Vol. 151, No. 1, 1988 February 29, 1988
BIOCHEMICALAND
BIOPHYSICALRESEARCH
COMMUNICATIONS Pages 201-206
INFECTION OF HUMANHEMATOPOIETICPROGENITOR CELLS USING A RETROVIRALVECTORWITH A XENOTROPIC PSEUDOTYPE Martin
A. EglitisI, R. Michael
1 Laboratory Blood Institute National Received
January
Donald B. Kahn*+, Blaese* and W. French
Robert C. MoenI, Andersonl*
of Molecular Hematology, National Heart, Lung, and and * Metabolism Branch, National Cancer Institute Institutes of Health, Bethesda, Maryland 20892
4,
1988
In an effort to determine if viral envelope type influences the infectivity of human hematopoietic progenitor cells with retroviral vectors, we have pseudotyped the retroviral vector N2, which confers G418-resistance, in either an amphotropic or xenotropic envelope. Vector titres obtained by the pseudotype procedure were nearly two orders of magnitude lower than the titer obtained when N2 was packaged using the amphotropic PA317 packaging cell line. Despite its low titer, xenotropically pseudotyped N2 generated G418-resistant hematopoietic colonies at levels approaching those observed after bone marrow was infected using vector packaged using PA317 cells. These results suggest that manipulations of vector envelope may lead to improvements in the level of infection of human hematopoietic stem cells. 0 1988 AcademicPm**, Inc.
Several
laboratories
hematopoietic
progenitors
resistance of the
To produce competent
proteins
envelopes
class,
* +
classes
not to
envelopes amphotropic
growth
drug.
In these
infection
infection
are
not
only present
envelopes,
which, upon
of other on the
Transfer between
either line
must
has a wide
1 and 20% of
to target viruses,
properties
and to a lesser
host
presence
be used to provide
leukemia
host-range species.
in the
a replication-
of retroviruses
species,
of a drug
resistant.
in murine
its
mammalian
also
drug
of human
in vitro
studies,
cell
of mouse cells
of other cells
(l-4).
virions,
The binding
protein
infection
of colonies were
a "packaging"
depending
cells
infect
by the
necessary.
envelope
permit
generally
or
successful vectors
vector-derived
virus
by the
the
retroviral
vector
infectious
one of three
these
from
helper
structural
viruses
selective
recovered
mediated
with
gene was detected appropriate
colonies
have reported
Xenotropic since
envelopes
the
receptors
cell
surface.
host
range,
To whom all correspondence should be addressed Current address: Division of Research Immunology/Bone Transplantation, Children's Hospital of Los Angeles, Los Angeles, California 90027
but
cells,
but
enable which
bind
The third
differs
Marrow 4650 Sunset
from
the
Boulevard,
0006-291X/88 201
is into
Ecotropic rat
species'
cells falls
(5). extent
the
$1.50
Copyright 0 1988 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 151, No. 1, 1988 xenotropic
class
homologous
and heterologous
envelope with
of viruses
classes
distinct
result cell
in that
amphotropic
cells.
The different
largely
surface
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from
molecules
the
viruses host
envelope
which
act
can infect ranges
both
of the
glycoproteins
different
interacting
as receptors
for
viral
binding
and uptake. To infect packaging
human cells systems
(amphotropic) cells
have
murine
obtained
from
with been
retrovirus
several of the type
4070A
compared
MATERIALS
it
is
envelope.
the
infectivity
of
in an amphotropic
murine
is
derived
from
virus
tests
was isolated
to
(8).
the
to date,
hematopoietic
Although
limited
indeed
investigate
the
infectivity
is
a specific
in a xenotropic
of of human
low role
4070A
cultured
capable
of human bone marrow packaged
the
amphotropic
infectivity
the
viruses,
from
as being
progenitors,
infectivity a vector
from
envelope
This
that
In an effort improving
derived
and was characterized
possible
reported
non-murine
in potentially
same vector
(6-7). mouse,
progenitors
different
the
and interference
human cells,
hematopoietic
vectors
used where
a feral
by cross-neutralization infecting
retroviral
of viral cells, envelope
of quality envelope we to the
coat.
AND METHODS
Media and Chemicals All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% fetal bovine serum (Hyclone) and supplemented with 6418 (Gibco) was prepared as a fresh glutamine (0.03% final concentration). stock solution in 50 mM HEPES at 50 mg (active 6418, approximately 50% of dry weight)/ml, and was added to culture medium at a final concentration of 400 ug (active G418)/ml (for fibroblasts) or 1200 ug/ml (for colony assays). Cell Lines and Viruses The PA317 packaging cell line, capable of packaging vectors in an amphotropic (4070A) coat, was kindly provided by Dr. A. Dusty Miller and has been described previously (9). The Mv 1 Lu (NBL-7) mink lung cell line (10) was the generous gift of Dr. Janet Hartley, who also provided the wild type 4070A amphotropic retrovirus and the 2776X xenotropic retrovirus. Vector Construction of the N2 retroviral vector, derived from the Moloney muri e leukemia virus and containing the bacterial neomycin phosphotransferase (neo R) gene, has been described previously (11). The N2 retrovirus was introduced as the plasmid pN2 into either PA317 cells or mink cells by calcium phosphate transfection (12). After transfection, cells were allowed to grow for 48 hr, then were selected continuously in 400 ug (active) G418/ml. Bone Marrow Infection Nucleated bone marrow cells, isolated by centrifugation over Ficoll-hypaque (LSM, Litton Bionetics), were infected as previously described (13) with the N2 vector by plating onto a nearly confluent monolayer After co-cultivation for 18 to of irradiated (24 Gy) vector-producing cells. 21.5 hr, the bone marrow cells were recovered by centrifugation and resuspended for plating into colony assays. Hematoooietic Colonv Assav Hematopoietic progenitors (CFU-E, BFU-E, and CFUGM) were grown as previously described (14) in Iscove’s Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 30% prescreened heat-inactivated bovine fetal serum, 10% phytohemagglutinin-stimulated lymphocyte conditioned medium as a source of colony-stimulating factors, 1% bovine serum albumin, 2.5 units/ml CFU-E were erythropoietin (TCepo, Amgen Biologicals) and 1.2% methylcellulose. counted after 7 days culture at 37O C in 5% C02. BFU-E and CFU-GM were counted after 14 days culture.
202
Vol.
151,
No.
BIOCHEMICAL
1, 1988
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS Mink medium
cells
transfected
obtained
from
retrovirus,
or the
populations
of NP-Mink
before
their
cells mink
cells,
mink
cell
itself, that
by N2-Mink
produced more
confirming
cells
then
on murine
cells
pN2 plasmid
preparation,
free
of any contaminating
capable
helper
cells
PA317
(Table
packaging the
Table
I
virus. were
were
The titre line
or on as the
resistance
titres
1A). titer
as well
6418
virus, cell
than
NIH-3T3
helper
of transferring with
of magnitude
and infection
either
were
orders
(cfu)
one month N2-Mink
the
as on mink
over
1, G418-resistant
titre,
infected
for
units
in Table
with
murine
Such infected
colony-forming
both
cells
by incubation
amphotropic
in culture
that
amphotropic
three
infected 4070A
retrovirus.
grown
As shown
viruses
as well
by the
than
were
the
murine
had no detectable
line
NIH-3T3
cells
were
either
xenotropic
determined.
establish
produced
pN2 (NP-Mink)
producing
of G418-resistant
were
themselves
To
2776X
titre
restrictions
with
cells
determined
A.
Host
Range
(PA317-N2)
was greater
of N2 vector
produced
Supernatantb
NIH-3T3
Cella
B. Interference Pattern PA317 4070A (NIH-3T3) (Mink)
Viral Mink
2776X (Mink)
NZ-Mink
CO
to
to
to
PA317-N2
6.70
5.70
3.70
2.60
6.28
4070A-N2
Mink
3.28
4.00
2.00
1.18
4.30
2776X-N2
Mink
to
4.00
3.60
0.90
a Target
Cells: NIH-3T3 = transformed murine fibroblast Mink = Mu 1 Lu mink lung cells PA317 = 4070A amphotropic retroviral NIH-3T3 cells 4070A Mink = mink cells infected with for over one month 2776X Mink = mink cells infected with for over one month
b Supernatants: PA317-N2 NZ-Mink 4070A-N2 2776X-N2
c Titers colony
per
line packaging
line
derived
as the logarithm supernatant.
base
203
10.
from
4070A
virus
and
passaged
2776X
virus
and
passaged
= PA317 helper cells transfected with pN2 = mink cells transfected with pN2, G418-selected population Mink = NE-Mink cells infected with 4070A virus passaged for over one month Mink = NZ-Mink cells infected with 2776X virus passaged for over one month
expressed ml viral
Limit
of
on
of N2 vector
HOST RANGE AND INTERFERENCE PATTERN OF PSEUDOTYPED N2 VECTOR AS DETERMINED BY TITERING ON VARIOUS TARGET CELLS
Target
being
sensitivity
and and
< 1
by
by N2-
Vol.151,
Mink
No.
cells
infected
infected titre
BIOCHEMICAL
I,1988
with
with
the
detectable
produced cells.
mink
that in
cells 4070A
do not still
the
its
similar of with
used
to
similar
(Table
mink
one another, with
2776X of
infect
measured
envelope established
restrictions
of
infections of
is
could
progenitors
were
4070A
or pseudotyped
to the
cells
by over
cells
three
titre
expressing
and a summary Using
2.
infected
the the
had the were
of the
proper performed.
infection
more primitive
PA317-N2,
and expressing
between neomycin
2
PSEUDOTYPED INFECTIONS OF HUMAN MARROW PROGENITORS N2 Packaged Using: PA317
4070A
(0.5%)
l/96:=1
(0.1%)
o/2095 (
BFU-E
28/3548 (0.8%) n=2
7/43e2
(0.2%)
o/4014 (<0.03%) n=2
CFU-GM
6/4234 (0.2%) n=2
CFU-E
6/1199 n=l
2776X
n, number of separate
4/36;X2(0.1%)
infections 204
very
of magnitude.
actually
as the
is
N2 was
the
of human bone marrow as well
on the packaged
expressing
orders
N2 vector
on both
titres
Conversely,
infect
Table
Colony Type
should
of
G418-resistant
of mink
in Table
envelopes
of magnitude
cells.
performed,
shown
expressing
with
PA317 cells
orders
the
mink
the CFU-E compartment,
CFU-GM compartments, of bone marrow
cells,
infections
using
2776X line
different
pre-infected
compared
a pseudotyped were
cell
when amphotropically
infection
was suppressed that
2776X-N2
performed
or the with
by using
cells
N2 vector
however,
infectivity,
Two independant efficiencies
mink
were
The infectivity
by three
on uninfected
pseudotyped envelope;
on NIH-3T3
vector.
In contrast,
2776X-producing
to that
cells
whether
mink
tests
Viruses
was found.
was suppressed cells.
vector
N2 had appropriate
amphotrope
1B).
pseudotyped
infection
and 4070A-producing
amphotropic Having
cells,
cells,
on mouse cells,
PA317 packaging
so that
N2 vector,
uninfected
xenotropically xenotropic
4070A
surface
PA317 cells respective
the
on its
amphotropically-packaged 4070A
either
envelope
restriction
had no
same vector
titre
interference
derived
be infectable
with
infection,
NIH-3T3
Specific
On mink
of the
pseudotyped
as the with
cells.
of detectable
cells
be expected,
to 4070A-N2.
as well interfere
N2-Mink
as would
10% that
xenotropically
range
(4070A-N2).
NIH-3T3
absence
cells
pre-infected
xenotrope,
amphotrope (2776X-N2),
had a titre
to the
on mink
To confirm
the
cells
In contrast
restrictions
4070A
xenotrope
on the mouse-derived
by PA317-N2
had a titer
the
2776X
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
O/3251 (~0.03%) n=2
BFU-E and 0.2
and 0.8%
BIOCHEMICAL
Vol. 151, No. 1, 1988
phosphotransferase selected
in
identical
1200
but
with
independent
that after
with
a titer mink
2776X-N2
as robust
PA317-N2
only
cells
vector
robust
colonies
same selection
generated
did
and were
the
2% of PA317-N2,
Even though
infections.
were
to generate Using
G418/ml.
4070A-N2
of 4070A-N2,
colonies
sufficient
ug (active)
envelope,
pseudotyping two
at levels
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
no 6418 titer
generate
generated
and
vector
produced
resistant
colonies
of 2776X-N2
by in
was as low
G418-resistant
at a rate
when
conditions
as
colonies.
nearly
equal
These
to that
seen
infection.
DISCUSSION We have
generated
pseudotyping
in mink
to investigate could
be obtained
range
with
infected
generated, nearly the
an envelope
with the
fact
of magnitude
amphotropic
pseudotyping
with marrow
the
4070A
colonies,
xenotropically-pseudotyped 2776X-N2
has an increased cells,
which
cells
studies
with
of both
its
packaged
of 6418 well
proportion
the
earlier.
of criteria could
obtained
infectivity
account
studies for
the for
which
transfer
scoring
in part
the
reduced
its
surface
of the
produced
using
by
is
of
possible
xenotropic
that
envelope
human target
to PA317. have
studies
described
inhibit
survival here
here,
infection
we have used
of uninfected
is
generally
selection
proportion
progenitor
reported
progenitors.
or possible
lower
by
to that It
because
vectors
of the
used to
successfully
no G418-resistant
cells.
relative
reported
colonies, for
produced
in infected
colonies
N2 was then were
its
pattern
of human hematopoietic
retroviral
The stringency
for
in these
These avenue
amounts
of gene
of G418-resistant
reported
choice
above
rate
titer
In the
interference
was identical
on mink
infection
in
N2 generated
titer
on the
lower the
BFU-E and CFU-GM (l-4).
levels
6418,
retrovirus its
colonies
4070A
same vector
Significantly,
lower
conditions variations
colonies
However,
the
than
used, in lots
of G418-resistant
the of
colonies
experiments. show that
development human target
alternative of retroviral cells.
packaging vectors Although
205
systems that these
it
was demonstrated
N2 has a titer
to the
used
cells
restricted
xenotropic
amphotropic
for
the
envelope
colonies
even though
describing
amphotropically
to determine those
line.
to receptors
compensates
Previous
This
in comparison
by
and have
than
proper
pseudotyped
G418-resistant
affinity
other
the
N2 when measured
may generate
retrovirus,
of the
exhibited
the
PA317 packaging
human bone
from
G418-resistant
that
(N2)
of human bone marrow
nature
viruses.
cells.
2776X
vector
N2 was appropriately it
different
marrow
two orders
derived
and that
retroviral
in infection
2776X-pseudotyped
despite
the
The xenotropic
that
human bone
with
improvements
of infection
on cells
packaged
infected
retrovirus.
by determining
infect
cells
whether
amphotropic host
a xenotropically
may be a fruitful
have a greater experiments
did
not
result
Vol. 151, No. 1, 1988
in
efficiencies
greater
amphotropic packaging higher
packaging cell
titres
for
the
already lines,
comparable
of vector
could
obtained the
yield
with
development improvements
Ultimately,
of gene
of genetic
AND BIOPHYSICAL
transfer
such to permit
RESEARCH COMMUNICATIONS
vectors
packaged
of helper-free
to PA317 and identification
stem cells.
of techniques treatment
than cell
lines
human hematopoietic refinement
BIOCHEMICAL
in the studies their
with
xenotropic of clones
infection
efficiency
may contribute clinical
producing of to the
application
disease.
ACKNOWLEDGEMENTS We gratefully acknowledge Drs. Robert Bassin, Sandra Ruscetti and Linda Wolff for sharing their insights. We are also appreciative of Dr. Janet Hartley for providing the wild type retroviruses, as well as for her advice. In addition, members of our gene transfer research group provided useful criticisms and suggestions. Sheri Bernstein, Martha Feller and Rinna Connol provided expert technical assistance.
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5. 6.
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