Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
218~64 1
S016573J.64
Methods of treating eukaryotic cells
The present invention relates to methods of treating
eukaryotic cells.
One problem which frequently occurs when working with
eukaryotic cells in culture or in vivo is the presence
of endotoxin.
Endotoxin (lipopolysaccharide, LPS), a major component
of the cell walls of gram-negative bacteria, is
frequently found, for example, as a cont~m;n~nt in
plasmid DNA preparations, as up to 40~ of the surface
LPS of E. coli are released with the methods
conventionally used to prepare plasmid DNA. As a result
of the negative charges, LPS behaves in a similar manner
to DNA on anion exchange chromatography resins, but
because of the size it has in its micellar form, LPS
behaves like a large DNA molecule on size exclusion
resins. The density of LPS in CsCl is similar to that
of plasmid/EtBr complexes, which means that the DNA in
CsCl bands can easily become contaminated. When
transfection with DNA is carried out the cells thus come
into contact with LPS.
Since the LPS molecule is toxic and is a powerful
stimulator of the m~mm~1ian imml~ne system, its presence
during the treatment of cells is undesirable. For this
reason there have already been numerous attempts at
detected LPS and finding methods of eliminating this
molecule or gram-negative bacteria or neutralising the
harmful effects caused by th~m (Cordle et al., 1993;
Elsbach and Weiss, 1993; Golenbock et al., 1993; Haziot
et al., 1993a; Lynn et al., 1991; Perera et al., 1993;
Rustici et al., 1993; Tobias et al., 1988; Tobias et
3~7
al., 1989; Ziegler-Heitbrock and Ulevitch, 1993).
The aim of the present invention is to overcome the
toxicity problems connected with the presence of LPS in
the treatment of eukaryotic cells, which arise in
particular in connection with the introduction of
foreign material, particularly DNA, into the cell.
In the course of the experiments carried out to find a
solution to this problem, it was first established that
toxicity problems occur when transferring plasmid DNA by
means of adenovirus-aided receptor-mediated endocytosis,
as described inter alia in WO 93/07283 and by Wagner et
al., 1992; Cotten et al., 1992; Curiel et al., 1991;
Cotten et al., 1993; Cristiano et al., 1993a, and
Cristiano et al., 1993b, into primary human skin
fibroblasts or into primary human melanoma cells. Using
experiments with LPS containing DNA or pure LPS, these
problems have been traced back to the presence of LPS.
Toxicity occurred with amounts of 100 ng/ml of free LPS
or, if LPS were incorporated in the poly-
lysine/adenovirus complexes, with amounts of 100 pg/ml.
Within the scope of the present invention, toxicity
which could be traced back to LPS was also found in gene
transfer methods independent of adenovirus; the
possibility of the toxicity being connected with
polylysine was also ruled out.
Starting from the findings obtained, first of all a
method was developed for removing the LPS impurities
from the DNA and thereby eliminating the toxicity. A
simple and effective method of removing LPS uses the
detergent Triton X-114. At temperatures below 20C,
Triton X-114 is miscible with aqueous solutions and at
temperatures above 20C it separates into a distinct
phase (Bordier, 1981). The phenomenon can be exploited
2183~47
in order to extract the lipophilic LPS molecule from
aqueous protein solutions (Aida and Pabst, 1990) or from
DNA preparations (Manthorpe et al., 1993).
An alternative method of separating off LPS uses
polymyxin B, a cyclic mould peptide antibiotic (Storm et
al., 1977), which binds with high affinity to the lipid
A/ketodeoxyoctolonic acid component of LPS (Ka = 1.15 x
107 M 1; Rustici et al., 1993; Lynn and Golenbock, 1992;
Schindler and Osborn, 1979). Polymyxin has hitherto
been used to remove LPS from proteins; to do this,
polymyxin is used in a resin-bound form in
chromatography columns.
The experiments with purified DNA showed that the
removal of LPS from the DNA preparations brings about an
increase in gene expression.
LPS is, however, not only an unwanted accompaniment to
DNA preparations but a ubiquitous cont~min~nt which
occurs, inter alia, in the cell culture media normally
used. The LPS content of serum preparations may
fluctuate over a wide range; unless special steps are
taken to exclude contaminated preparations, the content
may be up to 10 to 50 ng/ml. A problem also occurs in
connection with the glass apparatus conventionally used
which have come into contact with bacteria. Since it is
extremely laborious to monitor the LPS content of all
reagents and glass containers, in order to solve the
problem set out hereinbefore, attempts have been made to
find a method which combats, from the outset, the
problems connected with the presence of LPS in the
treatment of eukarotic cells, particularly when
transporting foreign material into the cells.
The present invention relates to a method of treating
eukaryotic cells, particularly for the incorporation of
~18:~647
foreign material, in which the cells are treated with a
substance which binds lipopolysaccharide and blocks its
toxicity to the cells, and/or the foreign material
introduced into the cell is one which has been purified
to remove LPS before the material is introduced into the
cells.
Substances having this property are hereinafter referred
to as "LPS binding substances" in the interests of
simplicity; the substance may occur as a single
substance or as a mixture.
The invention may be applied to all methods for
introducing foreign material into the cell where the
presence of LPS causes a toxicity which has a negative
effect on the efficiency of the method. These include,
inter alia, methods of importing drugs or drug
conjugates or toxins into the cell.
Preferably, the method is applied to transfection and
infection methods for introducing foreign DNA or RNA
into the cell, e.g. the calcium phosphate,
microinjection and DEAE method, methods which operate
with liposomes or cationic lipids, as well as methods
which use recombinant viruses; it is particularly
preferable to use the method in conjunction with gene
transfer methods based on receptor-mediated endocytosis.
A summary of such methods is provided, for example, by
Cotten and Wagner, 1993. The need to use the present
invention during transfection of the cells can easily be
determined by carrying out the method in the presence or
absence of LPS, the other conditions remaining
identical, and comparing the results obtained.
The LPS binding substances are required both to bind LPS
with sufficient avidity and also block its interaction
with cell components, which is responsible for the
~1836~
toxicity. (In contrast to substances which satisfy
these requirements, there are proteins, e.g. the
lipopolysaccharide binding proteins, an acute phase
protein which does indeed bind to LPS with a high
affinity but activates the response of the cell to the
LPS toxicity; such an effect would be undesirable with
the scope of the present invention).
In the embodiment of the invention in which the foreign
material to be imported into the cell, especially DNA,
is purified to remove LPS, the DNA is preferably treated
with polymyxin, particularly by chromatography through a
polymyxin resin, or by extraction with a suitable
detergent such as Triton X-114.
In order to treat the cells during transfection the LPS
binding substances are used in amounts which saturate
the LPS present at least to a degree such that its
toxicity with regard to the intended use is neutralised
and in which the substances themselves are not toxic.
In order to determine the suitability of the substance
and its optimum concentration, transfection experiments
are appropriately carried out, e.g. with a view to a
particular transfection to be carried out with the cell
type in question. By means of titrations, a suitable
concentration is found at which the substance brings
about an increase in gene expression without itself
having a detrimental effect on the cells. In addition
to the transfection system, the effect of the LPS
binding substance on the morphology of the cells may be
investigated under the microscope. Furthermore, assays
may be used to measure the release of cellular
components as a measurement of the necrosis or apoptosis
occurring as a reaction to the LPS toxicity. Example`s
of this are the commercially obtainable lactate
dehydrogenase assay which is used clinically on a large
2183~47
scale or the recently described epifluorescence method
after staining with calcein-AM and propidium iodide
(Lorenzo et al., 1994). Using such methods, it was
found, within the scope of the present invention, that
in the case of polymyxin B the medium should preferably
contain no more than 100 ~g/ml and the lower limit may
be 3 ~g/ml or below. The neutralisation achieved with
polymyxin began to deteriorate at 1 ~g/ml and below.
This corresponds to a requirement for 250 ng of
polymyxin to neutralise 10 to 100 ng of LPS. It should
be borne in mind that the LPS may be incorporated in the
transfection complexes and therefore need not absolutely
be available for binding to the LPS binding substances.
It is also possible for neutralisation by means of the
LPS binding substance to occur at an intracellular level
if the LPS containing transfection complexes are
released into the cytoplasm. Therefore, the excess
antibiotic in the medium might be necessary to ensure
sufficient intracellular concentrations of LPS binding
substances to achieve neutralisation. The minimum
concentration necessary to block the toxicity can be
determined by titration.
In one embodiment of the invention the LPS binding
substance is polymyxin B.
In another embodiment the substance is polymyxin E
(Storm et al., 1977), a derivative of polymyxin B which
differs from it only by one amino acid. Like polymyxin
B, it can bind LPS, but unlike polymyxin B it is not
capable of blocking the activation of protein kinase C.
Other examples which are known to bind and neutralise
LPS are: HDLs and LDLs (Levine et al., 1993; Flegel et
al., 1993; Roth et al., 1993), apolipoprotein A1 (Flegel
et al., 1993), BPI protein ("Bactericidal/Permeability
Increasing Protein") or fragments thereof (Dentener et
2183~4~
al., 1993; Elsbach and Weiss, 1993) which have the
properties required for the purposes of the invention,
Limulus proteins (Roth and Tobias, 1993), derivatives of
polymyxin which have a reduced cell toxicity, synthetic
peptides having an affinity for LPS (Rustici et al.,
1993), antibodies against LPS or lipid A (Burd et al.,
1992). In addition, suitable substances may be
designed, for example, by identifying the LPS binding
domain of larger molecules known to have an LPS binding
property and using the peptide, optionally in modified
form. Alternatively, analogously to the proposal made
by Rustici et al., 1993 for polymyxin B, the structure
of lower molecular molecules with a known LPS binding
property may be taken as a starting point for preparing
new molecules optimally designed to bind and detoxify
LPS.
The LPS binding substances for treating the cells are
simply used by preferably making them part of the
transfection medium. Generally, the LPS binding
substances are only required to be present during
transfection as the toxic effect of LPS manifests itself
primarily during transfection; consequently, when the
medium is changed after transfection, there is no need
to add this substance. However, the presence of the
substance even beyond transfection may be useful for the
growth of the cell if, for example, the cell type used
is sensitive to LPS; in this case, the fresh medium
applied to the cells after transfection or infection
will also contain the LPS binding substance.
In another embodiment of the process according to the
invention, the cells are pretreated, before application
of the transfection medium, with the LPS binding
substances in order to bind any existing LPS and remove
it if necessary.
2:183fi i7
- 8 -
The treatment of cells with LPS binding substances may
be advantageous even irrespective of their treatment
with foreign material which is to be introduced into the
cell, e.g. during the culturing of cells which are very
sensitive to LPS in their growth.
According to another aspect, the present invention
relates to compositions for treating higher eukaryotic
cells.
In the case of cell culture applications, this
composition is a medium which contains one or more LPS
binding substances in addition to the usual components.
The usual components include nutrients for the cells,
buffer substances, etc. If the LPS binding substance is
a component of a transfection medium, this also contains
the transfection components, i.e. the foreign material
which is to be introduced into the cell and the
components which mediate its transfer into the cell
(e.g. recombinant viruses, cationic lipids, liposomes,
as well as complexes for receptor-mediated gene
transfer, optionally in conjunction with endosomolytic
agents which increase the efficiency of gene transfer;
complexes of this kind are described, inter alia, in
WO 93/07283). When preparing a transfection medium of
this kind it is recommended to add the LPS binding
substance before the transfection components.
The present invention may be applied both to the
treatment of cells in cell culture systems and also to
therapeutic uses in vivo or ex vivo; in the latter case
the medium containing the LPS binding substance acts as
a drug.
The invention is beneficial, inter alia, in therapeutic
applications in which the presence of LPS causes
problems. One example of this in the field of gene
~183~7
therapy is the therapeutic use of recombinant adenovirus
vectors in order to administer intact CFTR ("Cystic
Fibrosis Transmembrane Regulator") genes to patients
with cystic fibrosis (CF) in whom these genes are
mutated. Since the lungs of CF patients may contain
large quantities of Pseudomonas-LPS (Pseudomonas
infections are an accompanying phenomenon of the
disease), one restriction to the use of this method,
which is carried out either through the nasal epithelium
or by instillation directly into the lungs, might
consist in toxicity to the pulmonary epithelium caused
by the joint entrance of recombinant adenovirus and LPS.
Within the scope of the present invention, experiments
were carried out with epithelial cells of the
respiratory tract, which show that polymyxin B is
capable of blocking the sharp decline in gene expression
caused by LPS. The addition of an LPS binding substance
to the medium containing adenovirus can thus achieve
significant improvements in the therapeutic treatment of
pulmonary epithelial cells.
Another example is the application to endothelial cells
which are known to be difficult to transfect; one
possible explanation for this difficulty might be in the
tendency of these cells to respond to tiny amounts of
LPS (Arditi et al., 1993; Haziot et al., 1993b; Pugin et
al., 1993). Whilst the secretion of different cellular
factors as a reaction of one cell to LPS influences
other cells as well which have not themselves come into
direct contact with LPS. Within the scope of the
present invention, transfections of umbilical vein
endothelial cells have shown that even very tiny amounts
of LPS contAminAnts will produce toxicity; as LPS-
cleansed DNA was used, these contAminAntS originated
exclusively from tissue culture reagents. Polymyxin was
able to neutralise even this toxicity caused by small
amounts of LPS.
~i836g~7
- 10 --
The present invention may also be advantageous in the
transfection of patient cells ex vivo when the cell
population is contaminated with small amounts of LPS.
One example of such an application is the preparation of
cancer vaccines in which tumour cells are taken from the
patient, transformed ex vivo with a DNA coding for an
immunostimulant polypeptide and given to the patient as
a vaccination.
In therapeutic applications, the LPS-binding substance
may be present as a component of the composition which
acts as the transfection or infection medium and is thus
applied to the cells together with the transfection
components. It may also be present as an active
component of a drug preparation which is added to the
transfection medium before transfection or administered
separately from the transfection composition, e.g.
before transfection. In its simplest form, a
preparation of this kind is a solution of the LPS
binding substance, and the preparation may also contain
conventional additives; methods of formulating
pharmaceutical preparations are known to those skilled
in the art. They may be found in the relevant
textbooks, e.g. Remington's Pharmaceutical Sciences,
1980.
Thus, according to another aspect, the invention relates
to pharmaceutical compositions containing an LPS binding
substance for use in therapeutic treatment in which
foreign material is introduced into the cell. The
foreign material is preferably a nucleic acid,
especially DNA.
According to another aspect, the present invention
relates to the use of LPS binding substances for
producing pharmaceutical compositions for use before
and/or simultaneously with and/or after the treatment of
2183fi~'7
the human or animal body by transfection or infection
with DNA or RNA.
These therapeutic treatments preferably include, in
addition to viral gene transfer methods, gene therapy
procedures as described in the summarising article by
Cotten and Wagner 1993, including those methods in which
inhibitory nucleic acid molecules such as antisense
RNAs, ribozymes or DNA molecules coding for them are
used specifically to inhibit cell function.
In another aspect the invention relates to a process for
treating DNA for incorporation in human or animal cells,
in which the DNA is cleansed of LPS.
Summary of Figures
ig. 1: Influence of the endotoxin content of the DNA
on the expression of IL-2 in human melanoma
cells
Fig. 2: Reduction in the toxicity of LPS during the
transfection of primary human fibroblasts by
means of polymyxin B
Fig. 3: Mode of activity of polymyxin B at
concentrations of 30 ~g/ml to 0.03 ~g/ml
Fig. 4: Effect of polymyxin B during and after
transfection
Fig. 5: Correlation between the release of lactate
dehydrogenase and transfection efficiency in
the presence of polymyxin B
Fig. 6: Blocking the toxicity caused by natural LPS
contamination of plasmid DNA, using polymyxin
B
ig. 7: Blocking the LPS toxicity in primary human
epithelial cells of the respiratory tract,
using polymyxin B
2 1~3647
- 12 -
ig. 8: Blocking the toxicity caused by LPS from
tissue culture reagents, by means of polymyxin
B in umbilical vein endothelial cells
Fig. 9: Blocking the LPS-induced toxicity using
polymyxin E
Fig. 10: Effect of polymyxin on the expression of DNA
when applying various methods of gene transfer
In the Examples which follow, which illustrate the
present invention, the following materials and methods
were used unless otherwise specified:
a) Plasmid constructs
i) pCMVL
The construction of the plasmid is described in
WO 93/07283.
ii) pGShIL-2tet
To obtain the plasmid which contains the sequence coding
for human IL-2, the vector pWS2 was used as starting
material: the plasmid pH~APr-1 (Gunning et al., 1987)
was cut with BamHI and EcoRI. By agarose gel
purification, a 2.5 kb fragment was isolated which
contains the ampicillin resistance gene and the
replication origin of pBR322 and the SV40
polyadenylation signal. This fragment was ligated with
the CMV promoter/enhancer which had been amplified as a
0.7 kb PCR fragment from vector pAD-CMVI (described in
EP-A 393 438) and digested with EcoRI/BamHI. The
resulting plasmid was called pWS. The cDNA coding for
human IL-2 was obtained as a PCR fragment from human
pIL2-50A (Taniguchi et al., 1983) which contains the
cDNA coding for human IL-2. The PCR fragment was
ligated into the vector pWS opened by SalI/BamHI
21836~7
digestion and in this way pWS2 was obtained.
An IL-2 cassette containing the CMV enhancer/promoter,
the sequence coding for IL-2 and the SV40-polyA
sequence, was obtained by PCR on the basis of pWS2. The
PCR product was subjected to restriction enzyme
digestion with EcoRI and cloned into the EcoRI/SmaI site
of the plasmid pUC19 (Pharmacia). The resulting plasmid
was known as pGShIL-2. The plasmid pBR327 (Soberon et
al., 1980) which had been digested with SspI and AvaI
was used as the source for the tetracyclin resistance
gene and parts of the "upstream" region of the ~-
lactamase gene (ampicillin resistance gene). Together
with an EcoRI/AvaI adaptor, the isolated tet sequence
was cloned into the EcoRI/SspI site of pGShIL-2. The
IL-2 cassette of the resulting clone pGShIL-2tet/amp was
sequenced; then the amp sequence was excised with
EamllO5I and SspI and the plasmid was religated. The
resultant plasmid was termed pGShIL-2tet.
b) DNA preparation
The plasmids were first cultured in bacterial strain
E. coli DH5~ in the presence of 100 ~g/ml ampicillin
(pCMVL) or tetracyclin (pGShIL-2tet) in LB medium. The
overnight culture was centrifuged and from it the DNA
was prepared as follows: the CsCl density gradient
centrifugation was carried out using the method
described by Cotten et al., 1993. In order to do this
the bacterial deposit from 1 litre of culture was
incubated in 10 ml of 20~ (weight/volume) sucrose, 10 mM
EDTA, 50 mM Tris, pH 7.5 (solution 1) on ice for 10
minutes. Then 2.2 ml of lysozyme (10 mg/ml in solution
1) were added for a further 10 minutes on ice, then 5 ml
of 0.2 M EDTA, pH 7, were added, the sample was
incubated on ice for 10 minutes and finally 10 ml of 2~
(volume/volume) Triton X-114, 60 mM EDTA and 40 mM Tris,
3~47
- 14 -
pH 7.5, were added followed by 15 minutes incubation on
ice. This lysate was then centrifuged for 30 minutes
(Sorvall SS34, 17K) and 28.5 g CsCl and 400 ~l of
ethidium bromide (10 ~g/ml) were added to the
supernatant (26 ml initial volume). This material was
centrifuged for 18 hours in a Beckman VTi50 rotor at
49,000 rpm at 20C. The lower one of the two ethidium-
rich bands was collected and centrifuged again, directly
in a Beckman VTi65 rotor, for 4 hours at 63,000 rpm and
at 20C. The ethidium-rich band was harvested again,
extracted with CsCl-saturated isopropanol until the pink
colour had disappeared, exhaustively dialysed against TE
(10 mM Tris, 0.1 mM EDTA, pH 7.4), mixed with 1/10
volume 3 M sodium acetate, pH 5, and precipitated with 3
volumes of ethanol at -20C. The DNA precipitate
obtained was further treated with RNase A, proteinase K,
phenol/chloroform and chloroform, precipitated once more
and the final DNA pellet was taken up in TE and
quantitatively measured by optical absorption, working
on the assumption that 0.05 mg/ml DNA has an absorption
of 1 at 260 nm.
c) DNA purification of LPS
i) Triton X-114 extraction
In order to obtain a homogeneous preparation of the
detergent, Triton X-114 (Sigma) was subjected to three
0C/30C temperature cycles as described by Bordier,
1981. The extraction of the lipopolysaccharides from
the DNA sample was carried out, in a modified version of
published methods (Aida and Pabst, 1990; Manthorpe et
al., 1993) as follows: the DNA sample (0.5 - 1.5 mg/ml
in 10 mM Tris, 0.1 mM EDTA, pH 7.4 (TE)) was applied to
0.3 M sodium acetate (pH 7.5). Then 3 ~1 of Triton
X-114 were added per 100 ~1 of DNA solution, the samples
were vigorously mixed in a vortex and incubated on ice
~1836~7
for 10 minutes. To allow the two phases to separate,
the samples were stored for 5 minutes at 30C,
centrifuged in a preheated Eppendorf centrifuge at
2,000 rpm for 2 minutes and the aqueous phase was placed
in a fresh Eppendorf test tube. This extraction was
carried out twice more and the aqueous phase finally
obtained was precipitated with 0.6 volumes of
isopropanol at ambient temperature, the precipitate was
obtained by centrifuging, washed twice with 80~ ethanol,
air-dried, taken up in TE again and the quantity was
measured. In order to do this, the sample was treated
with RNase A, proteinase K, phenol/chloroform and
chloroform, re-precipitated and the final DNA pellet was
suspended in TE and the absorption at 260 nm was
determined, working on the assumption that a
concentration of 0.05 mg/ml of DNA has an absorption
value of 1. (This method was used for the DNA used in
the Examples).
ii) Polymyxin chromatography
One volume of polymyxin resin slurry (Affi-Prep-
Polymyxin, Biorad) corresponding to the volume of the
DNA sample was briefly mixed with three volumes of 0.1 N
NaOH, then washed three times with five resin volumes of
TE. The pelleted resin was taken up again with the DNA
samples (in TE 0.8 - 1.2 mg/ml) and the mixture was
stirred overnight at 4C. Then the sample was placed on
a disposable column pretreated with 0.1 NaOH, and washed
with TE. The eluate was collected, the resin was washed
with another volume of TE and the eluate was combined
with the washing liquid. The DNA of this pooled sample
was precipitated with 1/10 volume of 3 M sodium acetate,
pH 5, and 2 volumes of ethanol. Further treatment of
the precipitate and DNA measurement were carried out as
described above. (This method was used in the
preliminary test.)
~1836'17
- 16 -
d) LPS preparation
A commercially available LPS preparation from
Escherichia coli was used (0111:B4, Sigma). The
preparation was dissolved in LPS-free water at the rate
of 10 mg/ml and before the preparation of serial
dilutions in LPS-free water it was sonicated for 5
minutes (SONOREX bath, 360 W). The final dilutions were
sonicated for 5 minutes before use.
The LPS assays were carried out using the BioWhittaker
assay (chromogenic Limulus assay; Iwanaga, 1993), and it
was found that all the reagents used were LPS-free (<0.1
endotoxin units/50 ~l of solutlon).
e) Adenovirus preparation
The E4-deficient adenovirus 5, dll014 (Bridge and
Ketner, 1989) was cultured in the complementary cell
line W162 (Weinberg and Ketner, 1983). Pellets of
infected cells were suspended in amounts of 2 ml/2 x 107
cells in 20 mM HEPES, pH 7.4, 1 mM PMSF (phenylmethyl-
sulphonyl fluoride) and subjected to three freeze/thaw
cycles (liquid nitrogen, 37C). The suspension was then
mixed with an equal volume of freon in the vortex and
centrifuged for 10 minutes at 3,000 rpm (Heraeus
Sepatech, 2705 Rotor). The aqueous (upper) phase was
removed and the freon phase was treated with 1/5 volume
20 mM HEPES, pH 7.4 in the vortex and centrifuged again.
The aqueous phases were combined, transferred into a
Beckman VTi50 centrifugal test tube (15 ml/test tube)
and underlayered with 15 ml of 1.2 g/cm3 CsCl/20 mM
HEPES, pH 7.4 and 7 ml of 1.45 g/cm3 CsCl, 20 mM HEPES
pH 7.4. The samples were centrifuged for 40 minutes at
20C in a Beckman VTi50 rotor at 49,000 rpm. The lowèr
opalescent band of mature virus particles at 1.34 to
1.35 g/cm3 (measured by means of the refractive index)
2183~47
- 17 -
and the upper band (immature particles at 1.31 to
1.32 g/cm3) were collected separately and subjected to
equilibrium centrifugation (more than 4 hours) at
63,000 rpm in a VTi65 rotor. The opalescent virus bands
(either 1.31 g/cm3 immature or 1.34 g/cm3 mature) were
harvested. Biotinylation of the resulting virus
particles with N-hydroxysuccinimide biotin (Pierce),
inactivation with 8-methoxypsoralene/WA and
purification by gel filtration using a Pharmacia PD10
column, equilibrated with HBS/40~ glycerol, were carried
out as described in WO 93/07283 or by Wagner et al.,
1992 and Cotten et al., 1992. The virus samples were
quantitatively determined by means of the protein
concentration (Biorad Bradford Assay using BSA as
standard) and analysed, using the equation 1 mg/ml of
protein = 3.4 x 1012 adenovirus particles/ml (Lemay et
al., 1980).
f) Transfection complexes
The modified adenovirus particles (8 ~l, 1 x 10l2
particles/ml) were diluted in 150 ~l of HBS and mixed at
room temperature with 1 ~g of StrpL (streptavidin-
modifed polylysine, prepared as described in
WO 93/07283) in 150 ~1 of HBS for 30 minutes. Aliquots
of 6 ~g of plasmid DNA were mixed with increasing
amounts of LPS in 100 ~l. The DNA solutions were then
mixed for 30 minutes with the adenovirus/StrpL solution
at room temperature. Finally, a 100 ~l aliquot of HBS
containing 5 ~g of TfpL, prepared as described in
WO 93/07283, was added to each sample, followed by 30
minutes at ambient temperature. Of the 500 ~l of
transfection medium thus obtained, 1/10 was used in each
well of a cell culture plate containing 20,000 cells.
~183~4~
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g) Cell cultures
i) Human fibroblasts
After surgical removal, skin biopsies were placed in 4C
DMEM, containing 10~ FCS, 2 mM glutamine and gentamycin.
The biopsies were finely comminuted in a tissue culture
device with forceps and a surgical blade in a l~m' n~r
air current in sterile 6 cm plastic dishes. Then 3 ml
of DMEM containing 20~ FCS, 2 mM glutamine and
antibiotics were added and the culture was placed in a
37C incubator. After 10 days the medium was replaced
by DMEM containing 10~ FCS. Then the medium was changed
twice a week. 4 weeks after the beginning of the
culture the cells which had grown out of the tissue
fragments were trypsinised and plated out into new
culture dishes for transfection. Primary cultures from
passage 5-10 were used. The fibroblasts were
transfected as described by Wagner et al., 1992.
ii) Human melanoma cells
Primary human melanoma cells were isolated and
cultivated in RPMI 1640 medium (Gibco/BRL) supplemented
with 100 I.U./ml of penicillin, 100 ~g/ml of
streptomycin, 2 mM L-glutamine, 1~ sodium pyruvate and
10~ heat inactivated FCS.
iii) Human respiratory tract epithelial cells
Human respiratory tract epithelial cells were isolated
from nasal polyps as described by Van Scott et al.,
1986. The cells were cultured on standard cell culture
dishes coated with human placental collagen (Sigma,
Catalogue No. C 7521) in bronchial epithelial cell
growth medium (BEGM, Promocell, Catalogue No. C-2106).
2183fi~ '7
- 19 -
iv) Human umbilical vein endothelial cells
Human umbilical vein epithelial cells (HWECs) were
obtained from cell systems (Kirklandt, Washington) and
cultured on 0.1~ gelatine-coated cell culture dishes.
h) Measurement of expression
i) Luciferase assay
The preparation of cell extracts, the standardisation of
the protein content and determination of the luciferase
activity were carried out as described by Zenke et al.,
1990 and Cotten et al., 1990 and in EP 388 758.
ii) IL-2 assay
The expression of interleukin-2 was determined using a
bioassay as described by Karasuyama and Melchers, 1988.
In addition, the IL-2 production was carried out using
the IL-2 ELISA kit made by Becton Dickinson (Catalogue
No. 30032) in accordance with the manufacturer's
instructions.
Example 1
Influence of the endotoxin content of DNA on the
expression of IL-2 in primary human melanoma cells
Primary human melanoma cells (2 x 105 cells/6 cm culture
dish) were transfected with 6 ~g of the plasmid purified
by various methods, according to the data in Fig. 1.
The endotoxin content of the plasmid preparation before
purification is shown in the drawing as a dark shaded
bar. After purification by means of polymyxin resin or
extraction with Triton X-114 all the preparations were
~I 83~ 7
- 20 -
given less than 0.1 EU lipopolysaccharide/6 ~g of DNA.
The IL-2 content was measured by ELISA in the cell
supernatant; the values shown in Fig. 1 indicate
units/106 cells and 24 hours.
Example 2
Reduction in the toxicity of LPS during transfection of
primary human fibroblasts by means of polymyxin B
First of all the toxicity of LPS during gene transfer by
means of adenovirus-aided receptor-mediated endocytosis
into primary human fibroblasts was investigated. The
content of LPS, based on 6 ~g of DNA, is shown in
Fig. 2. 24 hours after transfection the cells were
harvested and the luciferase measurement was carried
out. As the LPS content of the DNA increased, the gene
expression fell by nearly 2 powers of ten. The
disrupted morphology of the cells which had been exposed
to a high LPS content accorded with the low expression
values (Fig. 2, upper Table).
Next, the cells were subjected to the same
DNA/adenovirus/LPS complexes in the presence of
increasing amounts of polymyxin B (polymyxin B sulphate,
Sigma, Catalogue No. P 4932). Polymyxin was present in
the medium both during and after transfection in the
concentrations specified, i.e. the fresh medium changed
3 hours after transfection contained the specified
concentration of polymyxin. A typical antibiotic
concentration of polymyxin B is 1,000 units/ml. At a
specific activity of 7,500 units/mg this corresponds to
a concentration of 133 ~g/ml of polymyxin B. It was
found that there were virtually no differences in
expression in the presence of 3, 10 and 30 ~g/ml of
polymyxin B. This shows that the antibiotic is
tolerated by these cells. In all three concentrations
2183~7
- 21 -
used, polymyxin B allows successful transfections with
virus/DNA complexes containing LPS contamination, with
complete protection being achieved against 100 ng
LPS/6 ~g DNA (cf. samples 2, 5, 8 and 11) and with a
virtually total protective effect against 1,000 ng
LPS/6 ~g DNA (cf. samples 3, 6, 9 and 12). (The values
shown in the Figure are the averages of two
transfections.)
Example 3
Analysis of the method of activity of polymyxin at
concentrations of 30 ~g/ml to 0.03 ~g/ml
The minimum concentration of polymyxin necessary to
neutralise LPS toxicity was determined by titrations
similar to those in Example 1. It was found that total
neutralisation of a content of 100 or 1,000 ng of
LPS/6 ~g of DNA in the DNA complexes is achieved with
10 ~g/ml of polymyxin B. With 3 to 0.3 ~g/ml of
polymyxin B only partial neutralisation of the toxicity
was obtained and at lower concentrations only a very
slight neutralisation was observed. A content of 0.6 ~g
of DNA in the transfection mixtures shows that DNA
complexes containing LPS in concentrations of 100 or
1,000 ng/6 ~g DNA contain 10 to 100 ng of LPS in a
volume of 0.25 ml. The neutralisation brought about by
polymyxin began to deteriorate at 1 ~g/ml and below.
This corresponds to a requirement for 250 ng of
polymyxin to neutralise 10 to 100 ng of LPS. The excess
antibiotic in the medium would appear to be necessary in
order to ensure adequate intracellular polymyxin
concentrations for neutralisation. The results of the
titration are shown in Fig. 3: where shown in the Fig.,
polymyxin B was present, both during and after
transfection. In each case, double transfections were
carried out. The luciferase values (measured 24 hours
~J836~
after transfection) are expressed for each polymyxin
concentration as a percentage of the value of the
control sample.
Example 4
Investigating the effect of polymyxin B during and after
transfection
In the preceding Examples, polymyxin was present in the
culture medium both during transfection and afterwards,
until the cells were harvested for the luciferase assay.
Since it was assumed on the basis of preliminary tests
that the event responsible for the toxicity of LPS is
its entry into the cell together with the adenovirus,
the protective function of polymyxin B should only be
necessary during the period of contact of the cells with
the transfection complexes. To confirm this assumption,
primary fibroblast cultures were exposed to the LPS-
containing transfection complexes in the absence of
polymyxin (samples 1-4) and in the presence of polymyxin
B, on the one hand, only during transfection (in order
to do this, after transfection the samples were mixed
with fresh medium free from polymyxin B; samples 9-12)
and, on the other hand, also after incubation with the
transfection complexes (in order to do this, the samples
were mixed with fresh medium which contained identical
quantities of polymyxin B to the transfection medium;
samples 5-8). The luciferase expression obtained was
measured 24 hours after transfection. The results of
these experiments are shown in Fig. 4: it was found that
the presence of 30 ~g/ml of polymyxin B solely during
transfection has virtually the same protective effect as
the continued presence of the antibiotic beyond the time
after transfection. (The values shown in the Figure are
the averages of two transfections.)
~ 1 83~ 7
- 23 -
Example 5
Correlation between the release of lactate dehydrogenase
and transfection efficiency in the presence of polymyxin
B
In the tests carried out in Example 1 it was established
that the morphology of cells which had been transfected
with DNA complexes containing LPS accorded with the cell
toxicity which causes a reduction in gene expression.
Since the cytoplasmic enzyme lactate dehydrogenase (LDH)
is released into the surrounding medium when cells
undergo necrosis or apoptosis, simply measuring the LDH
activity in the cell medium is an indicator of cell
toxicity. An inverse correlation has been found between
the release of LDH into the medium and the successful
transfer of the luciferase gene (Fig. 5, samples 1-4),
whilst increases in LDH in the medium coincided with an
appreciable drop in luciferase gene expression. In
accordance with the results of Examples 1 and 2, the
presence of 10 or 30 ~g/ml of polymyxin B (polymyxin B
was present in the medium only during transfection)
blocked the LPS-induced reduction in gene transfer
(Fig. 5, samples 5-12). In accordance with the blocking
of the toxicity by polymyxin, the presence of polymyxin
blocks the LPS-induced release of LDH (Fig. 5, samples
5-12). (The values shown in the Figure are the averages
of two transfections.)
The LDH assay was carried out by removing aliquots of
the cell culture medium (5 to 50 ~l) at the specified
times after transfection and mixing them with 500 ~l of
LD-L reagent (Sigma, Catalogue No. 228-10). The samples
were incubated at 37C for 45 minutes and the absorption
at 340 nm was measured by comparison with an LD-L blind
value.
~1~.3~4 ~
- 24 -
Example 6
Blocking of the toxicity caused by natural LPS
contamination of plasmid DNA by means of polymyxin B
In the preceding Examples, LPS-free DNA was used to
which known quantities of defined LPS preparations had
deliberately been added. In this Example, an
investigation was carried out to find out whether the
toxicity of LPS contamination as typically found in DNA
preparations can be blocked by polymyxin B. For this
purpose, LPS-free luciferase plasmid was mixed with an
excess of DNA plasmid which had been obtained by the
Qiagen method with a plasmid DNA resin (Diagen) without
treatment to remove LPS. This DNA thus contains the
molecular form of LPS which is typically found in a
plasmid DNA preparation. The DNA sample used contained
about 16 ng LPS/6 ~g DNA; this corresponds to 6.4 ng/ml
during transfection. As can be seen from Fig. 6, only
weak DNA transfer was obtained with this preparation
(Fig. 6, sample 1), but an almost 10-fold increase was
achieved when 3 ~g/ml of polymyxin B was present during
the transfection (Fig. 6, sample 2). At higher
concentrations of antibiotic, no further improvement was
observed. The 3 ~g/ml of polymyxin represent an almost
500-fold excess by mass over the LPS concentration
obtained during transfection. (The values shown in the
Figure are the averages from two transfections.)
Example 7
Blocking the LPS toxicity in primary human respiratory
tract epithelial cells using polymyxin B
Primary human respiratory tract epithelial cells were
treated with LPS-containing transfection complexes as
described in the preceding Examples, on the one hand in
T~ 1 ~ 3 5 ~ 7
- 25 -
the absence of polymyxin B and on the other hand in the
presence of 10 ~g/ml of polymyxin B during transfection.
48 hours after transfection the cells were harvested and
the luciferase expression was measured. It was found
that the presence of LPS in the DNA complexes causes a
sharp drop in gene expression (Fig. 7, samples 1-4).
The toxicity caused by the LPS concentrations used can
largely be blocked by means of polymyxin B (Fig. 7,
samples 5-8). (The values shown in the Figure are the
averages from three transfections.)
Example 8
Blocking the toxicity caused by LPS from tissue culture
reagents by means of polymyxin B in umbilical vein
endothelial cells
Human umbilical vein endothelial cells were transfected
using the standard procedure. It was found that these
cells are sensitive to the presence of LPS in the
complexes; cf. Fig. 8, sample 1, in which the complex
contains no LPS, with samples 2, 3 and 4 in which LPS
was present in amounts of 10, 100 and 1,000 ng/6 ~g of
DNA. However, the transfection efficiency was low even
when LPS-free DNA was used. In order to establish that
this is caused by a toxicity which originates from the
tissue culture reagents, the quantities of polymyxin B
specified in the Figure were used, which were present in
the medium both during and after transfection. It was
found that the presence of 10 ~g/ml of polymyxin B
causes a 5-6-fold increase in gene expression (cf.
samples 1 and 5). Higher concentrations of polymyxin B
were less effective.
218~
- 26 -
Example 9
Blocking the LPS-induced toxicity using polymyxin E
The tests carried out in this Example were performed
chiefly as described in Example 2, except that polymyxin
E (colistin-methanesulphonate, Sigma, Catalogue
No. C 1511) was used instead of polymyxin B and it was
present only during the transfection. The results of
these experiments are shown in Fig. 9. It was found
that polymyxin E has the ability to block LPS in an
amount of 100 ng/6 ~g of DNA in a similar manner to
polymyxin B. However, unlike polymyxin B, polymyxin E
was unable to neutralise LPS contamination of
1,000 ng/6 ~g DNA (the apparent stimulation at 30 ~g
polymyxin E/ml (samples 4-6) can be put down to a
significant inhibition of the control sample (30 ~g
polymyxin E/ml, no LPS) rather than to stimulation of
the sample which contained 100 ng LPS/6 ~g DNA).
Example 10
Effect of polymyxin on the expression of DNA when using
different gene transfer methods
The experiments carried out in this Example serve as a
comparison in order to establish whether the toxicity
observed during gene transfer by means of adenovirus-
aided receptor-mediated endocytosis can be put down to
components of the transfection complexes.
a) Gene transfer with recombinant adenovirus
These experiments were intended to show whether the
toxicity which occurs in the presence of adenovirus and
LPS can be put down to polylysine. For this purpose, a
recombinant adenovirus which carries a genomic ~-
~1~3~
- 27 -
galactosidase gene (Stratford-Perricaudet et al., 1992)
was used as the gene transfer vehicle. The adenovirus
was applied to the cells (2 x lOq per well) in the
presence of LPS. After 24 hours the ~-galactosidase
expression was determined as a measurement of the
survival of cells which had taken up the adenovirus
particles. These experiments are shown in Fig. 10: an
LPS-dosage dependent reduction in ~-galactosidase
activity was found (Fig. lOA). Measurement of the
release of LDH (cf. Example 5) into the medium showed
that cell death and lysis could be responsible for the
reduction in ~-galactoside expression. It is thus found
that the presence of polylysine is not necessary in
order for the toxic reaction to occur, and this supports
the simple statement that toxicity is a consequence of
the internalisation of LPS. The toxic effect caused by
LPS was able to be counteracted by the addition of
polymyxin (Fig. 10, samples 5-8).
b) Gene transfer by means of non-viral systems
Next, investigations were carried out to discover
whether adenovirus might play a part in signal
transmission after the entry of LPS. If interactions
between adenovirus and the cell play a part in toxicity,
the transfer of DNA into the cells by non-viral methods
should not involve any LPS-induced toxicity. For this
purpose, glycerol on the one hand and cationic lipids on
the other hand were used as non-viral gene transfer
methods.
i) During the transfection of the fibroblasts (0.4 ~g pL
or StrpL in 75 ~l HBS were incubated with 3 ~g of
pCMVLuc in 75 ~l of HBS for 30 minutes, then 3 ~g of
TfpL or 2 ~g of pL in 75 ~l of HBS were added and
incubation was continued for a further 30 minutes.
After the addition of the medium and 13~ glycerol to the
21~3~ll7
- 28 -
complexes, the transfection medium was applied to the
cells for 4 hours) as a result of the addition of LPS in
various concentrations (see Fig. 10B, samples 5-8) a
reduction in luciferase expression was observed which
was almost as great as in the method based on
adenovirus-aided endocytosis.
ii) During transfection with the cationic lipid
Transfectam (DOGS; a commercially obtainable preparation
was used according to standard procedure) a reduction in
the luciferase activity was observed as a function of
the LPS content (Fig. 10B, samples 9-11). The presence
of 1000 ng LPS/6 ~g DNA reduced the luciferase
expression to about 10~ of the control value obtained
with LPS-free DNA.
~1~36~7
- 29 -
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