Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype

Infection of human hematopoietic progenitor cells using a retroviral vector with a xenotropic pseudotype

Vol. 151, No. 1, 1988 February 29, 1988 BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 201-206 INFECTION OF HUMANHEMATOPOIETICPROGENITOR ...

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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.

REFERENCES 1. 2. 3. 4.

5. 6.

Gruber, H.E., Finley, K.D., Hershberg, R.M., Katzman, S.S., Laikind, P.K., Seegmiller, J.E., Friedman, T., Yee, J.K., and Jolly, D.J. (1985) Science 230, 1057-1061. Hock, R.A., and Miller, A.D. (1986) Nature 320, 275-277. Hogge, D.E., and Humphries, R.K. (1987) Blood 69, 611-617. Eglitis, M.A., Kantoff, P.W., McLachlin, J.R., Gillio, A., Flake, A.W., Bordignon, C., Moen, R.C., Karson, E.M., Zwiebel, J.A., Kohn, D.B., Gilboa, E., Blaese, R.M., Harrison, M.R., Zanjani, E.D., O'Reilly, R., and Anderson, W.F. (1987) in Molecular Approachesto Human Polygknic Disease, Ciba Foundation Symposium 130 (G. Bock and G.M. Collins, eds.), pp. 229-246. Wiley, Chichester. Teich, N. (1982) in RNA Tumor Viruses (R. Weiss, N. Teich, H. Varmus, and J. Coffin eds.), pp. 25-207. Cold Spring Harbor Laboratory. Miller, A.D., Law, M.F., and Verma, I.M. (1985) Mol. Cell Biol. 5, 43 1

437. 7. 8. 9. 10.

Cone, R.D., and Mulligan, R.C. (1984) Proc. Natl. Acad. Sci. USA 81, 6349-6353. Chattopadhyay, S.K., Oliff, A.I., Linemeyer, D.L., Lander, M.R., and Lowy, D.R. (1981) J. Virol. 39, 777-791. Miller, A.D., and Buttimore, C. (1986) Mol. Cell Biol. 6, 2895-2902. Henderson, I.C., Lieber, M.M., and Todaro, G.J. (1974) 60, . _ Virology

282-287.

11. 12. 13. 14.

Armentano, D., Yu, S.-F., Kantoff, P.W., von Ruden, T., Ande son, W.F., and Gilboa, E. (1987) J. Virol. 61, 1647-1650. Wigler, M., Silverstein, S., Lee, L.S., Pellicer, A., Cheng, Y.C., and Axel, R. (1977) Cell 11, 223-232. Eglitis, M.A., Kantoff, P., Gilboa, E., and Anderson, W.F. ( 985) Science 230, 1395-1398. Gregory, C.J., and Eaves, A.C. (1977) Blood 49, 855-864.

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