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FIELD OF THE INVENTION
Compositions of cationic lipids and viral components useful for
transfecting eukaryotic cells with nucleic acids and for introduction of
other macromolecules into such cells are disclosed. Also disclosed are
methods of transfecting eukaryotic cells employing such compositions.
BACKGROUND OF THE INVENTION
Lipid aggregates such as liposomes can function to facilitate introduction
of macromolecules, such as DNA, RNA, and proteins, into living cells.
Recently, it has been shown that lipid aggregates comprising cationic
lipid components can be especially effective for delivery and introduction
of large anionic molecules, such as nucleic acids, into certain types of
cells. See Felgner, P. L. and Ringold, G. M. (1989) Nature 337:387-388.
Since the membranes of most cells have a net negative charge, anionic
molecules, particularly those of high molecular weight, are not readily
taken up by cells. Cationic lipids aggregate to and bind polyanions, such
as nucleic acids, tending to neutralize the negative charge. The
effectiveness of cationic lipids in transfection of nucleic acids into
cells is thought to result from an enhanced affinity of cationic
lipid-nucleic acid aggregates for cells.
A variety of types of lipid aggregates are known, including liposomes,
unilamellar vesicles, multilamellar vesicles, micelles and the like,
having particle sizes in the nanometer to micrometer range. As is
well-known in the art, the structures of lipid aggregates depend on the
lipid composition and the method employed to form the aggregate. Cationic
lipids can be used alone or in combination with non-cationic lipids, for
example with neutral phospholipids like phosphotidylethanolamines, to form
positively charged vesicles and other lipid aggregates which are able to
bind nucleic acids. The positively charged lipid aggregates bind to
nucleic acids, can then be taken up by target cells and thus facilitate
transfection of the target cells with the nucleic acid. (See, Felgner, P.
L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417: Epstein, D. et
al. U.S. Pat. No. 4,897,355.)
Cationic lipids are not universally effective for transfection of all cell
types. Effectiveness of transfection of different cells depends on the
cationic lipid composition used and the type of lipid aggregate formed. In
addition, a particular cationic lipid may be more or less toxic to a given
cell line, limiting the type or concentration of lipid that can be
employed for transfection. Certain types of higher eukaryotic cells are
not readily transfected employing presently available cationic lipids.
These hard to transfect cells generally include suspension cell lines and
primary human cell lines and more specifically include fibroblasts and
macrophage cell lines. Compositions and methods which would generally
enhance the efficiency of cationic lipid-mediated transfection and/or
broaden the range of cell types that can be efficiently transfected with
cationic lipid-DNA complexes would represent valuable improvements in the
art.
Many biological materials are taken up by cells by receptor-mediated
endocytosis. See: Pastan and Willingham (1981) Science 214:504-509. This
mechanism involves binding of a ligand to a cell-surface receptor,
clustering of ligand-bound receptors, and formation of coated pits
followed by internalization of the ligands into endosomes. Both enveloped
viruses, like influenza virus and alphaviruses, and non-enveloped viruses,
like adenovirus, infect cells via endocytotic mechanisms. See: Pastan, I.
et al. (1986) in Virus Attachment and Entry into Cells, (Crowell, R. L.
and Lonberg-Holm, K., eds.) Am. Soc. Microbiology, Washington, p. 141-146;
Kielian, M. and Helenius, A. (1986) "Entry of Alphaviruses" in The
Togaviridae and Flaviviridae, (Schlesinger, S. and Schlesinger, M. J.,
eds.) Plenum Press, New York p. 91-119; FitzGerald, D. J. P. et al. (1983)
Cell 32:607-617. Receptor-mediated endocytosis has been exploited to
deliver DNA into cells. Wu, G. Y. and Wu, C. H. (1987) J. Biol. Chem.
262:4429-4432; Wagner, E. et al. (1990) Proc. Natl. Acad. Sci. USA
87:3410-3414. These methods employ bifunctional conjugates having a
ligand, which binds to a specific cell-surface receptor, covalently linked
to a DNA-binding domain. Asialoglycoprotein-polylysine conjugates and
human transferrin-polylysine conjugates have, for example, been
demonstrated to mediate DNA entry into certain eukaryotic cells. (Wagner,
E. et al., 1990, supra).
Curiel, D. T. et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850-8854 and
Cotton, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6094-6098 have
recently reported that receptor-mediated transfection via
transferrin-polylysine/DNA complexes is enhanced by simultaneously
exposing the cells to defective adenovirus particles. These authors report
that adenovirus particles function to disrupt endosomes containing the
viral particle and the DNA complex. Replication-defective adenovirus
particles and psoralen inactivated adenovirus were reported to enhance
transfection. Adenovirus enhancement of transfection is limited, however,
to cells which have both a ligand receptor, i.e. transferrin receptor, and
an adenovirus receptor. Direct coupling of polylysine/DNA complexes to
adenoviruses has also been employed for transfection. Curiel, D. T. et al.
(1992) Hum. Gene Therapy 3:147-154; Wagner, E. et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6099-6103. In related work, Wagner, E. et al. (1992)
Proc. Natl. Acad. Sci. USA 89:7934-7938, report augmentation of
transfection in several cell lines when hemagglutinin HA-2 N-terminal
fusogenic peptides from influenza virus are included in
transferrin-polylysine-DNA complexes. The use of influenza peptide
conjugates was, however, reported to be less effective for enhancement of
transfection than defective adenovirus.
PCT patent applications WO 93/07283 and WO 93/07282, both published Apr.
15, 1993, relate to transfection of higher eukaryotic cells via
ligand/polylysine/DNA complexes and endosomolytic agents, such as
adenovirus and HA-2 fusogenic peptides.
Alphaviruses, mosquito-transmitted members of the family Togaviridae, are
RNA-containing enveloped viruses (also called membrane viruses).
Alphaviruses include, among others, Sindbis and Semliki Forest (SFV)
viruses, several equine encephalitis viruses (Eastern (EEE), Western (WEE)
and Venezuelan (VEE)), Chikungunya virus and Ross River virus. Sindbis and
Semliki Forest viruses are the least virulent and best characterized
alphaviruses. See generally: Schlesinger, S. and Schlesinger, M.J., eds.
(1986) The Togaviridae and Flaviviridae, Plenum Press. Alphaviruses in
general, and specifically SFV and Sindbis virus, have very broad host
ranges. SFV infects a wide variety of cultured cells including mammalian
(human, monkey, hamster, mouse, porcine), avian, reptilian, amphibian and
insect cell lines. Liljestrom P. and Garoff, H. (1991) Biotechnology
9:1356-1361 and references cited therein. Animal cell expression vectors
have been based on SFV (Liljestrom and Garoff (1991), supra) and Sindbis
virus (Xiong, C. et al. (1989) Science 243:1188-1191). The entry of
alphaviruses into cells has been studied using SFV as a model. Kielian and
Helenius (1986) supra. As with other viruses, SFV binds to the cell
membrane, and is internalized in coated vesicles. In contrast to
non-enveloped viruses, SFV (and other enveloped viruses) is released into
the cell cytoplasm by fusion of the viral envelope with the endosome
membrane. Acidic pH triggers the fusion process. The fusion process in SFV
is characterized as rapid, non-leaky and strictly dependent, both in in
vitro fusion with liposomes and in vivo infection, on the presence of a
3.beta.-OH sterol, such as cholesterol, in the membrane to which the virus
fuses. Kielian, M. and Helenius, A. (1985) J. Cell Biol. 101:2284-2291;
Kielian, M. and Helenius, A. (1984) J. Virol. 52:281-283; White, J. and
Helenius, A. (1980) Proc. Natl. Acad. Sci. USA 77:3273-3277; Phalen, T.
and Kielian, M. (1991) J. Cell. Biol. 112:615-623. Although the detailed
mechanism of fusion in alphaviruses (SFV and Sindbis virus) is not
completely understood, alphavirus fusion is reported to be distinct from
that of influenza virus. Kielian and Helenius (1985) supra; Wahlberg J. M.
et al. (1992) J. Virol. 66:7309-7318. As noted above, fusion of the
influenza virus is associated with influenza hemagglutinin (HA). An acidic
pH-induced conformational change in HA exposes a hydrophobic domain,
containing N-terminal sequences of the HA-2 subunits, which is thought to
bind to the target membrane facilitating fusion. SFV spike glycoprotein is
distinct in size, structure and amino acid sequence from HA and does not
have a hydrophobic domain linked to fusion as does HA.
In addition to the togaviruses (e.g., alphaviruses) and orthomyxoviruses
(influenza), enveloped viruses include the following major families of
animal viruses: Herpesviridae, Bunyaviridae, Paramyxoviridae,
Rhabdoviridae, Retroviridae, Arenaviridae, Coronaviridae and some members
of Iridoviridae. Although all enveloped viruses are released into the cell
cytoplasm by fusion of the viral envelope with the outer cell membrane,
the specific fusagenic component and thus mechanism of fusion may vary.
For example, vesicular stomatitis virus (VSV), a rhabdovirus, infects host
cells via adsorptive endocytosis. See, e.g., Dahlberg, J. E. (1974)
Virology 58:250-262; Dickson, R. B. et al. (1981) J. Cell Biol. 89:29-34;
Fan, D. and Sefton, B. (1978) Cell 15:985-992; and Matlin, K. S. et al.
(1982) J. Mol. Biol. 156:609-631. VSV fusion is thought to involve
interaction between the VSV glycoprotein (G protein) and specific membrane
lipids (Schlegel, R. et al. (1983) Cell 32:639-646). The VSV G protein
reportedly binds preferentially to "saturable receptors" such as acidic
phospholipid phosphatidylserine (Schlegel, R. and Wade, M. (1985) J.
Virol. 53(1):319-323. Unlike the fusion process for SFV, VSV fusion does
not require the presence of a 3.beta.-OH sterol. See: Young, J. D. E. et
al. (1983) Virology 128:186-194, and Phalen, T. and Kielian, M. (1991)
supra.
The present invention is based on the discovery that components of
enveloped viruses can significantly enhance the efficiency of cationic
lipid-mediated transfection of eukaryotic cells. Unlike prior art methods,
the enhanced transfection methods of this invention do not require
encapsulation of the nucleic acid within anionic phospholipid-based
liposomes. The present invention thus eliminates the need to construct
liposomes for each particular nucleic acid, an inconvenient and often
difficult procedure. Moreover, the enhanced transfection methods of this
invention do not require conjugation of a polycation to a ligand, nor to
the virus itself. The methods of this invention are applicable to a wider
range of cell-types than prior art methods. There is no requirement for
specific ligand receptors in target cell lines. Furthermore, since the
alphaviruses, at least SFV, require only cholesterol or closely related
sterols in target cells, the range of cells to which the methods of this
invention can be applied is much broader than prior art methods. In
addition, the methods of this invention can be combined with techniques
well-known in the art for introducing cholesterol into cell membranes or
enhancing the level of cholesterol in cell membranes to further enhance
transfection efficiency or further broaden the range of cell types to
which these methods are applicable.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for transfecting
eukaryotic cells, particularly higher eukaryotic cells, with nucleic
acids. Nucleic acids, both DNA and RNA, are introduced into cells such
that they retain their biological function. A composition for transfecting
eukaryotic cells comprising a cationic lipid and an enveloped virus or a
component of an enveloped virus is provided. Transfecting compositions
comprise cationic lipid which functions, alone or optionally in
combination with non-cationic lipid, to form cationic lipid aggregates
which complex nucleic acids. Cationic lipid aggregates, including
liposomes, vesicles and micelles, facilitate introduction of anionic
macromolecules, like nucleic acids, through cell membranes which are
typically negatively charged.
Transfecting compositions also comprise an active or inactive enveloped
virus capable of entry into the eukaryotic cell to be transfected or a
viral component of an enveloped virus that functions to facilitate entry
of cationic lipid aggregates into that cell. Viral components of enveloped
viruses useful in transfection compositions include viral proteins,
particular viral spike glycoproteins, multimers (i.e, dimers and trimers)
thereof, viral peptides of viral spike glycoproteins, and viral envelope
fragments containing embedded viral protein. Because of their very broad
host range, alphaviruses are preferred for transfection. Preferred
alphaviruses are Semliki Forest virus and Sindbis virus. Transfecting
compositions comprising viral components of influenza virus or vesicular
stomatitis virus are also preferred.
Inclusion of an enveloped virus in a transfection composition with cationic
lipid aggregates complexed with nucleic acids significantly enhances
transfection (2-fold or more) compared to transfection mediated by the
cationic lipid alone. Enhancement of transfection by enveloped viruses,
such as togaviruses, rhabdoviruses and orthomyxoviruses, is pronounced in
cell lines, including suspension cell lines, animal primary cell lines,
and human primary cell lines, that have been found to be hard to transfect
employing prior art cationic lipid-mediated transfection methods.
Enhancement of transfection by enveloped viruses occurs in any cell which
the virus can enter and infect. Enhancement of transfection with
alphaviruses, particularly SFV, occurs in cells which comprise cholesterol
or another 3.beta.OH-sterol in their cell membrane. Enhancement of
transfection by vesicular stomatitis virus occurs with a wide range of
cells, particularly cells which contain "saturable receptors" such as
acidic phospholipid phosphatidylserine.
Monovalent or preferably polyvalent cationic lipids are employed in
transfecting compositions. Preferred polyvalent cationic lipids are
lipospermines, specifically DOSPA
(2,3-droleylocy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini
um trifluoroacetate). Cationic lipids are optionally combined with
non-cationic lipids, particularly neutral lipids, for example lipids such
as DOPE (dioleoylphosphatidylethanolamine). A cationic lipid composition
composed of a 3:1 (w/w) mixture of DOSPA and DOPE is generally useful in
transfecting compositions of this invention. Cationic lipid aggregates can
also include 3.beta.OH-sterols, particularly cholesterol. Cationic lipid
aggregates that contain 3.beta.OH sterol are particularly useful in
combination with alphaviruses. Transfection compositions also optionally
contain agents which inhibit lysosomal enzymes or enhance release of
material from endosomes, such as chloroquine. Preferred transfection
compositions are those which induce substantial transfection of a higher
eukaryotic cell line. Substantial transfection is the introduction of
functional nucleic acid in 50% or more of the cells in a transfection
sample.
The methods of the present invention involve contacting a eukaryotic cell
with a transfecting composition containing a cationic lipid, an active or
inactive envelope virus or a viral component of such a virus, and a
nucleic acid. A preferred method employs an active or inactive alphavirus
or a viral component of an alphavirus. Methods employing alphavirus,
particularly Semliki Forest virus, are widely applicable to transfection
of eukaryotic cell types having cell membranes that contain cholesterol or
other 3.beta.OH-sterols. Methods employing rhabdoviruses, particularly
vesicular stomatitis virus, are widely applicable to transfection of
eukaryotic cell types having cell membranes that contain saturable
"receptors" such as phosphatidylserine. Methods of this invention are
applicable to transfection of adherent or suspension cell lines, in
general to animal cell lines, specifically to mammalian, avian, reptilian,
amphibian and insect cell lines and more specifically to animal primary
cell lines, human primary cell lines, stem cell lines, and fibroblasts.
These methods generally enhance transfection of hard to transfect cell
lines.
The lipids, viruses, and nucleic acid of transfecting compositions can be
combined in a variety of ways prior to contact with cells.
In one alternative transfection method, cationic lipid aggregates are
formed and complexed with nucleic acid. Virus or viral component is then
added to the nucleic acid complexes and the resulting mixture is employed
to transfect cells.
In a second alternative transfection method, cationic lipid aggregates are
formed and viral components are incorporated into the cationic lipid
aggregate, the resulting aggregate is complexed with nucleic acid and the
resulting nucleic acid complexes are employed to transfect cells. Viral
components can be incorporated into the lipid aggregate, for example by
application of a freeze-thaw cycle to a mixture containing lipid and
virus.
In a third alternative transfection method, cationic lipid aggregates are
complexed with nucleic acid, viral components are incorporated into the
nucleic acid/cationic lipid complexes and the resulting complexes are
employed to transfect cells.
In each of these alternative methods, cationic lipid aggregates can contain
non-cationic lipids and 3.beta.-OH sterols, like cholesterol. In each of
these alternative methods, agents which inhibit lysosomal enzymes or
enhance release of material from endosomes, such as chloroquine can be
added to the transfecting composition.
Transfection methods of this invention employing alphavirus can be further
improved by adding a step introducing a 3.beta.OH-sterol, preferably
cholesterol, into the cell membrane of the target cell to be transfected.
The 3.beta.OH sterol can be introduced into a target cell that contains no
3.beta.OH sterol, making the 3.beta.OH sterol-treated cell susceptible to
alphavirus entry. Alternatively, the level of 3.beta.OH sterol in a target
cell can be increased to enhance transfection of nucleic acids into the
cell in the presence of alphavirus and cationic lipid. Semliki Forest
virus are particularly useful in such methods. Cholesterol and other
3.beta.OH-sterols can be introduced into cell membranes prior to or
simultaneous with the step of contacting the cell with the transfecting
composition. Cells can be pretreated, for example, by growth on medium
containing high levels of cholesterol or other 3.beta.OH-sterol.
Alternatively, liposome fusion or exchange techniques can be employed to
introduce desired 3.beta.OH-sterols. Cholesterol or another
3.beta.OH-sterol can also be incorporated into the cationic lipid
aggregates in the transfecting composition. Transfection of eukaryotic
cells is enhanced when 3.beta.OH sterol-containing cationic lipid
aggregates are combined with alphaviruses and nucleic acids and used to
transfect those cells.
The transfection methods of the present invention can be applied to in
vitro and in vivo transfection of eukaryotic cells, particularly to
transfection of higher eukaryotic cells including animal cells. The
methods of this invention can be used to generate transfected cells which
express useful gene products. The methods of this invention can also be
employed as a step in the production of transgenic animals. The methods of
this invention are useful as a step in any therapeutic method requiring
introducing of nucleic acids into cells. In particular, these methods are
useful in cancer treatment, in in vivo and ex vivo gene therapy, and in
diagnostic methods. The transfection compositions of this invention can be
employed as research reagents in any transfection of eukaryotic cells done
for research purposes. Nucleic acids that can be transfected by the
methods of this invention include DNA and RNA from any source comprising
natural bases or non-natural bases, and include those encoding and capable
of expressing therapeutic or otherwise useful proteins in cell, those
which inhibit undesired expression of nucleic acids in cell, those which
inhibit undesired enzymatic activity or activate desired enzymes, those
which catalyze reactions (Ribozymes), and those which function in
diagnostic assays.
The compositions and methods provided herein can also be readily adapted in
view of the disclosure herein to introduce biologically-active anionic
macromolecules other than nucleic acids including, among others,
polyamines, polyamine acids, polypeptides, proteins, biotin, and
polysaccharides into eukaryotic cells. Other materials useful, for example
as therapeutic agents, diagnostic materials, research reagents, which can
be complexed by the cationic lipid aggregates and introduced into
eukaryotic cells by the methods of this invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides improved methods for transfecting eukaryotic
cells with nucleic acid employing cationic lipids. The improvement relates
to the use of an enveloped virus or a component of an enveloped virus to
either enhance the efficiency of transfection or to broaden the range of
types of cells that can be transfected. This invention has significant
advantages over prior art methods of transfection which employ viruses.
There is no limit on the size or composition of nucleic acid that can be
transfected and there is no requirement for chemical modification of the
nucleic acid. The method does not require the use of a ligand that binds
to cell-surface receptors and nucleic acid-complexing agents need not be
chemically linked to such ligands nor to viral particles. The methods are
useful for transfection of a wider range of cell types than prior art
methods. Methods of this invention employing alphaviruses, especially
Semliki Forest virus, are applicable to a wide range of higher eukaryotic
cells, both vertebrate and invertebrate, including but not limited to
mammalian, avian, reptilian, amphibian and insect cells. The range of cell
types that can be transfected by the methods of this invention can be
further expanded by introducing a 3.beta.OH-sterol, particularly
cholesterol, into the cell membrane of a cell to be transfected.
The following definitions are employed in the specification and claims.
Lipid aggregate is a generic term that includes liposomes of all types both
unilamellar and multilamellar as well as vesicles, micelles and more
amorphous aggregates. A cationic lipid aggregate is a lipid aggregate
comprising sufficient cationic lipid, optionally in combination with
non-cationic lipids, such that the lipid aggregate has a net positive
charge. Cationic lipids and lipid aggregates of this invention are capable
of complexing with nucleic acids.
The term transfection is used herein generally to mean the delivery and
introduction of biologically functional nucleic acid into a cell, i.e. a
eukaryotic cell, in such a way that the nucleic acid retains its function
within the cell. The term transfection includes the more specific meaning
of delivery and introduction of expressible nucleic acid into a cell such
that the cell is rendered capable of expressing that nucleic acid. The
term expression means any manifestation of the functional presence of the
nucleic acid within a cell, including both transient expression and stable
expression. Nucleic acids include both DNA and RNA without size limits
from any source comprising natural and non-natural bases. Nucleic acids
can have a variety of biological functions. They may encode proteins,
comprise regulatory regions, function as inhibitors of gene or RNA
expression (e.g., antisense DNA or RNA), function as inhibitors of
proteins, function to inhibit cell growth or kill cells, catalyze
reactions or function in a diagnostic or other analytical assay.
The transfection methods of this invention employing cationic lipids in
combination with alphaviruses can display significant enhancement of
transfection (2-fold or more) over transfection methods employing
comparable cationic lipids alone. Preferred transfection compositions and
methods of this invention are those that result in transfection of 50% or
more of the cells in a cell sample contacted with the transfection
composition.
Transfection activity or efficiency is measured by detecting the presence
of the transfected nucleic acid in a cell. This is often assessed by
measuring the biological function of the nucleic acid in the cell, and
most often assessed by measuring the level of transient or stable
expression of a reporter gene comprised in the transfected nucleic acid.
Reporter gene expression depends among other things on the amount of
nucleic acid transfected as well as promoter function in the cell.
Transfection activity can also be assessed by determining the percent of
cells in a sample that have been transfected, for example, by assessing
reporter gene expression using cell counting methods.
The methods of the present invention are particularly useful for
transfection of cells that have been found to be hard to transfect
employing prior art cationic lipid methods, for example the use of
cationic lipid reagents like "LIPOFECTAMINE". The term "hard to transfect"
refers to those eukaryotic cell lines in which, under transfection assay
conditions as described in Example 3, less than about 10% of the cells in
a sample are transfected employing the cationic lipid reagent
"LIPOFECTAMINE". Hard to transfect cells include animal primary cell lines
including human fibroblasts, animal embryo stem line cells, keratinocytes
and macrophage. Other hard to transfect cell lines can be identified
employing assay conditions as described in Example 3.
The method of this invention involves contacting a eukaryotic cell with a
transfection composition comprising a cationic lipid, a virus or viral
component and a nucleic acid. The transfection composition optionally
comprises a non-cationic lipid, preferably a neutral lipid. The virus or
viral component is an enveloped virus or a component thereof and is
preferably an alphavirus, an influenza virus or a vesicular stomatitis
virus or a component thereof. Enhanced transfection methods of this
invention have been demonstrated with the prototype alphavirus Semliki
Forest virus and the prototype fusagenic peptides from influenza (E5
amphiphilic peptide) and vesicular stomatitis virus (G protein).
The cationic lipid, alone or in combination with a non-cationic lipid,
forms a cationic lipid aggregate which can bind nucleic acid. A cationic
lipid aggregate may form spontaneously in an appropriate medium or various
well-known techniques may be employed to produce a desired type of lipid
aggregate. In particular, well-known techniques may be applied to form
cationic liposome or vesicles. The relative amounts of cationic lipid and
non-cationic lipid employed to form the lipid aggregate depends on the
type of aggregate desired, the toxicity of the lipids to the cell and the
environment (e.g. medium) in which the aggregate is to be employed. The
kinds and amounts of lipids employed are typically balanced to minimize
cell toxicity and maximize transfection efficiency. The cationic lipid
aggregate complexes the nucleic acid that is to be transfected into cells.
Nucleic acid complexes can be formed by combining nucleic acids with lipid
prior to aggregate formation or by adding nucleic acid to already-formed
cationic lipid aggregates. Nucleic acids can, for example, be complexed to
the outer surface of cationic liposomes or vesicles. Alternatively,
nucleic acids can be included within liposomes or vesicles. Transfecting
compositions of this invention may contain a mixture of structurally
distinct cationic lipid aggregates.
Alternative methods of combining cationic lipid, neutral lipid, virus or
viral component and nucleic acid can be used in the methods of this
invention. For example, transfecting compositions include, but are not
limited to, those in which:
cationic lipid aggregates are formed, complexed with nucleic acid and the
resulting complexes are combined with virus or viral components;
cationic lipid aggregates such as liposomes are formed, the aggregates are
fused with virus, the resulting virus-fused aggregates are complexed with
nucleic acids;
cationic lipid, neutral lipid and virus or viral components are combined
and subjected to treatments to form lipid aggregates and the resulting
aggregates are complexed with nucleic acid; or
cationic lipid, neutral lipid virus or viral component and nucleic acid are
combined and subjected to treatments to form lipid aggregates.
Transfection compositions contain active or inactive enveloped virus or
components thereof. The virus can include wild-type or mutant virus, so
long as the mutant virus remains capable of entering a cell. Wild-type
virus is generally not preferred due to safety concerns. The use of
replication-deficient virus and virus inactivated by various methods is
preferred. The terms inactive virus is used herein to refer to a virus
which, after exposure to certain chemical or physical conditions, is no
longer capable of expressing its viral RNA. Inactivation of SFV is
assessed herein by exposing viral particles whose RNA contains a reporter
gene under the control of the viral subgenomic promoter to potentially
inactivating conditions. Cells (BHK-21 cells) infected with the exposed
viral particles are then assayed for reporter gene expression. Cells
infected with inactive virus display no expression of reporter gene.
Viruses that are incapable of secondary infection are also useful in the
methods of this invention. Infectious SF particles incapable of secondary
infection because the structural protein region has been deleted from
their RNA can be formed as described in Liljestrom and Garoff (1991)
Biotechnology 9:1356. Such SF particles can enter cells, but are incapable
of producing infectious virus particles after entry into cells. Similar
methods can be employed to produce Sindbis particles incapable of
secondary infection (Xiong, C. et al. (1989) Science 243:1188-1191) and
can be readily adapted to other alphaviruses.
Inactive virus useful in this invention can be prepared by a variety of
methods. UV-inactivation, heat-inactivation and disruption of virus by
application of freeze-thaw cycles can be employed. Inactivation conditions
can be readily optimized for different virus and to obtain inactivation
without affecting the ability of the virus to enter cells.
Viral components can also be employed in the methods of this invention.
Useful viral components are those components, membrane fragments, spike
glycoproteins, multimers of spike glycoproteins (dimers and trimers) and
peptides of spike glycoproteins that function to enhance transfection of
cationic lipid aggregate/nucleic acid complexes into cells. Viral
proteins, multimers of viral proteins and peptides of viral proteins can
be incorporated into cationic lipid aggregates to achieve enhanced
transfection. Viral components can be isolated by a variety of well-known
techniques, for example using the cationic detergent DTAB as described in
Glushakova, S. E., et al. (1985) "Influenza viral glycoproteins isolation
using cationic detergent dodecylmethylammonium bromide and its subsequent
integration into liposomal membrane" Mol. Genet. Mikrobiol. Virol.
4:39-44. Alternatively, viral components can be produced by a variety of
standard chemical syntheses methods. Viral fusagenic peptides, for
example, can be synthesized using automated solid phase peptide synthesis
as described, e.g., in Stewart et al. (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Company, Rockford, Ill. Fusagenic peptides from
influenza and vesicular stomatitis virus, including the exemplified E5
amphiphilic peptide and G protein, are particularly useful in the methods
of this invention.
Cationic lipid aggregates which comprise viral components, such as viral
envelope fragments containing spike glycoprotein, can be produced by a
variety of well-known techniques. For example, methods such as those
described for the preparation of proteoliposomes, virasomes and
chimerasomes can be employed or readily adapted. Gould-Fogerite, S. et al.
(1989) Gene 84:429-438; Marsh, M. et al. (1983) J. Cell Biol. 96:455-461;
Tikchonenko, T. I. et al. (1988) Gene 63:321-330.
Media employed in transfections should preferably be free of components,
like serum or high salt levels, that can inhibit cationic lipid-mediated
transfection of cells or that can inhibit entry of the virus into a cell.
The SF viral stock used in transfection assays exemplified herein is
believed to contain inhibitory media components which inhibit transfection
as higher levels of viral stock are added. Viral stock free of such
inhibitory components is preferred. Methods for purifying viral stock are
well-known in the art.
A variety of cationic lipids is known in the art. Example structures of
cationic lipids useful in this invention are provided in Table 1.
Generally, any cationic lipid, either monovalent or polyvalent, can be
used in the compositions and methods of this invention. Polyvalent
cationic lipids are generally preferred. Cationic lipids include saturated
and unsaturated alkyl and alicyclic ethers and esters of amines, amides or
derivatives thereof. Straight-chain and branched alkyl and alkene groups
of cationic lipids can contain from 1 to about 25 carbon atoms. Preferred
straight-chain or branched alkyl or alkene groups have six or more carbon
atoms. Alicyclic groups can contain from about 6 to 30 carbon atoms.
Preferred alicyclic groups include cholesterol and other steroid groups.
Cationic lipids can be prepared with a variety of counterions (anions)
including among others: Cl.sup.-, Br.sup.-, I.sup.-, F.sup.-, acetate,
trifluoroacetate, sulfate, nitrite, and nitrate.
A well-known cationic lipid is
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
See Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417.
DOTMA and the analogous diester DOTAP
(1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), see Table 1 for
structures, are commercially available. Additional cationic lipids
structurally related to DOTMA are described in U.S. Pat. No. 4,897,355,
which is incorporated by reference in its entirety herein.
Another useful group of cationic lipids related to DOTMA and DOTAP are
commonly called DORI-ethers or DORI-esters. DORI lipids differ from DOTMA
and DOTAP in that one of the methyl groups of the trimethylammonium group
is replaced with a hydroxyethyl group, see structure in Table 1. The DORI
lipids are similar to the Rosenthal Inhibitor (RI) of phospholipase A
(Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem. 235:2202-2206).
The oleoyl groups of DORI lipids can be replaced with other alkyl or
alkene groups, such as palmitoyl or stearoyl groups. The hydroxyl group of
the DORI-type lipids can be used as a site for further functionalization,
for example for esterification to amines, like carboxyspermine.
Additional cationic lipids which can be employed in the compositions and
methods of this invention include those described as useful for
transfection of cells in PCT application WO 91/15501 published Oct. 17,
1991, Pinnaduwage, P. et al. (1989) Biochem. Biophys. Acta. 985:33-37;
Rose, J. K. et al. (1991) BioTechniques 10:520-525; Ito, A et al. (1990)
Biochem, Intern, 22:235-241.
Cationic sterol derivatives, like
3.beta.[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) in
which cholesterol is linked to a trialkyammonium group, see Table 1, can
also be employed in the present invention. DC-Chol is reported to provide
more efficient transfection and lower toxicity than DOTMA-containing
liposomes for some cell lines. (Goa, X. and Huang, L. (1991) Biochem.
Biophys. Res. Comm. 179:280-285.)
The polycationic lipid formed by conjugating polylysine to DOPE (Zhou, X.
et al. (1991) Biochem. Biophys. Acta 1065:8-14), as well as other
lipopolylysines, can also be employed in the methods and compositions of
this invention.
Polycationic lipids containing carboxyspermine are also useful in the
compositions and methods of this invention. Behr, J-P. et al. (1989) Proc.
Natl. Acad. Sci. 82.:6982-6986 and EPO published application 304 111
(1990) describe carboxyspermine-containing cationic lipids including
5-carboxyspermylglycine dioctadecyl-amide (DOGS) and
dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).
Additional cationic lipids can be obtained by replacing the octadecyl and
palmitoyl groups of DOGS and DPPES, respectively, with other alkyl or
alkene groups.
U.S. Pat. No. 5,334,761, issued Aug. 2, 1994, which is incorporated by
reference in its entirety herein, describes cationic lipids of formula A
which are useful in this invention:
##STR1##
where R.sub.1 and R.sub.2 separately or together are C.sub.1-23 alkyl or
alkenyl or (--CO--C.sub.1-23) alkyl or alkenyl, q is 1 to 6, Z.sub.1 and
Z.sub.2, separately or together, are H or an unbranched alkyl group having
one to six carbon atoms and where X can be a variety of groups including
haloalkyl, alkylamines, alkyldiamines, alkyltriamines, aklytertamines,
carboxyspermine and related amines, or polyamines including polylysine or
polyarginine.
Compounds of formula A in which X is a nitrogen containing group such as:
--(CH.sub.2).sub.n NH.sub.2, where n=0-6 (X1)
--NH--(CH.sub.2).sub.m --NH.sub.2, wherein m=2-6 (X2);
--NH--(CH.sub.2).sub.3 --NH--(CH.sub.2).sub.4 NH.sub.2 (X3);
--NH--(CH.sub.2).sub.3 --NH--(CH.sub.2).sub.4 --NH--(CH.sub.2).sub.3
--NH.sub.2 (X4);
##STR2##
where p is 2-5 and Y is H or a group attached by an amide or alkyl amino
group (X7) are particularly useful in the methods and compositions of the
present invention for complexation to nucleic acids. Polycationic lipids,
such as those of formula A where X is a spermine, like X5, are preferred.
In the transfection compositions of this invention cationic lipids can
optionally be combined with non-cationic lipids, preferably neutral
lipids, to form lipid aggregates that complex with nucleic acids. Neutral
lipids useful in this invention include, among many others: lecithins;
phosphotidyletahnolamine; phosphatidylethanolamines, such as DOPE
(dioleoylphosphatidylethanolamine), POPE
(palmitoyloleoylphosphatidylethanolamine) and
distearoylphosphatidylethanolamine; phosphotidylcholine;
phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine), DPPC
(dipalmitoylphosphatidylcholine) POPC (palmitoyloleoylphosphatidylcholine)
and distearoylphosphatidylcholine; phosphatidylglycerol;
phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG
(dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol;
phosphatidylserine; phosphatidylserines, such as dioleoyl- or
dipalmitoylphospatidylserine; diphosphatidylglycerols; fatty acid esters;
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