often as high as 80-90%. At higher DNA concentrations,
there was a small decrease in number of cells expressing. Although the
decrease appears insignificant in this experiment, more marked decreases
at the high DNA concentrations with some liposome preparations were
observed.
To test the effect of DNA concentration on the rate of protein synthesis
directed by the transfected DNA, vTF-7 infected cells were transfected
with the amounts of pARG DNA indicated (FIG. 3) and then pulse-labeled
cells with [.sup.35 S]-methionine at four hours after transfection. This
time was chosen because earlier experiments had shown that the rate of
protein synthesis was already maximal by 4 hours. The VSV G protein was
immunoprecipitated from the lysates and electrophoresed on an
SDS-polyacrylamide gel. To ascertain the effect of DNA concentration on
the rate of VSV G protein synthesis, six dishes containing
5.times.10.sup.5 Hela cells each were infected with vTF-7 and then
transfected with 30 .mu.l DDAB-liposomes and with certain amounts of DNA.
The rates of VSV G protein synthesis were assessed by pulse-labeling with
[.sup.35 S]-methionine, immunoprecipitation of the G protein, and
autoradiography after gel electrophoresis. The results are depicted in
FIG. 4.
Quantitation of the radiolabeled protein (FIG. 4) showed that the rate of
protein synthesis reached a maximum with 5 .mu.g of DNA and dropped off
above this level. These results thus correlate well with those in FIG. 3,
showing that transfection of 5 .mu.g of DNA with 30 .mu.l of DDAB
liposomes gave an optimal percentage of cells expressing.
EXAMPLE 9: COMPARISON WITH DOTMA AND CaPO.sub.4
To determine how effective liposomes composed of DDAB were compared to
those containing DOTMA, transfections of vTF-7-infected BHK cells with
pARG and quantitated cell surface VSV G protein by flow cytometry were
performed. A transfection with CaPO.sub.4 precipitated DNA was also
included for comparison. Four 6 cm dishes each containing 2.times.10.sup.6
BHK cells were infected with vTF-7 and were mock transfected (FIG. 5A),
transfected with 15 .mu.g of pARG DNA and 45 .mu.l of DDAB-liposomes (FIG.
5B), 15 .mu.g pARG DNA and 30 .mu.l "LIPOFECTIN".TM. reagent (DOTMA
liposomes) (FIG. 5C), and with 15 .mu.g pARG DNA as a CaPO.sub.4
precipitate (FIG. 5D). At six hours after transfection, cells were fixed
and stained for cell-surface G protein.
The flow cytometry profiles are shown in FIG. 5. Comparison with FIG. 5A
(negative control of mock-transfected cells, DDAB liposomes) showed that
DDAB-liposomes (FIG. 5B) gave 95% and DOTMA-liposomes (FIG. 5C) gave 85%
of the cells expressing. A CaPO.sub.4 -precipitate of the same amount of
DNA (FIG. 5D) yielded only 30% of the cells expressing. In other
experiments DDAB-liposomes gave expression frequencies that were as much
as two-fold higher than those obtained with DOTMA-liposomes.
RESULTS
Although liposomes containing all four lipids tested herein were effective
in HeLa cells at some concentration, only one, dimethyldioctadecylammonium
bromide, also mediated efficient DNA transfection into the cytoplasm of a
variety of other cells including BHK, RK13, AtT20 (a pituitary cell line),
and MDCK (Madin-Darby canine kidney), an epithelial cell line. The very
high frequency of transient expression that was observed herein (up to 95%
of the cells expressing) is presumably due to the requirement that the DNA
only reach the cytoplasm. In other highly efficient transient systems such
as monkey COS cells (Y. Gluzman, Cell, 23, 175-182, (1981)) the DNA must
also cross the nuclear membrane to be expressed.
The mechanism by which liposomes containing positively charged lipids
mediate transfection of DNA into animal cells is not presently understood.
The DNA undoubtedly binds to the positively charged surface of the
liposome, and residual positive charge then presumably mediates binding to
negatively charged sialic acid residues on cell surfaces. The decrease
that was observed in transfection frequencies at high DNA concentrations
(FIG. 4) might be attributed to saturation of the positive charge on the
liposomes.
Felgner et al, Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987) presented
evidence suggesting that liposomes containing DOTMA fuse with the plasma
membrane. Because the plasmid DNA should be located on the outside of the
liposomes, one would anticipate that fusion of the liposome with the
plasma membrane would leave the DNA on the cell surface. An alternative
possibility is that DNA bound to liposomes is taken up by endocytosis and
that some fraction of the DNA is then released into the cytoplasm by an
unknown mechanism. Given the uncertainty of how the DNA enters the
cytoplasm, it is premature to speculate on why three of the cationic
lipids tested worked well on HeLa cells, but not on BHK or other cells
that were tested. One or more of these lipids may well be more effective
than DDAB on some cell types.
The level of production of VSV G protein achieved in BHK cells was
quantitated using DDAB transfection and the vaccinia/T7 system. Using
immunoblots standardized with transfected with pARG synthesized the
equivalent of 3.times.10.sup.6 molecules VSV G protein per cell within six
hours after transfection. The level of translation of mRNA transcribed by
the T7 polymerase in vaccinia infected cells appears limited by the
efficiency with which vaccinia virus enzymes cap the transcripts. The
newly described use of the cap-independent translation initiation signal
from EMC virus in conjunction with the vaccinia/T7 system will undoubtedly
allow even higher levels of expression (O. Elroy-Stein, T. R. Fuerst and
B. Moss, Proc. Natl. Acad. Sci. USA, 86, 6126-6130, (1989)).
An important aspect of the vaccinia/T7 system combined with DDAB mediated
transfection, is that it permits a high efficiency of simultaneous
expression of two or more genes. Using indirect immunofluorescence to
monitor expression of two DNAs transfected simultaneously into HeLa cells,
it was found that all (>95%) of the expressing cells were expressing both
proteins at very similar levels. This feature of the system is very
important to quantitative studies on protein-protein interactions where
similar levels of each protein need to be made in all cells.
Given the high efficiency of DDAB containing liposomes in mediating
cytoplasmic transfection in the vaccinia/T7 system, it is surprising that
liposomes containing DDAB were reported to be ineffective in mediating
transfection into cell nuclei (Felgner et al, Proc. Natl. Acad. Sci. USA,
84, 7413-7417 (1987) and F.L. Felgner and G.M. Reingold, Nature, 337,
387-389 (1989)). It is conceivable that liposomes containing DDAB are
efficient only at delivering DNA into the cytoplasm. Arguing against this
possibility is the fact that transient nuclear expression using DDAB
containing liposomes and an SV40-based vector in monkey COS cells was
observed. Transient nuclear expression of DNA in BHK cells was also
observed. Transfection of DNA with liposomes containing DDAB is clearly
ideal for many applications involving the vaccinia/T7 hybrid system, and
will prove useful in many other applications as well.
It will be appreciated that the instant specification is set forth by way
of illustration and not limitation, and that various modifications and
changes may be made without departing from the spirit and scope of the
present invention.
* * * * *
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