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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to methods of inserting deoxyribonucleic acids and
fragments of deoxyribonucleic acids into living cells, including mammalian
cells.
2. Description of the Prior Art
Prior to our invention, lipid vesicle encapsulation techniques have not
been available for the efficient encapsulation of large macromolecules
such as deoxyribonucleic acid. The deoxyribonucleic acid molecule is
relatively large; i.e.; on the order of about 20 angstroms in thickness
and having lengths of circa 30,000 angstroms. By the method of our
invention, more fully described in our copending patent application Ser.
No. 881,116 filed Feb. 24, 1978, and issued as U.S. Pat. No. 4,235,871,
these large macromolecules are efficiently encapsulated in oligolamellar
lipid vesicles.
In addition, the structure of the prior art lipid vesicles has not been
conducive to utilization of the encapsulated deoxyribonucleic acids.
Cellular uptake of multilamellar vesicles and/or their contents appears to
be less efficient than has been experienced with monolamellar or
oligolamellar structured lipid vesicles.
SUMMARY OF THE INVENTION
The invention comprises a method of inserting deoxyribonucleic acid (DNA)
or fragments thereof into a living cell, which comprises; encapsulating
the acid or fragment in a lipid vesicle and bringing the vesicle in
contact with said cell, whereby insertion occurs. By the method of the
invention, encapsulated DNA or fragments thereof can be inserted into
living cells, both plant and animal through the delivery vehicle of the
lipid vesicle. The advantage is in labor and time saving over present
methods which comprise the well known plasmid and splicing techniques.
Also, the vesicle encapsulated DNA is protected from degradation by
certain enzymes, providing further advantages in the method of the
invention.
The term "living cell" as used herein means a cell of a living organism,
plant or animal, unicellular such as a microorganism like E. coli and like
microorganisms or multicellular including mammals such as humans and the
like.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
Deoxyribonucleic acid (hereinafter referred to at times as "DNA" for
convenience) or fragments thereof may be encapsulated within a lipid
vesicle by the method of our pending patent application, Ser. No. 881,116
filed on Feb. 24, 1978 and issued as U.S. Pat. No. 4,235,871. The method
comprises:
(1) providing a mixture of a vesicle wall forming compound in organic
solvent and an aqueous mixture of the DNA material to be encapsulated, the
ratio of organic phase to aqueous phase being that which will produce an
emulsion of the water-in-oil type;
(2) forming a homogeneous emulsion of said mixture, of the character
produced by ultra-sonic radiation;
(3) evaporating organic solvent from the emulsion, whereby a mixture is
obtained having a gel-like character; and
(4) converting the gel-like mixture to synthetic, oligolamellar vesicles
encapsulating the DNA material.
The term "synthetic, oligolmellar lipid vesicles (liposomes)" as used
herein means man-made lipid vesicles, created in the laboratory and
characterized in part by few or single bimolecular lipid layers forming
the vesicle walls.
The first step in the preparation of encapsulated DNA is to provide a
mixture of a lipid vesicle wall forming composition in organic solvent and
an aqueous mixture of the DNA material to be encapsulated in the vesicle.
Vesicle wall forming compounds are generally well known as are the methods
of their preparation. For example, any number of phospholipids or lipid
compounds may be used to form the vesicle walls. Representative of such
wall forming compounds are; phosphatidylcholine (hereinafter referred to
as "PC"), both naturally occurring and synthetically prepared,
phosphatidic acid (hereinafter referred to as "PA"),
lysophosphatidylcholine, phosphatidylserine (hereinafter referred to as
"PS"), phosphatidylethanolamine (hereinafter referred to as "PE"),
sphingolipids, phosphatidyglycerol (hereinafter referred to as "PG"),
spingomyelin, cardiolipin, glycolipids, gangliosides, cerebrosides and the
like used either singularly or intermixed such as in soybean phospholipids
(Asolectin, Associated Concentrates). In addition, other lipids such as
steroids, cholesterol, aliphatic amines such as long chain aliphatic
amines and carboxylic acids, long chain sulfates and phosphates, dicetyl
phosphate, butylated hydroxytoluene, tocophenol, retinol, and isoprenoid
compounds may be intermixed with the phospholipid components to confer
certain desired and known properties on the formed vesicles. In addition,
synthetic phospholipids containing either altered aliphatic portions such
as hydroxyl groups, branched carbon chains, cycloderivatives, aromatic
derivatives, ethers, amides, polyunsaturated derivatives, halogenated
derivatives or altered hydrophillic portions containing carbohydrate,
glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate,
carboxy, amine, sulfhydryl, imidazole groups and combinations of such
groups can be either substituted or intermixed with the above mentioned
phospholipids and used in the process of the invention. It will be
appreciated from the above that the chemical composition of the lipid
component of the vesicles prepared by the method of the invention may be
varied greatly without appreciable diminution of percentage capture
although the size of the vesicle may be affected by the lipid composition.
A convenient mixture we have used extensively and which is representative
of lipid mixtures advantageously used in the method of the invention is
composed of PS and PC, or PG and PC as identified above (advantageously at
a 1:4 molar ratio in each instance). The PC, PG, PA and PE, may be derived
from purified egg yolk. Saturated synthetic PC and PG, such as dipalmitoyl
may also be used. Other amphipathic lipids that may be used,
advantageously also at 1:4 molar ratios with PC, are gangliosides,
globosides, fatty acids, stearylamine, long chain alcohols, and the like.
The liposome wall forming composition may be initially provided dissolved
in any inert solvent that can be substantially removed from the lipid or
phospholipid compound when desired. Representative of such solvents are a
wide variety of eithers, esters, alcohols, ketones, hydrocarbons (aromatic
and aliphatic including fluorocarbons), and silicones in which an aqueous
phase does not have an appreciable solubility. The solvents may be used
either alone or in admixture. For each solvent or mixture of solvents
however, the optimal ratio of lipid, aqueous space, and solvent is
different and must be determined for each case by trial and error
techniques as will be appreciated by those skilled in the art. The term
"inert solvent" as used herein means a solvent for the lipid or
phospholipid, which will not interfere with or otherwise adversely affect
the desired course of the method of the invention.
The phospholipid or lipid along with any lipid-soluble additives, are
advantageously evaporated from their solvent on to the sides of a suitable
reaction vessel. The organic phase, in which the "reverse phase
evaporation vesicles" of the invention will be formed is then added to the
vessel, i.e., an inert organic solvent for the lipids and phospholipids as
described above. With mixing, dissolution of the lipid component of the
vesicles to be formed, previously deposited on the vessel walls is
obtained. A number of inert organic solvents are preferred for forming the
organic phase according to the method of the invention, depending on the
following conditions of the method employed. For low temperature
conditions, i.e., removal subsequently of the organic phase at relatively
low temperatures, we find diethyl ether most advantageous, although
chloroform, or tetrahydrofuran may also be used advantageously. For higher
temperature processing, isopropyl ether is a preferred inert organic
solvent, particularly for preparing lipid vesicles containing saturated
phospholipids as the lipid component. Following dissolution of the
phospholipid or lipid to form the organic phase, an aqueous phase is added
to obtain a heterogeneous 2-phase mixture. The aqueous phase contains in
dissolution/suspension the DNA materials to be encapsulated in the
synthetic lipid vesicles. Preferably the aqueous phase is buffered to a pH
suitable to maintain stability of the DNA material for encapsulation. The
ionic strength of the aqueous phase has a bearing on the encapsulation
efficiency obtained in the method of the invention. As a general rule, the
higher the ionic strength of the aqueous phase, the lower the percentage
of entrapment. For example, with 15 mM sodium chloride present, one can
encapsulate circa 60 percent of the aqueous phase, while with 500 mM
sodium chloride present, only about 20 percent of the aqueous phase may be
encapsulated. Thus, to maximize the encapsulation of macromolecules, a
buffer of low ionic strength (less than 0.3) is preferably employed. The
encapsulation efficiency is also dependent to some degree on the
concentration of lipid or phospholipid present in the 2-phase system.
Preferably the proportion of lipid or phospholipid component is within the
range of from about 0.5 mg to about 50 mg/ml. of the inert organic
solvent. Preferably the ratio of organic phase to aqueous phase is within
the range of from about 2:1 to about 20:1 v/v, most preferably about 4:1
to form a water-in-oil emulsion.
The heterogeneous 2-phase mixture obtained as described above is then
emulsified to obtain an emulsion of the character produced by ultrasonic
radiation. Preferably this is accomplished with the use of a bath type
sonicator, or for large volume preparations in an industrial size
emulsifier. Generally, the 2-phase mixture is sonicated for about 3 to 5
minutes, or until either a clear 1-phase mixture or a homogeneous emulsion
forms. This is achieved by simply placing the container vessel in the
sonicating bath at an optimal level. Emulsification may be carried out
over a wide range of temperatures, i.e., from about 10.degree. to about
50.degree. C., advantageously at a temperature of from
0.degree.-20.degree. C. The optimum conditions under which emulsification
is carried out depends upon the solvent, phospholipid, and volume of
aqueous phase used in the preparation. It will be appreciated that trial
and error techniques may be used to determine the optimum conditions for
emulsification. The emulsion mixture is then treated to remove a
substantial portion of the inert organic solvent. This may be carried out
conveniently by use of a rotary evaporator, at a temperature of circa
20.degree. C. to 60.degree. C. and under a reduced pressure, i.e.; under
vacuum (10 mm to 50 mm Hg). The temperature employed for evaporation of
the organic solvent from the emulsion depends on the boiling point of the
particular organic solvent used in the emulsion and the stability of the
DNA material being encapsulated. During evaporation, the emulsion first
becomes a viscous gel, which is an intermediate product. The gel is stable
and can be stored in this state for short periods of time, up to a week
(at least), at 4.degree. C. under an inert atmosphere such as nitrogen
gas. A small amount of water or buffer can then be added to the gel and
the resulting mixture evaporated for an additional period (circa 15
minutes) to help remove residual traces of the gel into a
homogeneous-appearing, suspension to oligolamellar lipid vesicles. The gel
may be converted by agitation or by dispersion in a aqueous media such as
a buffer solution. The vesicles obtained range in diameter from 2,000 to
4,000 Angstroms (average). A significant proportion of the DNA materials
for encapsulation contained in the aqueous buffer is captured within the
lipid vesicles (up to circa 60 percent, depending on the amount of lipid,
volume of the aqueous phase, ratio of the organic phase to aqueous phase
to lipid, type of inert organic solvent(s) and, type of lipid(s) used in
the process). The non-incorporated aqueous material may be removed of
necessary by appropriate and known techniques such as by repeated
centrifugations, column chromatography, ion exchange chromatography,
dialysis and like procedures. The lipid vesicles with their encapsulated
DNA contents can then be suspended in any isotonic buffer for use. The
vesicles may be sterilized by passage through a 0.4 micron filter
(nucleopore) when sterility is desired.
Advantageously the method of preparing the DNA containing vesicle is
carried out under an inert atmosphere. The term "inert atmosphere" as used
herein means a non-oxidizing atmosphere such as an atmosphere of nitrogen
gas, argon and like inert gases.
The encapsulated DNA material is inserted into the living cell by contact
of the encapsulating lipid vesicle with the cell plasma membrane. In the
case of cells associated with multi-cellular organisms, contact may be
in-vivo or in-vitro. The encapsulated DNA materials are passed into the
cell (inserted) when the encapsulating lipid vesicle contacts the cell and
is taken up by the living cell through fusion of the lipid vesicle with
the cellular plasma membrane or by endocytosis. There is evidence that
vesicle uptake may be by both mechanisms. In any event the DNA material is
taken up by the living cell and the encapsulated DNA material incorporated
within the living cell.
Generally, the rate of uptake by the living cell is influenced by a number
of factors including ambient temperature, lipid concentration and the
charge on the lipid vesicle surface. A positive charge on the surface of
the lipid vesicle enhances uptake. The rate of uptake is directly
proportional to the ambient temperature and increases with temperature.
Generally, cellular uptake is satisfactory within a temperature range of
from about 0.degree. to 40.degree. C. Optimum temperature may be
determined by trial and error techniques. Of course, selection of a given
temperature will also depend upon whether the insertion of DNA material is
being made in-vivo or in-vitro to living cells.
Techniques of bringing the lipid vesicles into contact with the living
cells are conventional and include previously known techniques. For
example if the contact is to be effected in-vitro, a simple admixture of
cells and lipid vesicles is brought about. In-vivo, the lipid vesicles may
be injected intravenously into the host organism, in a pharmaceutically
acceptable carrier such as a saline suspension of appropriate pH.
The following examples describe the manner and process of making and using
the invention and represent the best mode contemplated by the inventors,
but are not to be construed as limiting.
EXAMPLE 1
Encapsulation of Mammalian Chromosomes in Phospholipid Vesicles
Chinese hamster chromosomes are prepared by the method of Matsui et al J.
Cell Biol. 75, 121a, (1977) in a solution containing 10 mM
tris(hydroxymethyl)aminomethane hydrochloride (pH 7.5), 2.5 m MgCL.sub.2,
2.0 mM CaCl.sub.2, and 0.5 mM phenylmethylsulfonyl fluoride (buffer T).
A suspension of the chromosomes in 60 .mu.l. of buffer T is added to 1 ml.
diethylether containing 2.mu. moles of total lipid composed of
phosphatidylglycerol/phosphatidylcholine/cholesterol/N-4-nitro-benz-2-oxa-
1, 3-diazole phosphatidylethanolamine (NBD-PE; mole ratios 10/40/50/5) and
sonicated in a bath-type sonicator under nitrogen for 3 minutes at
0.degree.-5.degree. C. The diethyl ether is removed on a rotary evaporator
under reduced pressure and the resulting suspension is removed from the
flask with 1.0 ml buffer T. The chromosomes are stained with 5 .mu.l of an
ethidium bromide solution and the vesicle-chromosome suspension is
examined under a fluorescent microscope (excitation 460 nm, emission 520
nm). The bilayer of the vesicles appears intense yellow due to the
inclusion of the fluorescent phospholipid NBD-PE. The chromosomes appear
as intense red structures. The color difference between the two
fluorescent probes conveniently allows one to determine whether or not a
chromosome has a lipid bilayer surrounding it.
EXAMPLE 2
Transfer of an Encapsulated Chromosome
Chromosomes extracted from HELA cells by the technique of S. Matsui et al,
supra., and encapsulated as detailed in Example 1 supra., are used to
transform mouse cells A9 (obtained from Dr. T. Shows, Roswell Park
Memorial Institute, Buffalo, New York) that are lacking the enzyme
hypoxanthine quanine phosphoribosyltransferase (HGPRT). A vesicle
suspension containing 100 nmoles of the encapsulated chromosomes is mixed
with 1.times.10.sup.6 A9 mouse cells in 1 ml Dulbecco's minimal essential
medium without serum and incubated at 37.degree. C. for 4 hours. At the
end of this time the mouse cells are exposed to
hypoxanthine/aminopterin/thymidine (HAT) medium and the clonal growth
monitored. Assay shows the presence of HGPRT, indicating that encapsulated
chromosomes are effective for transferring genetic information to a cell
lacking the necessary information to form the HGPRT enzyme.
EXAMPLE 3
Encapsulation of a Plasmid and its Transfer to a Bacterium
The plasmid pBR 322 which carries tetracycline resistance is encapsulated
in phosphatidyl glycerol vesicles as follows. 1 .mu.g of pBR 322 in 0.1 ml
of Dulbecco's phosphate buffered saline (PBS) is added to 0.3 ml diethyl
ether containing 10.mu. moles of phosphatidylglycerol. The
plasmid-containing lipid vesicles are prepared following the procedure in
Example 1 supra. The vesicle preparation is then treated with 10 .mu.l of
a solution containing 5 .mu.g/ml DNase to degrade unencapsulated DNA. The
vesicles with encapsulated DNA are added to tetracycline sensitive E. coli
RRI after they had been prepared by the method of Cohan et al., Proc.
Natl. Acad. Sci., USA, 69, 2110, (1972). The transformed E. coli are
selected on a tetracycline containing agar medium and observed to be
resistant to tetracycline. The use of lipid vesicle encapsulated DNA leads
to a substantial increase in the efficiency of plasmid transfer to the E.
coli when compared to unencapsulated plasmid DNA uptake by the bacteria.
EXAMPLE 4
Encapsulation of Mouse DNA and its Transfer to Mouse Liver in vivo
Unfractionated mouse DNA isolated by the procedure of Maniatis et al., Cell
15, 681-701, (1978) from C57 BL/6J mice (black 6) is encapsulated in lipid
vesicles composed of lactosyl
cerebroside/phosphatidylserine/phosphatidylcholine/.alpha.tocopherol/chole
sterol (mole ratio 1/1/4/0.1/4) as follows. 2 mg of black 6 DNA in 1 ml of
buffer T is added to 3 ml of diethylether containing 30.mu. moles of the
lipid. The encapsulation is carried out by the procedure described in
Example 1, supra. Unencapsulated DNA is separated from the encapsulated
DNA by centrifugation at 10,000.times.G for 10 minutes. The pellet
containing the encapsulated DNA is resuspended and the separation
procedure is then repeated two times. The encapsulated DNA is resuspended
in PBS at a lipid concentration of 30.mu. moles per ml. Six female, 21-28
day old, balb/cB.sub.Y J (Balb-c) mice are injected in the tail vein with
0.2 ml of the liposome encapsulated DNA. The animals are placed in a
metabolic cage with free access to water and animal chow and the total
urine excreted in twenty-four hours is collected for 3 consecutive 24 hour
periods. The successful transfer of a mouse gene is observed by analyzing
the mouse urine for the appearance of mouse urinary protein 2 (mup 2).
Balb-c mice lack the gene for synthesizing this protein, while Black 6
mice contain it (Szoka and Paigen, Genetics 90; 591-612, 1978). The
appearance of this protein in urine from Balb-c mice which had received
the liposome encapsulated DNA is quantitated by acrylamide electrophoresis
following the method of Szoka and Paigen, Genetics 90; 597-612 (1978). The
inclusion of lactosyl cerebroside in the lipid vesicle is advantageous to
obtain high levels of gene transfer to the Balb-c mice. The technique of
this example may also be used to transfer specific cloned DNA sequences to
other species in vivo.
EXAMPLE 5
Encapsulation of a Large Plasmid and its Transfer to a Plant Cell
Protoplast
The plant tumor plasmid Ti (Mol. weight >100.times.10.sup.6) isolated from
Agrobacterium tumifaciens is encapsulated in liposomes as follows: 33
.mu.g DNA (Ti) in 0.33 ml of sorbitol buffer is added to 1.0 ml diethyl
ether containing 10.mu. moles phosphatidylserine and 10.mu. moles
cholesterol. The liposomes are prepared by the general procedure set forth
in Example 1, supra. The non-encapsulated DNA is either degraded by DNase
or separated on Ficoll density gradients by centrifugation. The amount of
DNA encapsulated in liposomes is found to be 14.9 .mu.g or 45% of the
total amount added.
The liposomes containing the Ti plasmid are subsequently incubated with
Nicotiana Tabacum (tobacco) protoplasts. The growth characteristics of the
plant cells on hormone-free solid media indicate that liposome
encapsulated Ti plasmid is at least 50-100 times more efficient than free
Ti DNA in enhancing the ability of these cells to sustain non-self
limiting growth on the hormone free media.
These results indicate that liposome-encapsulated DNA is extremely useful
in plant cell genetics by enhancing the incorporation and expression of
foreign DNA. The large enhancement could be explained by increased
protection of the encapsulated DNA from degradative enzymes and/or
enhanced delivery into the cellular interior. The method of encapsulation
is found to be very efficient (45%) even for such large macromolecules as
the Ti plasmid (M.W. >100.times.10.sup.6).
The Ti plasmid is carried by virulent strains of Agrobacterium tumifaciens
which is known to induce a neoplastic disease (Crown Gall) of
dicotyledenous plants. During transformation, a portion of the Ti plasmid
is transferred and incorporated in the recipient cells. Transformation
confers the ability to synthesize the novel arginine derivatives nopaline
and octopine to the recipient cells, as well as permitting non-self
limiting growth on hormone-free solid media. Since the Ti plasmid is a
natural vector which can promote the transfer, integration, and expression
of foreign DNAs into plant cells, it is an extremely useful vehicle for
introducing other genes into plant cells. Free Ti plasmid (without
encapsulation into liposomes) can transform tobacco protoplasts only with
a very low frequency (1.times.10.sup.-5) probably due to enzymatic
degradation. The method for Ti encapsulation in liposomes therefore
provides a powerful and unique new tool for enhancing the cellular
incorporation of such large DNA molecules.
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