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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 401-410
Efficient Gene Delivery to Quiescent Interleukin-2 (IL-2)-Dependent
Cells by Murine Leukemia Virus-Derived Vectors Harboring IL-2 Chimeric
Envelopes Glycoproteins
By
Marielle Maurice,
Stéphane Mazur,
Frances J. Bullough,
Anna Salvetti,
Mary K.L. Collins,
Stephen J. Russell, and
François-Loïc Cosset
From the Laboratoire de Vectorologie Rétrovirale et
Thérapie Génique, Unité de Virologie Humaine, INSERM
U412, Ecole Normale Supérieure de Lyon, Lyon, France; the Centre
de Génétique Moléculaire et Cellulaire, CNRS UMR5534,
Université Claude-Bernard Lyon-1, Villeurbanne, France; the
Cambridge Centre for Protein Engineering, MRC Centre, Cambridge, UK;
the Laboratoire de Thérapie Génique, CHU Hotel-Dieu,
Nantes, France; the Department of Immunology, Windeyer Institute for
Medical Science, University College London, London, UK; and Molecular
Medicine Program, Guggenheim 18, Mayo Clinic, Rochester, MN.
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ABSTRACT |
Interleukin-2 (IL-2) is a cytokine that induces the proliferation of
certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused
to the amino-terminus of amphotropic murine leukemia virus (MLV)
envelope glycoproteins. Retroviral vectors were pseudotyped with both
the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope.
The chimeric IL-2 glycoproteins were incorporated on retroviral vectors
and the IL-2-displaying vector particles could bind specifically to
cell surface IL-2 receptors. In addition, the IL-2-displaying vectors
could infect proliferating cells through amphotropic receptors
irrespective of whether the cells expressed the IL-2 receptor.
IL-2-displaying vector particles could also transiently stimulate the
cell cycle entry and proliferation of several IL-2-dependent cell
lines. Finally, retroviral vectors displaying IL-2 could efficiently
transduce G0/G1-arrested cells expressing the IL-2 receptor at a
34-fold higher efficiency compared with vectors with unmodified
envelopes. This new strategy, whereby C-type retroviral vector
particles display a ligand that activates the cell cycle of the target
cells at the time of virus entry, may represent an alternative to
lentivirus-derived retroviral vectors for the infection of quiescent
cells. In addition, upon infection of an heterogeneous population of
nonproliferating cells, MLV-retroviral vectors that display
cytokines/growth factors will allow the transgene of interest to be
integrated specifically in quiescent cells expressing the corresponding
cytokine/growth factor receptor.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GENE THERAPY PROTOCOLS will benefit from
the design of vectors that permit the transfer of therapeutic genes
directly to target cells and tissues in vivo. This strategy requires
that the therapeutic transgene should be efficiently and accurately delivered and stably expressed in the target cells. To date, retroviral vectors derived from murine leukemia viruses have been used in the
majority of gene transfer protocols,1 because, in addition to the simplicity of their manipulation, the transgene is stably integrated into the target cell DNA. However, these vectors suffer a
number of drawbacks that need to be overcome before they can be of use
for in situ gene transfer approaches. In particular, in a quantitative
and specific manner, they have to be optimized such that they reach the
target cells and are integrated in the genome of cells that are often quiescent.
Integration of murine retroviruses requires cell division, and in
nonproliferating cells the block in the integration process occurs at
the level of entry of the viral preintegration complex into the
nucleus. This complex, containing the proviral DNA and an assembly of
viral proteins necessary for its integration in the host DNA, can only
enter the nucleus after the disruption of the nuclear membrane, ie,
during mitosis.2 This characteristic is not common to all
retroviruses. Indeed, lentiviruses are able to infect certain types of
quiescent cells.3 Several characteristics specific to the
lentivirus subfamily, including the expression of certain viral
accessory proteins such as vpr, as well as distinct features harbored
by their matrix and integrase proteins appear to play an important role
in their ability to infect quiescent cells, particularly
macrophages.4-8
Strategies to obtain retroviral vectors capable of infecting quiescent
cells have therefore been based on the development of
lentivirus-derived packaging cell lines and vectors or, alternatively, on the introduction of human immunodeficiency virus (HIV) determinants into murine leukemia virus (MLV) retroviral vectors to permit their
integration in quiescent cells. Several groups have reported the
generation of lentivirus-derived vectors that are capable of infecting
quiescent cells,7 but there are still concerns regarding
the safety of these vectors for human gene therapy. Furthermore, some
experiments suggest that not all quiescent cells can be transduced by
lentiviral vectors. For example, HIV cannot integrate in certain cells
blocked at the Go phase of the cell cycle (such as T lymphocytes in
vivo), and the cells must be in an active metabolic state to permit
completion of reverse transcription and integration.3,9-11
An alternative strategy for the stable transduction of quiescent cells
is to engineer growth factor domains onto the surface of the retroviral
vector particles so that they can stimulate receptors on the target
cell surface, inducing transient proliferation of the target cells at
the time of gene delivery.
We describe here the construction of a novel chimeric MLV retroviral
envelope glycoprotein containing the interleukin-2 (IL-2) polypeptide.
IL-2 is a cytokine that induces the proliferation of certain IL-2
receptor-expressing quiescent cells. The IL-2 chimeric envelopes were
coexpressed in retroviral vector particles with wild-type envelopes to
allow infection to proceed through the natural retroviral receptor. We
found that IL-2 receptor-expressing target cells were transiently
activated when they were incubated with retroviral vectors displaying
the IL-2 chimeric envelope glycoprotein. Moreover, the IL-2
receptor-positive target cells were transduced at a 34-fold higher
efficiency by vectors pseudotyped with the IL-2 chimeric envelope than
by vectors carrying wild-type amphotropic envelopes.
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MATERIALS AND METHODS |
Cell lines.
The TELCeB6 cell line12 was derived from the TELac2
line13 after transfection and clonal selection of TE671
cells (ATCC CRL8805; American Type Culture Collection, Rockville,
MD) containing a plasmid expressing Moloney murine
leukemia virus (MoMLV) gag and pol proteins. TELCeB6 cells produce
noninfectious viral core particles carrying an nlsLacZ reporter
retroviral vector, whereas TELac2 cells express only the nlsLacZ
reporter retroviral vector. TELCeB6 were grown in Dulbecco's modified
Eagle's medium (DMEM; Life Technologies,
France) supplemented with 10% fetal bovine serum.
The F7 cell clone (kind gift of T. Taniguchi), derived
from the BAF-BO3 bone marrow murine pro-B-cell line,14
expresses the three IL-2 receptor subunits ( ,  , and  ) as well
as bcl-2, an antiapoptotic protein.15 Proliferation of F7
cells is dependent on the addition of exogenous either IL-3 or IL-2
cytokines. F7 cells were grown in RPMI 1640 (Life Technologies)
supplemented with 10% fetal bovine serum and 2 ng/mL of recombinant
human IL-2 (R&D Systems, France).
The Bclp75 and W4E9 cells were derived from the IL-3-dependent BAF3
cell line, murine pro-B-cell line.16 They
have been rendered responsive to IL-2. The Bclp75 cells express the
human IL-2 receptor subunit as well as Bcl-2. The W4E9 cells
express high-affinity human IL-2 receptors ( and IL-2 receptor
subunits). These cells were grown in DMEM supplemented with 10% fetal
bovine serum and 0.1% IL-3-conditioned medium.
The IL-2-dependent T-helper murine cell line HT-2 (kind gift of S. Zurawski) was grown in RPMI 1640 supplemented with 10% fetal bovine serum and 10 10 mol/L of murine IL-2
(mIL-2).17
Kit225 cells (kind gift of P. Lecine) are an
IL-2-dependent T-cell line derived from a patient with chronic
T-lymphocytic leukemia.18 They were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mmol/L of L-glutamine, and
20 ng/mL of IL-2.
J3-13 cells (kind gift of T. Taniguchi) are derived from the NIH3T3
murine fibroblasts and express the human IL-2 receptor as well as jak3
Janus kinase that renders them responsive to IL-2.19 J3-13
cells were grown in DMEM (Life Technologies) supplemented with 10%
fetal bovine serum.
Plasmids and transfection.
The unmodified wild-type amphotropic 4070A MLV envelope20
was encoded by the expression plasmid FBASALF that also expresses the
phleo selectable marker conferring resistancy against
phleomycin.12
The human IL-2 cDNA (396 bp) was obtained by polymerase chain reaction
(PCR) by using the following pair of primers: OUIL2SfiI, 5'-CAT
AAT GGC CCA GCC GGC CAT GGC CGC ACC TAC TTC AAG TTC TAC; and OLIL2NotI,
5'-TGT CCA GCG GCC GCA GTT AGT GTT GAG ATG ATG C, which contain
Sfi I and Not I sites at the 5' and 3'
ends, respectively. The Sfi I/Not I PCR fragment,
containing the IL-2 cDNA without the IL-2 signal peptide sequence and
stop codon, was cloned in the +1 position of the 4070A MLV envelope
glycoprotein in FBASALF (Fig 1) by using
the EXA1 adapter plasmid21 that provides Sfi I and
Not I sites at the 5' end of the sequence corresponding to the surface subunit (SU) of the amphotropic envelope glycoprotein. To reduce steric hindrances between IL-2 and the receptor binding domain of the amphotropic envelope, a 7-amino acid linker (FX) was
inserted between these two domains. The resulting plasmid FBIL2A1FXSALF
codes for a chimeric envelope (IL2-SU).
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| Fig 1.
Schematic representation of envelope expression
constructs. The human IL-2 cDNA was fused to the 5' end of the
amphotropic MLV env gene at the first codon of the SU envelope subunit.
A schematic diagram of the IL2-SU polypeptide encoded by this chimeric
gene is shown. A 7-amino acid-long interdomain spacer was inserted
between the cytokine and amphotropic receptor binding domain to reduce
steric hindrances and optimize the display of IL-2. In the IL2-SUX
chimeric envelope derived from IL2-SU, mutations were introduced in the
SU/TM cleavage site to prevent cleavage of the envelope precursor and,
thus, SU shedding. The amino acid sequence of the interdomain spacer
between IL-2 and the SU protein as well as that of the SU/TM cleavage
site are shown. L, leader-signal-peptide; IL2, IL-2; SU, surface
envelope subunit; TM, transmembrane envelope subunit.
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Two PCR fragments were generated on the matrix FBASALF and cloned in
the FBIL2A1FXSALF backbone digested with BamHI and Cla I to introduce a mutation between the SU and TM subunits of the envelope glycoprotein without changing the reading frame. This mutation
results in the inactivation of the cleavage site located between the SU
and TM subunits. The first PCR fragment (229 bp) was obtained with a
5' oligonucleotide located upstream of the BamHI
restriction site (UpBamHI, 5'-AGG CCT TAT GTA ACA CCA CC) and a
3' oligonucleotide providing the point mutation and a Pvu I restriction site (LowFX, 5'-TCC TTC GAT CGT ACG CTG TTC AAG CTG
ACC). The second PCR fragment (554 bp) was generated with a 5'
oligonucleotide overlapping LowFX (UpFX, 5'-AAG TAC GAT CGA AGG
AAG AGA GCC AGT ATC ATT GAC C) providing the mutation and a Pvu
I restriction site, and a 3' oligonucleotide located downstream of the Cla I restriction site (LowClaI, 5'-AGC CTG GAC
TAC TGA GAT CC). The first PCR fragment was digested with
BamHI and Pvu I and the second with Pvu I and
Cla I, and they were cloned together into
BamHI-Cla I-digested FBIL2A1FXSALF plasmid, resulting
in the plasmid FBIL2A1SUXSALF. The chimeric envelope encoded by
FBIL2A1SUXSALF plasmid has been named IL2-SUX (Fig 1).
Chimeric envelope glycoproteins expression plasmids were either
transfected or cotransfected with the expression plasmid FBASA (plasmid
encoding the amphotropic 4070A-MLV envelope deprived of selection
marker) by calcium phosphate precipitation into TELCeB6 cells, as
previously described.22 Transfected cells were selected with phleomycin (50 µg/mL; CAYLA, Toulouse, France), and
phleomycin-resistant clones were isolated and screened for the
coexpression of both envelopes by Western blot analysis.
Antibodies.
Anti-gp70 (Quality Biotech Inc, Camden, NJ), a goat antiserum raised
against the Rausher leukemia virus gp70, was diluted 1/2,000 for
Western blots. Anti-CA (Quality Biotech Inc), a goat antiserum raised
against the Rausher leukemia virus p30 capsid protein (CA), was diluted
1/10,000 for Western blots. 83A25, a rat monoclonal antibody against
MLV SU, was used for binding assays.23 Dichlorotriazinyl
amino fluorescein (DTAF)-conjugated affinity-purified F(ab)2 fragment goat antirat IgG (Immunotech, Marseille,
France) was diluted 1/50 for binding assays
Immunoblots.
Lysates of virus producer cells and virus samples were prepared as
previously described.22 Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein transfer
onto nitrocellulose, and immunostaining were also performed as
previously reported.22
Binding assays.
Target cells (Kit225; 800,000 cells/point) were incubated for 1 hour at
4°C with 3 mL of nontransfected TELCeB6 cell supernatant and 3 mL
of viral supernatants supplemented with 8 µg/mL of polybrene. After
virus binding, cells were washed twice with PBA (phosphate-buffered saline [PBS] with 2% fetal bovine serum and 0.1% sodium azide) and
incubated with 83A25 antibody23 supplemented with 0.1%
sodium azide for 30 minutes at 4°C. Cells were washed with PBA and
incubated for 30 minutes at 4°C with DTAF-conjugated
affinity-purified F(ab)2 fragment goat antirat IgG. Five
minutes before the two final washes in PBA and the final resuspension
in PBS, cells were counterstained at 4°C with 20 µg/mL of
propidium iodide. Fluorescence of living cells was analyzed with a
fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson,
France). For the competition binding assay with human recombinant IL-2
(rIL-2; R&D Systems), target cells (Kit225) were preincubated for 30 minutes at 37°C with 100 ng of rIL-2 to provoke IL-2 receptor
downregulation and then exposed to virus. Cells were then processed as
previously described.
[3H]-Thymidine incorporation assay.
Twenty thousand to 40,000 IL-2-dependent cells per well (96-well
plates) were cultivated overnight in medium without IL-2. The cells
were then stimulated, in triplicate, for 24 hours with IL-2 (50 U;
Boehringer Mannheim, Mannheim, Germany) or by adding 70 µL of
filtered viral supernatant (confluent producer cells were cultivated
overnight in DMEM-0% fetal bovine serum). Four hours before the end of
the stimulation, the cells were pulsed with 1 µCi/mL of
[3H]-Thymidine. The 96-well plates were passed in a cell
collector to harvest cells on filters (cell collector TOMTEC MACH III;
Wallac, EG&G Instruments, Finland), and incorporated
radioactivity was measured in a scintillation counter (LS 6000SC;
Beckman Instruments, France).
Cell proliferation and quantification of cell viability.
Cell proliferation and cell viability were quantified by a colorimetric
assay using the cell proliferation reagent WST-1 as per the
manufacturer's instructions (Boehringer Mannheim). This method is
based on the cleavage of the tetrazolium salt WST-1 by mitochondrial
dehydrogenases in viable cells. After cultivation of 5,000 F7 cells for
24 hours without IL-2 on 96-well plates, filtered viral supernatants
(13 µL, produced from confluent producer cells during 8 hours in DMEM
free of serum) or 10 U of recombinant human IL-2 were added. Cell
proliferation was quantified several days later. Quantifications were
performed after 4 hours of incubation with the cell proliferation
reagent, WST-1. The absorbance was measured at 450 nm.
Cell cycle stimulation assays.
F7 cells were arrested in their cycle by IL-2 deprivation when they
were at a confluency of 600,000 to 700,000 cells/mL. To remove IL-2,
cells were washed once with PBS (Life Technologies) and seeded in
24-well plates at a density of 600,000 to 700,000 cells per well. Cells
were deprived of IL-2 for 24 hours. During IL-2 deprivation, the cells
were grown in RPMI 1640 supplemented with 10% fetal bovine serum.
J3-13 cells, seeded in 24-well plates at confluency, were arrested in
their cycle by 48 hours of incubation in DMEM supplemented with 1%
fetal bovine serum. F7 cells and J3-13 cells were stimulated for
different time periods by adding 1 to 2 mL of filtered viral
supernatants, equivalent quantities of ultracentrifuged supernatants,
or 2 ng/mL of IL-2.
At the end of the stimulation period, the cells were centrifuged (F7
cells) or trypsinized and centrifuged (J3-13 cells) and then fixed in
70% ethanol-30% PBS. The cells were stored at 4°C until
fluorescence-activated cell sorting (FACS) analysis. Just before FACS analysis, the fixed cells were pelleted and resuspended in
1 mL of PBS containing 50 µg of RNase and 10 µg/mL of propidium iodide. The cells were incubated for 30 minutes at 37°C and the cell cycle was then analyzed with an FACS (FACSCalibur; Becton Dickinson).
For the kinetics with F7 cells, after the different times of
stimulation, the cells were cultured in medium deprived of IL-2 until a
time period of 24 hours (the longest period of stimulation) and then
fixed in 70% ethanol-30% PBS to be analyzed.
Infection assays.
The confluent virus producer cells were incubated for 3 days at
32°C. Overnight virus production was performed in a medium deprived
of serum. Target cells were seeded in 24-well plates at a density of 5 × 104 cells per well for infection assays on
proliferating cells. For assays on nonproliferative cells, cells were
cultured in DMEM supplemented with 1% fetal bovine serum for 48 hours
once they had reached confluency. Viral supernatant dilutions
containing 5 µg/mL of polybrene were added and cells were incubated
with viruses for 5 hours at 37°C. Viral supernatant was then
removed and cells were incubated in complete medium (for proliferating cells) or in DMEM supplemented with 1% of fetal bovine serum (for nonproliferative cells) for 48 hours. X-Gal staining and viral titer
determination were performed as previously described22 and
expressed as LacZ infectious units (IU)/mL.
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RESULTS |
Construction of mutant envelopes.
Two IL-2-displaying envelope glycoproteins were generated. In the
first envelope, termed IL2-SU, the human IL-2 was fused at the
amino-terminus of the amphotropic MLV SU envelope subunit (Fig 1). This
position of insertion was previously shown to allow the functional
display of various polypeptides at the surface of
virions.22 To optimize its functionality, the IL-2 moiety was separated from the SU by a small linker containing 7 amino acids
(Fig 1), as previously described.24,25 In the second recombinant envelope glycoprotein, termed IL2-SUX, the cleavage site
between the SU and TM envelope subunits of the IL2-SU chimeric envelope
was inactivated by PCR mutagenesis (Fig 1). Thus, in this latter
chimera, the IL-2-displaying chimeric SU subunit is covalently
attached to the TM subunit of the envelope glycoprotein and is
therefore not shed from the surface of the viral particles.
Expression and incorporation of envelopes into virions.
Expression vectors encoding the IL2-SU or IL2-SUX envelope chimeras or
the control amphotropic MLV envelopes, A, were transfected into TELCeB6
cells that express MLV gag-pol core particles and an nlslacZ retroviral
vector. In a separate set of transfections, plasmids encoding the
wild-type amphotropic envelope were cotransfected with either the
IL2-SU or IL2-SUX envelope expression vectors. The expression of a
wild-type envelope glycoprotein was necessary because retroviruses
expressing only IL2-SU or IL2-SUX demonstrated impaired infectivity,
whereas retroviruses pseudotyped with a combination of amphotropic and
chimeric envelopes were fully infectious.22
Lysates of transfected-TELCeB6 cells were analyzed for envelope
expression using antibodies against MLV SU
(Fig 2A). For IL2-SU envelopes, both a
precursor and a processed SU product were detected, suggesting that the
chimeras underwent normal envelope maturation and were correctly
expressed. As expected, a band corresponding to the envelope precursor
was detected for the IL2-SUX chimera but was not processed as a mature
SU protein. A second band, which was more diffuse and exhibited a lower
mobility, was detected and most likely corresponded to a fully
glycosylated form of the IL2-SUX envelope glycoprotein. Expression of
both chimeric envelopes was approximately 20-fold lower than that of
wild-type amphotropic envelopes.
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| Fig 2.
Expression of IL-2 chimeric envelopes. (A) Immunoblots of
lysates of TELCeB6 cells expressing wild-type amphotropic envelopes
(A), IL2-SU, and IL2-SUX chimeras. (B) Immunoblots of viral pellets
obtained by ultracentrifugation of supernatants of TELCeB6 cells
expressing wild-type amphotropic envelopes (A), IL2-SU, and IL2-SUX or
coexpressing wild-type amphotropic envelopes and either IL2-SU
(IL2-SU/A) or IL2-SUX (IL2-SUX/A) chimeras. All blots were stained with
an MLV-SU antiserum. The immunoblot of viral pellets was separated at
the position of 46-kD marker, and the lower portion of the membrane was
stained with a p30-CA antiserum. The positions of the IL-2 chimeric
envelope glycoproteins and the wild-type amphotropic SU are
indicated.
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To demonstrate incorporation of the chimeric envelope glycoproteins
into retroviral vector particles, supernatants from the different
transfected-TELCeB6 cells were ultracentrifuged to pellet the vector
particles. Pellets were then analyzed on immunoblots for the presence
of capsid (p30-CA) and envelope proteins (Fig 2B). Envelope
glycoproteins could be detected for both IL2-SU and IL2-SUX mutants,
albeit at 20- to 50-fold reduced levels, respectively, compared with
wild-type amphotropic envelopes, thus indicating a weaker incorporation
of the chimeras into vector particles. As expected, no envelope
glycoprotein was detected in pellets of IL2-SU- and
IL2-SUX-transfected TElac2 cells that do not express gag and pol
proteins (data not shown). Altogether, these results demonstrate that
the envelope glycoproteins detected in the pellets of env-transfected
TELCeB6 cells were stably associated with gag-pol viral particles.
TELCeB6 cells coexpressing wild-type envelope and either of the two
chimeric envelope glycoproteins (IL2-SU/A or IL2-SUX/A) exhibited a
stronger incorporation of the former in the generated vector particles
(Fig 2B), consistent with the weaker expression of the latter (Fig 2A).
IL-2 receptor binding of chimeric envelopes.
Kit225 human cells expressing the three subunits comprising the
high-affinity IL-2 receptor were used for binding assays. The cells
were incubated with vector-containing supernatants and binding of viral
envelopes to the cell surface was analyzed by flow cytometry using
antibodies against the MLV SU (Fig 3). To assess binding to the IL-2 receptor, binding assays were performed at
4°C for 1 hour. Under these temperature conditions, binding to the
Pit-2 amphotropic receptor is very weak.26 As expected, no
binding was detected with vectors carrying wild-type amphotropic envelopes (Fig 3). In contrast, vectors carrying the IL2-SU envelopes bound specifically to IL-2 receptor-positive human cells (Fig 3).
Although clearly detectable, binding of vector particles generated with
IL2-SUX envelopes was 5 times weaker than that of vectors carrying
IL2-SU envelopes (Fig 3). This result is likely due to the weaker
expression and incorporation of the former chimera as compared with the
latter (Fig 2).
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| Fig 3.
Envelope binding assays to IL-2 receptor-expressing
target cells. Kit225 cells were used as an IL-2 receptor-expressing
target cell. The background fluorescence was determined by incubating
cells with supernatant from nontransfected TELCeB6 packaging cells
(white area). Binding assays were performed with viruses pseudotyped
with the wild-type amphotropic envelope, A, the IL2-SU, or the IL2-SUX
chimeric envelopes. Target cells were either treated (broken line) with
recombinant IL-2 (100 ng for 30 minutes at 37°C) or not treated
(black area) before binding assays with the various virions.
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To demonstrate the specificity of IL2-SU and IL2-SUX binding to cells,
IL-2 receptors on Kit225 cells were blocked by preincubation with
recombinant soluble IL-2 (rIL-2). Although this treatment did not
affect the binding of control envelope chimeras targeted to a receptor
other than the IL-2 receptor (data not shown), there was a consistent
decrease in the binding of vectors carrying either IL2-SU or IL2-SUX
envelopes (Fig 3). These data indicated that IL-2 was correctly
displayed on retroviruses and could specifically retarget the binding
of vector particles to cells expressing the IL-2 receptor.
Cell cycle activation of IL-2-dependent cells by IL-2-expressing
retroviral envelopes.
To determine whether retroviruses carrying the IL-2-fusion envelopes
could stimulate cell cycle progression, DNA synthesis was measured by
3H-thymidine incorporation assays in several G0/G1-arrested
IL-2-dependent cell types (Fig 4). Cells
incubated with media containing either nonenveloped retroviruses or
retroviruses with unmodified amphotropic MLV envelopes did not
demonstrate an increase of 3H-thymidine incorporation as
compared with cells incubated with unconditioned medium. In contrast,
incubation of G0/G1 arrested cells in media containing vectors
pseudotyped with IL2-SU chimeric envelopes resulted in a fivefold to
12-fold increase in 3H-thymidine incorporation (Fig 4),
indicating that binding of these latter vectors to the IL-2 receptor
induced the IL-2 signaling cascade resulting in DNA synthesis. The
level of response to IL-2 of the different cell types is related to the
number of IL-2 receptors expressed by these cells and to the affinity
of these receptors for IL-2.
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| Fig 4.
Induction of DNA synthesis in cells incubated with
IL-2-displaying retroviruses. 3H-thymidine incorporation
was measured in four different IL-2-dependent cell types: F7, Bclp75,
HT-2, and W4E9 cells. Cells, which were arrested in Go/G1 by overnight
deprivation of IL-2, were either incubated in 96-well plates for 24 hours in media conditioned with 50 U of recombinant IL-2 (rIL-2), 70 µL of viral supernatant containing nonenveloped retroviruses ( ),
70 µL of viral supernatant containing retroviruses coated with
amphotropic envelopes (A), or 70 µL of viral supernatant containing
retroviruses coated with IL-2 chimeric envelopes (IL2-SU). The levels
of 3H-thymidine incorporation are expressed as percentages
relative to 3H-thymidine incorporation in rIL-2-stimulated
cells. Experiments were performed in triplicate, and the means ± standard deviations are shown.
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Because 3H-thymidine incorporation is a measure of DNA
synthesis but does not provide any information regarding the capacity of the cells to undergo a productive division, we also assessed cell
proliferation using the WST-1 reagent. The F7 IL-2-dependent cell line
was arrested in GO/G1 and cultured in media containing vector particles
carrying wild-type MLV envelopes or IL2-SU chimeric envelopes, and cell
proliferation was then quantified 3 and 7 days later. The results shown
in Fig 5 indicate that, whereas media
containing retroviruses devoid of envelope glycoproteins or carrying
wild-type MLV envelopes could not sustain cell multiplication, the
IL-2-dependent cells replicated in media containing retroviruses with
IL-2 fusion envelopes in a manner similar to that observed in
rIL-2-conditioned media (Fig 5). Similarly, viral particles carrying
the IL2-SUX chimera alone or viral particles coexpressing wild-type
amphotropic envelopes with either IL2-SU or IL2-SUX chimeras could
stimulate the cell cycle progression and division of IL-2-responsive
target cells (data not shown). Collectively, these data therefore
indicated that IL-2-displaying retroviral envelope glycoproteins were
fully functional with respect to IL-2 receptor stimulation and
activation of the cell cycle leading to DNA synthesis, mitosis, and
cytokinesis.
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| Fig 5.
Proliferation of IL-2-dependent cells induced by
IL-2-displaying retroviruses. F7 cells were arrested in Go/G1 by
overnight deprivation of IL-2. Cell proliferation was then quantified 3 or 7 days later after culture in media conditioned with either 10 U of
recombinant IL-2 (rIL-2), 13 µL of viral supernatants containing
nonenveloped retroviruses (), 13 µL of viral supernatants
containing retroviruses coated with amphotropic envelopes (A), or 13 µL of viral supernatants containing retroviruses coated with IL-2
chimeric envelopes (IL2-SU). The values shown are the means ± SD of
four separate experiments.
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To determine the minimal period of exposure to IL-2-displaying
retroviruses required to stimulate the cell cycle, F7 cells were
arrested in G0/G1 by overnight IL-2 starvation. At that time, G0/G1-arrested cells were incubated for different time periods with
serum-free media containing rIL-2 or, alternatively, with retroviruses
harboring wild-type or IL2-SU fusion envelopes. After the stimulation
period, cells were washed and reincubated in media without IL-2 until
T24 hours, at which time the proportion of cells in the different cell
cycle stages, G0/G1, S, and G2/M, was measured by FACS analysis
(Fig 6). In contrast to media containing viral particles generated with wild-type MLV envelopes, media containing rIL-2 or containing retroviruses with IL2-SU chimeric envelopes stimulated cell cycle progression of G0/G1-arrested cells
(Fig 6). This was demonstrated by the diminution of the percentage of
cells in G0/G1 and the concomitant increase in the proportion of cells
in the S phase. In addition, these results indicate that only a brief
exposure of cells to IL2-SU carrying retroviruses was necessary to
stimulate cell division. Indeed, after 6 hours of exposure to
IL-2-displaying viruses, the proportion of cells in the G0/G1 phase
decreased by about 10%, resulting in an increased number of cells in
the S phase (Fig 6).
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| Fig 6.
Stimulation of cell cycle progression in Go/G1-arrested
cells by IL-2-displaying retroviruses. F7 cells, arrested in Go/G1 by
an overnight IL-2 deprivation, were stimulated for different time
periods with 2 ng/mL of recombinant IL-2 (rIL-2), viral supernatants
containing retroviruses coated with amphotropic envelopes (A), or viral
supernatants containing retroviruses coated with IL-2 chimeric
envelopes (IL2-SU). The percentage of cells in the different phases of
the cell cycle (Go/G1; S; G2/M) was measured by FACS analysis after
propidium iodide staining. At the onset of stimulation, 72.33% of F7
cells were in the Go/G1 phase. Experiments were performed in duplicate
and the means are shown.
|
|
Retroviral envelope glycoproteins are not very stable structures,
because the SU subunits of MLV are not covalently attached to the
membrane-bound TM subunit and are easily shed from viral particles or
from the surface of the vector producer cells.27 Thus, it
is probable that the viral supernatants generated with IL2-SU chimeric
envelopes are contaminated by soluble, non-virion-associated, IL2-SU
fusion envelopes. This evidence was supported by the finding that
supernatants of cells producing vector particles with IL2-SU envelopes
could efficiently stimulate GO/G1-arrested target cells, regardless of
whether the viral particles were removed from the supernatant by
ultracentrifugation (Fig 7). Thus, it is
likely that soluble-shed IL2-SU accounted for a significant part of the cell activation measured in our experiments. In addition, the activation observed upon incubation with nonultracentrifugated viral
supernatants bearing IL2-SUX envelopes, in which cleavage of the
envelope precursor was prevented, was significantly less than that
observed with the IL2-SU envelopes (data not shown). This was probably
due to the inability of the IL2-SUX envelope to accumulate in the
supernatant as soluble material and to its less efficient incorporation
on viral particles. Indeed, no activation of cell cycle was observed
upon culture of cells in the presence of supernatant in which IL2-SUX/A
vector particles were removed by ultracentrifugation (data not shown).
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| Fig 7.
Cell cycle activation by virion-shed and
virion-associated IL2-SU chimeras. Go/G1-arrested J3-13 cells were
incubated for 24 hours in media conditioned with either rIL-2
(recombinant IL-2; bottom right panel) or with viral supernatants
containing retroviruses coincorporating IL2-SU and wild-type
amphotropic envelope glycoproteins (top left panel). Viral particles
were removed by ultracentrifugation before the addition of supernatants
to cultures of Go/G1 arrested J3-13 cells (top right panel). Cells were
stained with propidium iodide to assess the DNA content of the treated
cells. Before stimulation, 80% of J3-13 cells were in the Go/G1 phase
of the cell cycle (bottom left panel).
|
|
IL-2-displaying vector particles efficiently infect GO/G1 arrested
cells.
Vectors generated with wild-type amphotropic, IL2-SU, or IL2-SUX
envelopes were used to infect IL-2-responsive J3-13 cells. These cells
were chosen because they are adherent and are easily infected by
wild-type amphotropic retroviruses. As expected, vectors bearing
IL2-SUX envelopes were not infectious because of the lack of cleavage
between their SU and TM subunits that is necessary for fusion
activation.27 In contrast, vectors bearing IL2-SU envelopes
were infectious, and although titers reached 103 IU/mL,
this titer was more than 4 orders of magnitude lower than that obtained
with viruses carrying wild-type amphotropic envelopes (data not shown).
As previously shown for other chimeric envelopes, the reduced capacity
of IL2-SU envelopes to mediate infection via PiT-2 amphotropic
receptors was either due to a steric hindrance of envelope
binding/fusion by the displayed polypeptide domain24,25 and/or to the sequestration of retroviruses displaying a growth factor/cytokine upon its interaction with the corresponding tyrosine kinase receptor.22,28 To rescue or enhance the infectivity of IL-2-displaying envelope glycoproteins, retroviruses were
pseudotyped with both amphotropic envelopes and either of the two
IL-2-fusion envelopes. The infectivity of the two resulting
retroviruses, IL2-SU/A and IL2-SUX/A, was greater than 106
IU/mL on J3-13 proliferating cells, which was only 1 log lower than
that of amphotropic-pseudotyped retroviruses (data not shown).
Infectivity of the IL2-SU/A and IL2-SUX/A viruses was then compared
with that of control retroviruses carrying wild-type amphotropic envelopes on J3-13 cells that were GO/G1-arrested by serum starvation. A low residual infectivity was detected on the latter cells with the
control amphotropic viruses (1.4% ± 1% [n = 8] of the infection obtained on proliferating J3-13 cells) and was most likely due to a
small number of J3-13 cells that were not arrested in G0/G1 (Fig 8). In contrast, the relative ability
of IL2-SU/A and IL2-SUX/A viruses to infect nonproliferating J3-13
cells was significantly higher than that of control amphotropic
retroviruses and reached 12.8% ± 4.2% (n = 8) and 48.4% ± 16.3% (n = 8), respectively (Fig 8). It is likely that the increased
ability of IL2-SU/A retroviruses to infect the nonproliferating J3-13
cells was due to cell activation mediated by both soluble-shed IL2-SU
and virion-associated IL2-SU envelopes. In contrast, because
soluble-shed IL2-SUX envelopes were not found in viral supernatants,
our data indicate that viruses carrying IL2-SUX/A envelope
glycoproteins activate the cells into which they enter, thus favoring
their integration after mitosis.
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| Fig 8.
Infectivity of retroviruses carrying IL-2-fusion
envelopes. Retroviral infections were performed on proliferating and
Go/G1-arrested J3-13 cells. The latter cells were obtained by 48 hours
of incubation in medium containing 1% serum, which resulted in 80% of
cells being arrested in the Go/G1 cell cycle phase, as shown by
propidium iodide staining. Proliferating or quiescent (Go/G1-arrested)
J3-13 cells were infected with lacZ retroviral vectors carrying the
indicated envelope glycoproteins. The retroviruses, which were
harvested in serum-free medium, were diluted in medium containg 1%
fetal calf serum and deposited on the cells. After 5 hours of
infection, virus-containing media were removed and replaced by fresh
media supplemented with 1% fetal calf serum only. Cells were then
cultured for 48 hours before X-gal staining. Results are expressed as
the percentage (mean ± SD; n = 8) of titers on quiescent cells
relative to titers on proliferating J3-13 cells.
|
|
| |
DISCUSSION |
We report here a novel strategy whereby retroviral particles displaying
a polypeptide growth factor can transiently activate cell division at
the time of virus entry, allowing integration of a transgene in
quiescent cells. This strategy is based on the development of
retroviral vectors that bear bifunctional chimeric envelope
glycoproteins. Such recombinant envelopes are able to stimulate the
proliferation of resting cells via the activation of a specific
cytokine or growth factor receptor at the cell surface. This
stimulation results in a mitosis that allows the penetration of the
retroviral capsid and viral genome into the nucleus with subsequent
integration of the transgene into the host cell DNA. Because the
cytokine/growth factor harbored by the bifunctional retroviral
envelopes is not encoded by the genome of the retroviral vector itself,
the activation is only transient. Therefore, the stimulation of the
target cells is not expected to result in more than one or two cell
divisions and the activated cells should return to their initial
nonproliferative state after having integrated the transgene.
Our results provide a proof of concept for the approach that may have
utility in various gene transfer applications. In view of the
complexity of T-cell activation pathways, we anticipate that the
display of IL-2 on the vector particles may not be sufficient to give
highly efficient transduction of primary T cells. For example, primary
CD4+ T cells, which exhibit increased responsiveness to
IL-2 only after preactivation with other stimuli, such as
phytohemagglutinin (PHA) or multivalent anti-CD3 and
anti-CD28, may not be optimal.10,29 However, we anticipate
that it will be easy to generate bifunctional chimeric envelope
glycoproteins able to activate various cell surface molecules involved
in transduction of a mitogenic signal. Indeed, over the last few years,
we have extensively characterized a great number of chimeric envelope
glycoproteins generated by amino-terminal extensions of MLV envelopes
with various polypeptides,30 such as growth
factors,22,28 single-chain antibodies,24,31-33 or other ligands.22,25,34 Viral particles carrying these
recombinant envelopes could efficiently and easily retarget virion
binding and, in some cases, were able to redirect infection, although at low levels.30 Moreover, preliminary results from our
laboratory indicate that virions displaying epidermal growth factor
(EGF) or stem cell factor (SCF) are also able to activate cells
expressing EGF or c-kit receptors, respectively (F.-L.C. and S.J.R.,
unpublished results). Therefore, we believe that
recombinant retroviruses displaying polypeptide growth factors and
other cell signaling polypeptides hold promise for several in vivo gene
transfer situations in which the target cells are quiescent. For
example, airway epithelial cells are important targets for diseases
such as cystic fibrosis. However, gene transfer in such primary cells
using MLV-derived retroviral vectors can only be achieved if the cells
are induced to divide by in vivo or in vitro prestimulation with
keratinocyte growth factor.35,36 The display of KGF, a
single chain 162 aa-long polypeptide,37 at the surface of
retroviral particles through molecular engineering of the viral
envelope glycoprotein might allow the generation of MLV-derived
retroviral vectors that can activate these airway cells, resulting in
efficient integration of the therapeutic transgene.
Several aspects of the current study point to the need for improvements
to optimize the strategy for clinical gene transfer applications. Although the incorporation of IL-2-chimeric
envelopes on viral particles was reasonably efficient, we found that a
substantial part of the IL-2-dependent cell cycle activation was not
mediated by IL-2-displaying vector particles, but by soluble IL-2
chimeric SU envelope subunits shed from vector particles or from the
packaging cells. Indeed, the association between the SU and the TM
retroviral envelope subunits is known to be weak,27
resulting in the accumulation of free SU in vector supernatants. To
overcome this problem, we designed the IL2-SUX recombinant envelope
glycoproteins in which the cleavage site between the SU and the TM
subunits was inactivated, preventing shedding of the IL-2-displaying
SU subunit. Thus, in the IL2-SUX envelope, the displayed IL-2
polypeptide was covalently attached to the envelope complex and was
tightly anchored to the viral particles by the transmembrane anchoring
domain located at the carboxy-terminus of the envelope glycoprotein.
Despite a lower incorporation of IL2-SUX compared with IL2-SU on viral particles, gene transfer to growth arrested cells was significantly increased upon infection with viral particles pseudotyped with the
former envelope. One possible explanation for this phenomenon is that,
because the IL2-SUX envelope is always associated with virions, cell
activation through interaction with the IL-2 receptor occurred
concurrently with viral entry. In contrast, upon infection of cells
with virions pseudotyped with IL2-SU, activation of cells with
soluble-shed IL2-SU subunits was not necessarily associated with viral
entry. Our results suggest that the backbone of the IL2-SUX vector will
be superior for the display of other cytokines/growth factors.
Although for some quiescent cells activation of the cell cycle can be
achieved using a single cytokine or growth factor, the proliferation of
most other resting cell types requires stimulation with two or more
cytokines. For example, most gene transfer protocols involving
hematopoietic progenitors use a combination of SCF, IL-3, and IL-6 to
induce cell proliferation and allow subsequent integration of
MLV-derived retroviral vectors.38 Similarly, optimal
activation and proliferation of T lymphocytes is obtained by
stimulation with anti-CD3 and anti-CD28 antibodies in combination with
IL-2.10 Therefore, gene delivery to such cells using the strategy described in this report will require MLV-derived retroviral vectors that display, in addition to wild-type envelope glycoproteins, at least two chimeric envelopes able to activate different cell surface receptors.
In contrast to MLVs, lentiviruses such as HIV are able to infect
nonproliferating cells such as macrophages and resting CD4+
T cells. However, particularly for primary T lymphocytes, completion of
reverse transcription and subsequent integration of the HIV genome does
not occur unless the cells have been activated, and it is quite
probable that the gp120 envelope glycoproteins of HIV can activate
certain target cell types through their interaction with CD4 or
CXCR4.39 Morevover, the integration of the HIV genome in
resting cells, such as macrophages, requires several accessory proteins, such as vpr, which are specifically encoded by the lentivirus genome.40 Thus, although retroviral vectors derived from
HIV seem to exhibit the same biological properties as wild-type HIV, allowing integration of the transgene in the nonproliferating target
cells,5,41-43 some particular quiescent cell types may not
be transduced by such vectors (D. Trono, personal
communication, November 1998). Additionally, because the
expression of accessory lentivirus proteins in HIV-derived vectors is
best avoided for biosafety, retroviral vectors derived from either HIV
or MLV that incorporate stimulating envelope chimeras may represent an
interesting alternative for the design of safe gene delivery vectors
for in vivo applications.
Finally, because of their inability to integrate in the genome in
nonproliferating cells, MLV-derived vectors displaying polypeptide growth factors will facilitate selective gene transfer to cells that
express the corresponding cytokine receptor. Indeed, although they
penetrate most resting cell types (depending on the presence of the
corresponding retroviral receptor), such MLV vectors will only
integrate in cells in which they have concomitantly and specifically activated the cell cycle. Therefore, this strategy may also allow the
delivery of a transgene in a tissue-specific manner.30
| |
ACKNOWLEDGMENT |
The authors are grateful to Jacqueline Marvel and Naomi Taylor for
stimulating discussions and for critical reading of the manuscript. We
are also grateful to T. Taniguchi, S. Zarawski, and P. Lecine for
kindly providing the IL-2 receptor expressing cell lines.
| |
FOOTNOTES |
Submitted December 9, 1998; accepted March 21, 1999.
Supported by Agence Nationale pour la Recherche contre le SIDA
(ANRS), Association pour la Recherche contre le Cancer (ARC), Association Française de Lutte contre la Mucoviscidose (AFLM), Centre National de la Recherche Scientifique (CNRS),
Ministére de l'Enseignement Supérieur et de la Recherche
(ACC-SV2), and Institut National de la Santé Et de la Recherche
Médicale (INSERM). S.J.R. and F.J.B. were supported by the
Medical Research Council. M.M. was supported by a fellowship from the
Association Française contre les Myopathies.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to François-Loïc Cosset,
PhD, U412, ENS de Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France; e-mail: Francois-Loic.Cosset{at}ens-lyon.fr.
| |
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Interleukin-13 Receptor-targeted Cancer Therapy in an Immunodeficient Animal Model of Human Head and Neck Cancer
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August 1, 2001;
61(16):
6194 - 6200.
[Abstract]
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S. Snitkovsky, T. M. J. Niederman, B. S. Carter, R. C. Mulligan, and J. A. T. Young
A TVA-Single-Chain Antibody Fusion Protein Mediates Specific Targeting of a Subgroup A Avian Leukosis Virus Vector to Cells Expressing a Tumor-Specific Form of Epidermal Growth Factor Receptor
J. Virol.,
October 15, 2000;
74(20):
9540 - 9545.
[Abstract]
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith
Gene Therapy 2000
Hematology,
January 1, 2000;
2000(1):
376 - 393.
[Abstract]
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ABSTRACT
INTRODUCTION