Note: Descriptions are shown in the official language in which they were submitted.
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Suspension packaging cell lines for retroviral vectors
The invention concerns suspension packaging cell lines for retroviral vectors
and their use
for producing infectious replication-incompetent retroviral vectors.
Viruses are composed of a genome coding for the viral genes and a virus capsid
which, as in
the case of the retroviruses, can be surrounded by a membrane envelope.
With retroviruses one distinguishes between the cis elements 5'-LTR, 3'-LTR,
packaging
sequence Psi, + and - strand primer binding site and the trans elements gag
(capsid
proteins), pol (reverse transcriptase) and env (envelope proteins). The trans
elements can
be replaced by foreign genes on the viral genome; however, in this case the
virus is
dependent on so-called helper viruses which can either be complete infectious
viruses or
l0 non-infectious viruses e.g. viruses without packaging sequences. Cells
which contain such
non-infectious helper viruses andlor helper genes of one or various helper
viruses that are
present transiently, cpisomally or integrated into the genome are referred to
as packaging
cell lines. I-fence, packaging cell lines do not release infectious,
replication-competent virus
particles. If the helper genes are derived from different helper viruses then
the vectors that
are formed are referred to as chimeric retroviral vectors; this process is
referred to as
pseudo typing. Such ehimeric packaging systems are usually used e.g. G('+env
Aml2
(Markowitz, D.G. et al. in Trans. Assoc. Am. Physicians 101 (1988) 212 - 218)
carries the
gag-pol genes of the ecotropic MoMuLV and the env gene of the amphotropic 4070
Ampho virus. It is even possible that a mirture of env genes from several
viruses (even with
genetically modified env genes) are present in the packaging cell lines.
Packaging cell lines can also contain the incomplete viral helper genome in a
fragmented
form in which case they are referred to as packaging cell lines with a split
up virus genome
and these are a variant with greater safety against undesired release of
infectious,
replication-competent viruses by recombination events.
However, if they are transfected or transduced with a retroviral vector which,
in addition to
the cis elements, can also carry foreign genes, then these virus genomes are
packaged and
released as infectious but not replication-competent retroviral vectors. This
is referred to as
a retrovirus producer cell line.
All previously produced retroviral packaging cell lines are based on adherent
cells i.e. on
cells which grow adhering to surfaces:
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The packaging cell lines GP+E86 and GP+envAml2 based on adherent NIH3T3 cells
are
described by Markowitz, D.G. et al. in Trans. Assoc. Am. Physicians 101 (
1988) 2I2 - 218.
In Mol. and Cell Biol. 6 (1986) 2895 - 2902 Miller, A.D. et al. describe the
packaging cell
line PA317 based on adherent NIH3T3 cells. Miller, A.D. et al. describe in J.
Virol. 65
S ( 1991 ) 2220 - 2224 the GALV packaging cell line PG13 based on NIH3T3
cells. Danos, O.
et al describe in PNAS USA 85 (1988) 6460 - 6464 the packaging cell lines
~rCre and ~Crip
based on NIH3T3 cells.
Rigg, R.J. et al. describe in Virology 218 (1996) 290 - 295 the packaging cell
line Propack-A
which is based on a human cell line. Cosset, F.-L. et al. describe in J.
Virol. 69 (1995) 7430 -
7436 the packaging cell lines FLY A13 and FL.Y RD18 which are based on a human
fibrosarcoma line (HT10 80). Packaging cell lines based on D17 and other
adherent dog cell
lines are described in W092/OS266.
The majority of the generally described cell lines also grow adherently. An
advantage of
adherent cell lines is that they are easy to handle. Thus adherent cells are
relatively easy to
1 S clone and viruses, especially retroviruses can be used i.e, a direct
clonal selection of the cells
after transfection or transduction is possible, individual colonies can be
easily recognized,
counted and isolated by picking, dead cells simply become detached and can
therefore not
be mistaken for living cells, in order to change the medium it is only
necessary to simply
remove the supernatant by pipette and add new medium. Seeding and keeping the
cells is
usually not critical since adherent cells form colonies of cells which
stimulate each other in
the tissue unit. When transferring the cells an additional trypsin treatment
is necessary for
adherent cells in order to detach them which is usually not critical.
Cells growing in suspension require a different spectrum of methods for
cloning and for
transduction e.cperiments. Hence, clonal selection requires the use of
immobilizing
2S additives such as soft agars, a dilution series of the cells referred to as
limited dilution until
theoretically only 1 cell per vessel is present or the use of a surface marker
or a fluorescent
marker (such as GFP) which enables the direct selection of recombinant cells
by means of
an FAC sorter. Living and dying cells can only be differentiated by staining.
A change of
medium always requires a centrifugation step.
Cell lines growing in suspension as retroviral packaging cell Lines have not
been previously
known. Also no e:cperiments have been described up to now in which established
packaging cell lines growing adherently have been brought into suspension.
~ trademark
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Surprisingly it has turned out that it is indeed possible to adapt adherent
packaging cell
lines to growth in suspension and that the resulting suspension packaging cell
Lines are able
to produce viruses with high titres. Moreover, it was demonstrated that it is
also possible to
produce cell lines growing in suspension that produce retroviruses from
classical
S suspension cell lines such as the human erythroleukemia cell line K562, the
rat hepatoma
line N1-S1 and the rat lymphoma line C58 by cotransfection of retroviral
packaging gene
plasmids with retruviral vector plasmids i.e. these cell lines release
retroviral vector particles
which are able to transduce other cells.
The technical procedure for producing suspension packaging cell lines is
similar to
methods described many times for adherent cells by introducing helper
sequences (in one
piece or preferably split up) which alone are not able to form an infectious
virus e~ccept for
the already mentioned difference that suspension cells require other cloning
and culture
techniques.
Once a suspension packaging cell line has been prmiuced, it can be converted
into a
suspension-producer cell lim which releases infectious but replication-
incompetent
retroviral vectors by transductiun with a compatible retroviral vector (e.g.
an amphotropic
packaging cell line containing a vrctor derived from an ecutropic packaging
cell line), and
preferably by transfection with a retroviral vector plasmid.
Mammalian cell lines growing completely in suspension as retroviral packaging
cell lines
have not been known previously. Also no method has previously been known in
which
established packaging cell lines growing adherently can be brought completely
into
suspension.
The object of the invention is to provide packaging cell lines for retroviral
vectors growing
in suspension and methods for their production and use.
The invention concerns a packaging cell line for retroviruses which grows
completely in
suspension which is preferably transduced with a retroviral vector.
A further subject matter of the invention is a method for producing retroviral
vectors
characterized in that a packaging cell line growing in suspension which is
transduced with
the said vector, is cultured in suspension, the cells are separated from the
cell supernatant
and the vector is isolated from the supernatant.
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A packaging cell line is understood as a mammalian cell line which contains
the genes gag,
pol and env (helper genes) necessary for the production of viral particles but
not
functionally active packaging sequences (y). Such a cell line cannot alone
form a virus
particle containing genomic RNA.
S A functionally active packaging sequence is understood as the region of a
retroviral genome
which functionally causes the packaging of the RNA genome. Its function can be
deactivated by complete or partial deletion or by mutation.
According to the invention a retroviral packaging cell line growing completely
in
suspension (no insoluble matrir (vessel wall or microcarrier to which the
cells adhere) is
required for growth even for long periods e.g. days) is preferably produced
from a
retroviral packaging cell line growing adherently by the stepwise adaptation
of such an
adherent packaging cell Line to growth in suspension over a period of at least
three months.
The steps comprise a continuous reduction of the FCS content in the medium
until it is
free from serum, repeated treatment of the cells with trypsin andloc DNAse,
the use of
medium for suspension cell lines (for example DMFO?4A), preferably tissue
culture flasks
for suspension cells and hybridoma cells (Sarstedt), continuous shaking of the
culture
flasks on a shaker and a cell density between 5 x 10° and 1 x
10° cells/ml (hence it is
necessary to split the cells much more frequently than in a comparable
adherent culture).
The cell line according to the invention which grows completely in suspension
and is
transfected with a retroviral vector plasmid or transduced with a retroviral
vector is
preferably produced by transfecting a suspension cell line with a retroviral
vector plasmid
or transducing it with a retroviral vector, selecting the transfected or
transduced cells by
means of a co-transfected or co-transduced surface marker by limited dilution
andlor the
addition of immobilizing additives and isolating the suspension cells
according to the
invention selected in this manner. The suspension cell line is preferably a
retroviral
packaging cell line. In order to produce a retroviral packaging cell line
which grows in
suspension, a retroviral packaging cell line which grows adherently and is
transfected with
nucleic acids which code for helper sequences and with a retroviral vector
plasmid or is
transduced with a retroviral vector and is cultured in a serum-containing
medium is
preferably converted into a cell fine growing in suspension by continuous
reduction of the
serum content in the medium until it is essentially free of serum, repeated
treatment of the
cells with trypsin and DNAse to detach the cells from the carrier during which
the cell
density in suspension is maintained in a range between S r 10° to 1 x
106 cells/ml. This
process is particularly preferably carried out over a long period preferably
for at least three
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months. In a further preferred embodiment the transfected or transduced cells
are selected
by means of a co-transfected or co-transduced surface marker by limited
dilution and/or
the addition of immobilizing additives.
It has surprisingly turned out that packaging cell lines growing completely in
suspension
that are treated in this manner are still able to produce retroviruses despite
the massive
long-term manipulation over several months after they have been transfected
with
retroviral vector plasmids or transduced with retroviral vectors.
It is also surprising that such packaging cell lines growing completely in
suspension can be
cloned by transfection with retroviral vector plasmids or transduction with
retroviral
vectors by isolation steps such as limited dilution in which the cells are
diluted until
practically only single cells per vessel are present in suspension or by the
use of
immobilizing additives such as an overlay agar or methylcelluloses (Metho Cult
H4100 /
Stem Cell Technologies) or cell sorting by means of a cell surface marker, and
retroviral
prouucer cell lines which grow completely in suspension are formed from this
which are
able to produce rrtroviral vectors to the same e:ctent as adherent retroviral
producer cell
lines.
Rctroviral producer cell lines produced in this manner allow a much simpler
large-scale
culture compared to adherent retroviral producer cell lines in stirred
fermenters or high
density dialysis culture methods.
In addition the methods for isolating the vector viruses also differ. Whereas
in the case of
adherent retroviral producer cell lines the culture supernatants containing
retroviruses only
have to be removed and a sterile filtration step, although not absolutely
necessary, is
nevertheless always carried out for safety reasons, in the case of retroviral
producer cell
lines growing completely in suspension the much smaller, lighter retroviral
vector particles
are separated from the larger heavier producer cell lines by a filtration and
centrifugation
step or at least two filtration steps.
Retroviral vectors which contain a therapeutic gene are used as retroviral
vector plasmids
or retroviral vectors. These vectors contain no packaging sequences like gag,
pol, env in
order to prevent release of replication-competent retroviral vectors during
production in
the suspension producer cell line. The therapeutically relevant gene is
usually inserted
between 5'-LTR and 3'-LTR and it is e:cpedient that selection genes such as
the neomycin
or hygromycin resistance gene are present.
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A replication-incompetent (replication-deficient) vector is understood as a
vector which
contains no retroviral gene functions (gag, env, pol) and hence cannot
independently form
virus particles. However, the vector usually contains a packaging function
(psi). In order to
form infectious retroviral particles a packaging cell line which contains the
gene functions
gag, env and pol in a stable form (as an episome or integrated into the
genome) is
transduced with a replication-deficient DNA vector. RNA transcripts are formed
which are
packaged into the virus particles as a result of the gag and env functions.
These virus
particles are replication-deficient because the RNA genome that they contain
carries no
retroviral gene functions.
lU Abbreviations
MoMuLV moloney murine leukaemia virus
FCS foetal calf serum
~ LNGFR low affinity nerve growth factor receptor in which the intracellular
domain is deleted (cf. WO 95/06723)
SV40-PlE promoterlenhancer unit of SV40
neon neomycin resistance gene
pBR 322 E. coli base vector plasmid
'1' 25 cell culture flask size
CMV-PlE promoter/enhhancer unit of CMV (cf. e.g. EP-B 0 173 177)
TK-P herpes simple:c thymidine kinase promoter
Poly-A polyadenylation site
hygR hygromycin resistance gene
gpt gpt selection gene
pUCl9 base vector
pUC20 base vector
5'LTR MoMuLV 5'LTR from MoMuLV
cfu colony forming units
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The invention is further elucidated by the following examples and publications
the scope of
which is derived from the patent claims. The described methods are to be
understood as
examples which still describe the subject matter of the invention even after
modifications.
Example 1 Adaptation of adherent retroviral packaging cell lines to growth in
suspension
The adherent packaging cell lines FLY A13 (amphotropic env from MoMuLV) and
FLY
RD18 (env from cat endogenous virus RD114) (Cosset, F.-L. et al., J. Virol. 69
(1995) 7430
- 7436) which are both based on the human fibrosarcoma line HT1080, were
adapted to
suspension growth by the following process steps:
a Adaptation of the adherent packaging cell lines to growth in suspension:
I. Continuous and slow FCS reduction in the medium; duration ca. 5 months;
10 % FCS ->S °/~ ->2.5 % ->2 % -> 1 % ->0.5 % ->scrum free
II. Use of a medium for suspension cell lines
III. Use of culture flasks for suspension cell lines (Sarstedt)
IV. Continuous gentle shaking of the culture flasks on a shaker
V. The maintenance requires a frequent detachment of the clumps of cells that
form by
trypsin and
DNAse treatment and frequent splitting so that they remain in suspension.
The cell density is always a critical factor (5.10' - 1.106 cells/ml) i.e. it
must not be
too high or too diluted.
b) Determination of the retrovirus titre of transient cultures
The packaging cell lines growing in suspension were compared with the
corresponding
packaging cell lines growing adherently with regard to production properties.
For this all cell lines were transfected with the retroviral vector plasmid
and the retrovirus
23 titre of the transient cultures was determined.
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Materials:
*retroviral vector pLONSN (6853 bp)
(-5'LTR MoMuLV - ur - ~LNGFR - SV40-PIE - neoR - 3'LTR MoMuLV - pBR322 -
plasmid part-]
*transfection reagent: DOSPER; in each case 20 pg DOSPERImixture
(manufacturer: Boehringer Mannheim GmbH, GER)
*plasmid DNA cone: 5 pg DNAlmirturelplasmid
Methods:
1" day: seed cells for the transfection:
adherent cells: 6x105 cellsldish in 60 mm petri dishes
suspension cells: 3x105 cellslml; in culture flasks for suspension cells (T25,
Sarstedt,
upright); total of 2 ml.
incubation overnight at 37°C, 5 % C02, suspension cells are shaken
gently.
2"'~ day: change of medium (by centrifugation and resuspension in the case of
suspension
cells), DNA/DOSPER mirture is prepared and added dropwise onto the cells (100
pl
DNA/DOSPER mirture in each case).
incubation for 5-6 h, 37°C, S % COz. subsequently addition of medium.
3"~ day: the supernatant is removed 24 h after the transfection, centrifuged
(only in the case
of suspension cell lines!), filtered (0.45 ~m filter) and titrated on NIH3T3
cells
(G418 selection).
titration result ca. 10 - 14 days later
Titre:
adherent cell lines FLY A13: 8x105 cfulml
2x105 cfulml
FLY RD18: previously not determined
cell lines brought into suspension: FLY A13: 2.4x10' cfu/ml
FLY RD18: lxl0i cfu/ml
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Result:
The packaging cell Lines brought into suspension are in principle able to
produce
recombinant retroviruses. Differences in the titre are due to the different
e.~cperimental conditions in the transfection and in the titre determination
e.g.
different transfection efficiencies in suspension cells and adherent cells and
differences in the stability and infectiousness of retroviruses in FCS-free
and FCS-
containing medium.
Example 2: Retrovirus production after transient triple transfection of the
two helper
plasmids and the retroviral vector plasmid into suspension cell lines
An adherent cell line and several cell lines growing in suspension were
compared in
transient triple transfection preparations with regard to their ability to
form infectious
retroviral vectors.
Cell lines used:
adherent cell line (control) NII-13T3 (mouse fibrosarcoma)
suspension cell lines: K562 (human erythroleukaemia cell line)
N1-S1 (rat hepatoma)
C58 (rat lymphoma)
Material:
a) Helper plasmids:
*gag-pol expression plasmid pGAPOGPT ( 10202 bp)
(-CMV-P/E-gag-pol-polyA-SV40-PIE-gpt-polyA-pUCl9-plasmid part-]
*ENV e.~cpression plasmid pENVCHT (7933 bp)
(-TK-P-hygR-polyA-CMV-P/E-env (amphotropic)-IpolyA-pUC20-plasmid part-J
b) retroviral vector plasmid:
*retroviral vector pL~.NSN (6853 bp)
[-5'LTR MoMuLV - y~ - ~LNGFR - SV40-PIE - neon - 3'-LTR MoMuLV - pBR322-
plasmid part-]
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*transfection reagent: DOSPER (BM); 20 Pg DOSPER/mixture in each case
*plasmid DNA cone: 5 ~g DNA/mixture/plasmid
Methods:
ls~ day: seed cells for the transfection:
adherent cells (NIH3T3): 6x105 cells/dish in 60 mm petri dishes
suspension cells: 3x105 cells/ml; in T25 culture flasks (for suspension cells;
upright); total of 2 ml,
incubation overnight at 37°C, 5 % COz, suspension cells are shaken
gently.
2"'~ day: change of medium (by centrifugation and resuspension in the case of
suspension
cells), DNAIDOSPER mixture is prepared and added dropwise onto the cells ( 100
pl
DNAIDOSPER mix in each case).
incubation for S-6 h, 37°C, 5 % CO~. subsequently addition of medium.
3"~ day: the supernatant is removed 24 h after the triple transfection,
centrifuged (only in
the case of suspension cell lines!), filtered (0.45 Pm filter) and titrated on
NIH3T3
cells (G418 selection).
titration result ea. 10 - 14 days later
Titration result on NIH3T3 (number of 6418 resistant colonies):
NIEI3'r3 (adherent control cells): 3 colonies per 1.4 ml virus supernatant
(titre: 2
cfu/ml)
K562: 6 colonies per 0.8 ml virus supernatant (titre: 7.5 cfulml)
N1-Sl: 6 colonies per 2 ml virus supernatant (titre: 3 cfu/ml)
C58: 1 colony per 2 ml virus supernatant (titre: 0.5 cfu/ml)
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List of References
Cosset, F.-L. et al., J. Virol. 69 (1995) 7430-7436
Danos, O. et al., PNAS USA 85 ( 1988) 6460-6464
Markowitz, D.G. et al., Trans. Assoc. Am. Physicians 101 (1988) 212-218
Miller, A.D. et al., J. Virol. 65 (1991) 2220-2224
Miller, A.D. et al., Mol. and Cell Biol. 6 ( 1986) 2895-2902
Rigg, R.J. et al., Virology 218 ( 1996) 290-295
WO 92/05266
