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Journal of Virology, August 1999, p. 6923-6929, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Retrovirus Targeting by Tropism Restriction to
Melanoma Cells
F.
Martin,1
S.
Neil,1
J.
Kupsch,2
M.
Maurice,3
F.-L.
Cosset,3 and
M.
Collins1,*
Department of Immunology, Windeyer Institute
for Medical Science, University College London,
London,1 and Mount Vernon Hospital,
RAFT Laboratories, Northwood, Middlesex,2 United
Kingdom, and Centre de Genetique Moleculaire et Cellulaire,
CNRS UMR5534, UCB Lyon-1, France3
Received 22 February 1999/Accepted 10 May 1999
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ABSTRACT |
Targeted vectors will be necessary for many gene therapy
applications. To target retroviruses to melanomas, we fused a
single-chain variable fragment antibody (scFv) directed against the
surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins. The modified viruses bound only to
HMW-MAA-expressing cells, as inclusion of the proline-rich hinge
prevented viral binding to the amphotropic viral receptor. Following
attachment to HMW-MAA, MMP cleavage of the envelope at the melanoma
cell surface removed the scFv and proline-rich hinge, allowing
infection. Complexing of targeted retroviruses with
2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]N,N-dimethyl-1-propanaminium trifluoroacetate-dioleoyl phosphatidylethanolamine liposomes greatly increased their efficiency without affecting their target cell specificity. In a cell mixture, 40% of HMW-MAA-positive cells but less
than 0.01% of HMW-MAA-negative cells were infected. This approach can
therefore produce efficient, targeted retroviruses suitable for in vivo
gene delivery and should allow specific gene delivery to many human
cell types by inclusion of different scFv and protease combinations.
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INTRODUCTION |
Retroviral vectors have several
features which make them attractive for clinical gene delivery. In
particular, integration of the vector genome allows stable expression
of the transduced gene in the infected cell and its progeny. Retroviral
vectors can infect a wide range of cell types, including nondividing
cells, following the development of vectors based on human
immunodeficiency virus (20). Because viral coding regions
are deleted from the vector, viral proteins are not expressed in
infected cells, which avoids stimulation of an inappropriate antiviral
immune response.
In gene therapy clinical trials, retroviral vectors have been used for
in vitro infection, followed by transfer of modified cells to the
patient. Such modification of cells is time consuming and costly and
may not always be possible. It is therefore desirable to develop
retroviruses suitable for in vivo gene delivery. Such vectors should
efficiently infect specific target cells. Nontarget cells should not be
infected, as gene delivery could be deleterious to their function and
they would deplete the pool of viral particles.
The host range of retroviruses is partly determined by the surface
domain (SU) of the envelope glycoprotein, which binds to a cell surface
receptor (37). For murine leukemia viruses (MLVs), the
receptor binding domain has been mapped to the N-terminal portion of SU
(1). Previously described strategies for targeting of
retroviruses have incorporated ligands (10, 29, 39) or single-chain variable-fragment antibodies (scFvs) (12, 15, 16,
31) recognizing targets on human cells into the SU protein of
ecotropic MLV (MLV-E), which infects only rodent cells. Although viruses bound to human cells, infection was generally poor or not observed.
The aim of this study was to achieve efficient, specific targeting of
human melanomas by limiting the tropism of amphotropic MLV (MLV-A),
which can infect cells of many mammals, including humans and rodents.
The receptor for MLV-A on human cells is RAM-1, a phosphate transporter
expressed on most cell types (11). High-molecular-weight melanoma-associated antigen (HMW-MAA; also called melanoma-associated chondroitin sulfate proteoglycan) was selected as the target molecule. This integral membrane proteoglycan is expressed in more than 90% of
human melanomas but not in most normal adult tissues (21, 25). Its expression by melanomas is associated with a poor
prognosis (9). HMW-MAA has been used successfully as an in
vivo target for radioimaging (6, 14, 19, 30) and
immunotherapy of melanoma (2, 17, 18).
An scFv which recognizes HMW-MAA was linked to the extreme N terminus
of MLV-A SU. A proline-rich spacer was used to link the scFv to SU in
order to block MLV-A SU binding to RAM-1. Inclusion of this spacer in a
chimeric envelope has been shown to block MLV-E binding to its receptor
(35). A cleavage site for matrix metalloproteases (MMPs) was
inserted after the proline-rich spacer. MMPs are highly expressed on
the cancer cell surface (3) and are critical for tumor
invasion of normal tissue (27, 33, 38). Cleavage of a
chimeric retroviral envelope by exogenous factor X or cell surface MMP
to remove an epidermal growth factor (EGF) domain has previously been
reported (7, 22, 23). In these experiments, the EGF domain
efficiently targeted virus to the EGF receptor, which destroyed its
infectivity, so MLV-A SU binding to RAM-1 did not need to be blocked.
The rationale for our approach was that attachment of viruses to
HMW-MAA would lead to MMP removal of the scFv and proline spacer at the
cell surface, allowing infection following MLV-A interaction with its receptor, RAM-1. Retroviruses carrying these targeted envelopes selectively infected HMW-MAA-positive cells in culture; their efficiency was increased by complexing with
2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-di methyl-1-propanaminium
trifluoroacetate (DOSPA)-dioleoyl phosphatidylethanolamine (DOPE) liposomes.
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MATERIALS AND METHODS |
Chimeric envelopes.
The scFv which recognized HMW-MAA was
derived from monoclonal antibody LMH2. The scFv coding sequence was
removed from the vector pCantab.5 (13) by digestion with
SfiI and NotI. pEGFPRO4070A was constructed by
inserting a proline spacer (isolated from the 4070A envelope by using
the primers 5'-atcgaggtcaccgcggccgcgggaccccgagtccccatagggccc-3' and 5'-ataatcggccgggggtggctgtgggac-3' into the
amphotropic chimera EA (4).
ScLPA was made by inserting the scFv coding sequence into pEGFPRO4070A
after removal of the EGF coding fragment by digestion with
SfiI and NotI. Insertion of the peptide
Pro-Leu-Gly-Leu-Trp-Ala as an MMP cleavage site between the PRO linker
and the env protein was made by PCR site-directed mutagenesis
(Quikchange site-directed mutagenesis kit; Stratagene). The
oligonucleotide primers used were
5'-cacagccacccccggccgcacccctgggcctgtgggccccccatcaggtctttaatgtaacctgg-3' and
5'-ccaggttacattaaagacctgatggggggcccacaggcccaggggtgcggccgggggtggctgtg-3', where the PLGLWA coding sequence is underlined. The 4070A
envelope expression plasmid (ALF) were described previously
(5).
Cell culture, virus production, and virus concentration.
TELCeB6 cells (5) are derived from the human
rhabdomyosarcoma TE671 cell line (ATCC CRL-8805) and harbor the
MFGnlslacZ vector genome and an MLV-Gag-Pol expression plasmid, CeB
(5). BOWES (ATCC CRL-9607) and A375m (ATCC CRL-1619) are
human melanoma cell lines. B-1 is a human melanoma cell line
established in our laboratory. Ecv304 are a spontaneously transformed
immortal human endothelial cell line (ATCC CRL-1998). All cells were
grown in Dulbecco modified Eagle medium (DMEM; GIBCO-BRL) supplemented with 10% fetal calf serum (FCS) at 37°C and 10% CO2.
Envelope expression plasmids scLPA, scLPMA, and ALF were transfected
into TELCeB6 cells by using Lipofectamine (GIBCO-BRL). Transfected
cells were selected with phleomycin (50 µg/ml), and pools of
phleomycin-resistant clones were used for virus production. The
TELCeB6-scLPMA bulk population was also cloned by serial dilution, and
the clone which produced the highest virus titer was identified. To
harvest viruses, producer cells were grown at 37°C until they became
confluent and then cultured at 32°C for 4 to 7 days with feeding of
fresh DMEM supplemented with 10% FCS every 2 days. The medium was then
replaced with serum-free Optimem (GIBCO-BRL), and supernatant was
collected 12 to 16 h later. The harvested virus was filtered
through 0.45-µm-pore-size filters and, in some cases, concentrated by
centrifugation at 2,500 × g and 4°C for 12 h.
Concentrated virus was kept frozen at 
70°C.
Envelope incorporation and gelatinase A cleavage.
Virus
pellets were subjected to Western blot analysis using goat antisera
against the Rauscher leukemia virus gp70 (SU) and p30 (CA) proteins as
described previously (4). Accessibility of the MMP cleavage
site to protease was demonstrated by treatment of pelleted viruses with
activated gelatinase A (Boehringer Mannheim). scLPMA viruses were
centrifuged at 100,000 × g for 1 h at 4°C, resuspended in 50 µl of 100 mM Tris (pH 7.5)-200 mM NaCl-1 U of activated gelatinase A, incubated for 6 h at 37°C, and then
subjected to Western blot analysis using anti-RLV gp70 antibody.
Protease activity.
To determine protease activity on target
cells, the 2,4-dinitrophenol
(DNP)-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2
peptide (Bachem) was used. Target cells were grown to
semiconfluency and then washed twice in buffer A (50 mM Tris [pH
7.5], 10 mM Ca2Cl, 0.2 M NaCl). The DNP-peptide was
diluted to 20 µM in buffer A, added to the different target cells,
and incubated for 1 h at 37°C. Substrate hydrolysis was
determined by monitoring the increase in fluorescence emission at 346 nm using an excitation wavelength of 280 nm.
HMW-MAA expression, envelope binding, and virus binding.
Expression of HMW-MAA on the target cells was determined by using LMH2
antibody (13) and CP/Me1.2 (Immune Systems Ltd.). For
envelope binding, cells were incubated with viral supernatants and
washed and envelope binding was then determined by use of a
fluorescence-activated cell sorter (FACS) and goat anti-RLV gp70
antibody (16). For detection of virus binding, a
fluorescence microscopy method was used (24). Briefly, cells
were grown to confluence on glass coverslips and then incubated in
concentrated virus for 30 min at 37°C. Cells and viruses were fixed
in 4% paraformaldehyde for 5 min, washed once with phosphate-buffered
saline (PBS), permeabilized in 0.02% Triton X-100 for 2 min, washed
once with PBS, and then incubated with PBS containing 1% (wt/vol)
bovine serum albumin (PBA) for 15 min at room temperature (RT). For
detection of gp70 and p30, samples were incubated for 1 h with
goat anti-RLV p30 polyclonal antibody and rat anti-gp70 83A25
monoclonal antibody, washed three times with PBA, incubated with a
mixture of anti-rat immunoglobulin G (IgG)-tetramethyl rhodamine
isocyanate (TRITC) and anti-goat IgG-fluorescein isothiocyanate (FITC)
for 45 min, washed four times with PBS, mounted, and analyzed by
confocal scanning microscopy (Bio-Rad).
Analysis of viral infection.
Target cells were seeded in
24-well plates at a density of 5 × 104 cells/well
24 h before infection. Viruses were incubated with 4 µg of
Polybrene (PB) per ml or 10 µg of Lipofectamine per ml, as indicated
in Results, for 10 min at RT before being added to the target cells.
Cells were incubated in the presence of the viruses for 1 h at
37°C, washed once in Optimem and once in DMEM-10% FCS, and then
cultured for 24 to 48 h.
5-Bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal)
staining was performed as previously described (34). For
infection of mixed target cells, HMW-MAA-negative and -positive cells
were mixed at a 10:1 ratio and then seeded on glass coverslips. After
24 h, the mixed population was infected, washed once with PBS
after a further 48 h, incubated with PBA at RT for 15 min, incubated with LMH2 antibody for 45 min at 4°C, washed three times with PBS, fixed with 4% paraformaldehyde, and permeabilized in 0.02%
Triton X-100. To detect -galactosidase, cells were washed once with
PBS, incubated with PBA for 15 min at RT, and then incubated with
rabbit anti--galactosidase antibody in PBA for 3 h. After three
washes in PBA, samples were incubated with a combination of anti-rabbit
IgG-TRITC and anti-mouse IgG-FITC. After five washes in PBS, cells were
analyzed by using a confocal scanning microscope (Bio-Rad).
To inhibit targeted virus infection, cells were preincubated with 50 µg of LMH2 per ml for 5 min at 37°C and then incubated
with virus
in the presence of 50 µg of LMH2 per ml and 10 µg of
Lipofectamine
per ml for 30 min at 37°C. To inhibit MMP activity,
cells were
incubated with virus in the presence of 4 µg of TIMP-2
(Boehringer)
per ml for 30 min at 37°C. After incubation, viruses
were removed and
the cells were washed, cultured for 24 to 48
h, and then X-Gal
stained.
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RESULTS |
scLPA and scLPMA chimeric envelopes.
To construct the modified
retroviral envelope scLPA (Fig. 1A, top),
an scFv which recognizes HMW-MAA (13) was inserted at amino
acid +5 of the MLV-A 4070A envelope with a proline-rich spacer (PRO)
(36). In scLPMA, the MMP cleavage site PLGLWA
(40) was introduced between the PRO linker and the envelope
protein (Fig. 1A, bottom). Plasmids expressing scLPA and scLPMA were
transfected into TELCeB6 cells, which express the MLV gag and pol
proteins and carry a provirus encoding -galactosidase. Virions from
bulk populations of TELCeB6-scLPA and TELCeB6-scLPMA producer cells were pelleted and analyzed for the presence of chimeric envelopes by
using a polyclonal anti-SU antibody. The 100-kDa scLPA and scLPMA
envelope proteins were incorporated into virions, although at a lower
level than the MLV-A 4070A envelope (Fig. 1B).
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FIG. 1.
(A) An scFv derived from the antibody LMH2, which
recognizes HMW-MAA, was fused to the N terminus of MLV-A 4070A SU by
using the proline-rich region from MLV-A 4070A env (nucleotides 751 to
927) (P) as a spacer to make scLPA. An MMP cleavage site (PLGLWA) was
introduced between the P linker and the SU of the MLV-A envelope to
make scLPMA. TM, transmembrane protein. (B) Detection of unmodified
MLV-A 4070A envelope (lane 1A), scLPA, and scLPMA in viral pellets from
producer cell lines. p30 gag was detected in the same pellets to
quantitate viral particles. (C) Cleavage of scLPMA by gelatinase A (Gel
A) as described in Materials and Methods. Equal amounts of unmodified
MLV-A 4070A and scLPMA envelopes were loaded in each lane. kd,
kilodaltons.
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To assess whether the cleavage site was accessible to MMPs, scLPMA
viruses were incubated with activated gelatinase A. This
treatment
reduced the size of the chimeric scLPMA envelope to
the size of
unmodified SU (70 kDa) (Fig.
1C), demonstrating that
the MMP cleavage
site was cleaved. Some cleavage of scLPMA was
observed without
gelatinase A, probably due to production of MMPs
by the TELCeB6 cells
(Fig.
1C).
scLPA and scLPMA enveloped virions bind only to HMW-MAA-positive
cells.
Binding of scLPA, scLPMA, and unmodified MLV-A 4070A (A)
viral envelopes to HMW-MAA-positive or -negative cell lines was
measured. HMW-MAA expression was defined by using LMH2, the monoclonal
antibody from which the scFv used for viral targeting was derived
(13). Producer cell supernatants were incubated with target
cells, and envelope bound to cells was then detected with a polyclonal
anti-SU antibody using a FACS. The scLPA and scLPMA envelopes bound to HMW-MAA-positive cell lines A375m and BOWES but not to HMW-MAA-negative cell lines B1 and ECV304 (Fig.
2A, black and green
lines), demonstrating that the RAM-1 binding domain of scLPA and scLPMA
was indeed masked. HMW-MAA-positive cells bound approximately 10-fold
more targeted envelopes than unmodified MLV-A 4070A (Fig. 2A, blue
lines). scLPA and scLPMA binding was blocked by LMH2 antibody (Fig.
2B), demonstrating that binding of the scLPA and scLPMA envelopes was
dependent on interaction with HMW-MAA.
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FIG. 2.
Specific binding of scLPA- or scLPMA-enveloped virus to
HMW-MAA-positive cells. (A) Concentrated supernatants from producer
cells expressing the MLV-A 4070A (blue), scLPA (black), or scLPMA
(green) envelope were incubated with each target cell line for 45 min
at 37°C. TELCeB6 supernatant was used to define control fluorescence
(red). Envelopes were detected as described in Materials and Methods.
HMW-MAA status of each target cell is shown (HMW-MAA + VE or VE). (B)
HMW-MAA-positive BOWES cells were incubated in TELCeB6 (red), scLPA
(black), or scLPMA (green) concentrated supernatant in the absence
(top) or presence (bottom) of LMH2 antibody. (C) HMW-MAA-positive BOWES
or HMW-MAA-negative B-1 cells were incubated with concentrated scLPMA
or MLV-A 4070A enveloped virus. Samples were stained for p30 and gp70
by using a goat anti-RLV p30 polyclonal antibody and rat anti-gp70
monoclonal antibody 83A25 as described in Materials and Methods. Viral
particles appear as yellow dots as a result of the colocalization of
FITC-labelled anti-goat and TRITC-labelled anti-rat signals.
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Producer cell supernatants contain both virus particles and shed SU
proteins, and FACS measurement does not discriminate between
virus
binding and SU binding. To directly measure cell attachment
of
scLPMA-enveloped viruses, we stained viruses bound to cells
with
antibodies against the SU and p30 gag proteins. Virions were
detected
by confocal microscopy as spots of colocalized SU and
p30 staining.
scLPMA-enveloped viruses attached efficiently to
HMW-MAA-positive cells
and showed little binding to HMW-MAA-negative
cells (Fig.
2C). Virions
with the unmodified MLV-A 4070A envelope
bound to both HMW-MAA-positive
and -negative cells (Fig.
2C).
Thus, scLPMA enveloped virions had lost
the ability to bind to
RAM-1 but could efficiently attach to HMW-MAA.
scLPMA enveloped virions infect HMW-MAA-positive cells.
HMW-MAA-positive and -negative cells were infected with scLPMA-,
scLPA-, or unmodified MLV-A 4070A-enveloped viruses and with nonenveloped virions. Infections were performed either in the absence
of any enhancing reagent or with PB or DOSPA-DOPE liposomes (Lipofectamine). Nonenveloped or scLPA-enveloped viruses failed to
infect either cell line. Viruses with unmodified MLV-A 4070A envelopes
infected both cell lines, and their efficiency was increased by PB or
DOSPA-DOPE liposomes. The scLPMA-enveloped viruses infected the
HMW-MAA-positive cell line A375m when mixed with PB, but their efficiency was greatly enhanced when they were complexed with DOSPA-DOPE liposomes. They did not infect the HMW-MAA-negative cell
line B1 (Fig. 3). In this experiment,
centrifugation at low speed was used to concentrate all of the viral
preparations (see Materials and Methods). In the absence of
concentration, the scLPMA-enveloped viruses complexed with DOSPA-DOPE
liposomes (virosomes) could still infect 20% of the A375m cells (data
not shown). The fact that scLPA-enveloped viruses were unable to infect
HMW-MAA-positive cells demonstrated that the MMP cleavage site in
scLPMA virosomes was necessary for infectivity.
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FIG. 3.
Percentage of HMW-MAA-positive or -negative cells
infected. A375m and B-1 cells were incubated with virus with no
envelope (Non) or an scLPA, scLPMA, or unmodified MLV-A 4070A (A)
envelope concentrated by centrifugation (see Materials and Methods)
without any treatment (N) or after mixing with PB or
Lipofectamine (Lip) (see Materials and Methods).
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The titer of scLPMA virosomes on HMW-MAA-positive and -negative cell
lines is shown in Fig.
4A (top). On the
HMW-MAA-negative
cell line B-1, the titer was similar to that of
nonenveloped virosomes
(50 IU/ml) while the titer on Ecv304 cells (also
HMW-MAA negative)
was slightly higher (300 IU/ml). The scLPMA virosomes
infected
HMW-MAA-positive cells efficiently with titers of up to
10
6 IU/ml on A375m cells and 10
5 IU/ml on BOWES
cells. Titers of the targeted virosomes showed
that they were only
10-fold less efficient than virosomes with
unmodified MLV-A 4070A
envelopes.
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FIG. 4.
Infection by targeted virosomes (A) The graph at the top
shows titers of scLPMA-enveloped virus (best clone virus; see Materials
and Methods) and unmodified MLV-A 4070A on HMW-MAA-negative (B-1 and
Ecv304) cells and HMW-MAA-positive melanoma (A375m and BOWES) cells.
Infections were performed after complexing of unconcentrated viruses
with DOSPA-DOPE liposomes to produce virosomes. Titers are
expressed as the mean infectious units (iu) per milliliter (± the
standard error) of triplicate determinations. The lower graph shows
cleavage of the PLGLWA peptide by B-1, Ecv304, BOWES, and A375m cells
(see Materials and Methods). (B) Inhibition of scLPMA virosome
infection of BOWES cells by competition for HMW-MAA or inhibition of
MMP activity. scLPMA-enveloped virus and MLV-A 4070A were used to
infect BOWES cells with LMH2 antibody, TIMP-2, or a combination of both
as described in Materials and Methods. Data are expressed as mean
percentages of the titer of untreated viruses (triplicate
determinations ± the standard error).
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We used the dansylated peptide DNP-PLGLWADR-NH
2
(
32) to measure the level of MMPs capable of cleaving scLPMA
present at
the surface of each cell line. The cleavage of the peptide
at
its MMP site separates the DNP group (which acts as a quencher)
from
the tryptophan, leading to a fluorescence increase with excitation
at
280 nm. A DNP-peptide buffer was incubated with the different
target
cells for 1 h at 37°C, and the change in fluorescence was
measured. All of the cell lines showed similar levels of DNP-peptide
cleavage (Fig.
4A, bottom). No change in fluorescence was observed
in
the absence of cells or without incubation at 37°C (data not
shown).
Thus, the ability of cells to cleave scLPMA was not sufficient
to
permit infection; expression of HMW-MAA to allow virosome attachment
was also necessary. This conclusion was supported by the blocking
of
scLPMA virosome infection by LMH2 (60%), the MMP inhibitor
TIMP2
(50%), or a combination of LMH2 and TIMP2 (80%) (Fig.
4B).
scLPMA virosomes selectively infect HMW-MAA-positive cells in
mixtures.
Cocultures of HMW-MAA-positive and -negative cells were
infected with scLPMA or unmodified MLV-A 4070A virosomes. Cells were then stained for expression of HMW-MAA (green) and for nuclear -galactosidase (red) and analyzed by confocal microscopy (Fig. 5A). In both cocultures, almost all of
the cells infected with scLPMA virosomes were HMW-MAA positive (Fig.
5A), as shown by the colocalization of red nuclei and green surface
fluorescence. Unmodified MLV-A 4070A virosomes infected all of the
cells (Fig. 5A). In the B-1-A375m mixed population, scLPMA virosomes
infected 39% of the A375m cells but only 0.008% of the B-1 cells. In
the Ecv-A375m mixed population, scLPMA virosomes infected 37.5% of the
A375m and 0.01% of the Ecv304 cells (Fig. 5B). In both mixtures, the
HMW-MAA-negative cells were present in excess (40 B-1 cells to 1 A375m
cell and 60 Ecv304 cells to 1 A375m cell). This shows that scLPMA
virosomes became activated at the HMW-MAA-MMP-positive target cells
membrane, resulting in infection of these cells but not neighboring
cells.
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FIG. 5.
Infection of mixed target populations. (A) Mixed
populations were infected with unconcentrated MLV-A 4070A or
scLPMA-enveloped virus complexed with DOSPA-DOPE liposomes. Two days
after infection, cells were stained for -galactosidase (red) and
HMW-MAA (green) expression as described in Materials and Methods. (B)
Percentage of HMW-MAA-positive and -negative cells infected in
cocultures using unmodified MLV-A 4070A or scLPMA-enveloped virus.
Final cell ratios at confluency were 40:1 Ecv304 to A375m cells and
60:1 B-1 to A375m cells. Data are means (± the standard errors) from
two separate experiments in which 20 randomized fields (~3,000 cells)
were counted for each infection. To determine the infection of B-1 or
Ecv304 cells by scLPMA-enveloped virus, 200 fields (~30,000 cells)
were counted.
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DISCUSSION |
Previous attempts to target retroviruses have tried to extend the
tropism of MLV-E to specific human cells by incorporation of ligands or
scFvs recognizing various human cell surface proteins into SU (10,
12, 15, 16, 29, 31, 39). Although retargeted binding was
achieved, infection of target cells tended to be inefficient. In
contrast, insertion of the RAM-1 binding domain of MLV-A at the N
terminus of MLV-E SU allowed efficient infection of human cells
(4). Thus, attachment to the natural retrovirus receptor RAM-1 allowed infection whereas attachment to a variety of other cell
surface proteins did not. Probably only a very limited number of human
cell surface proteins can function as retrovirus receptors and can
therefore be used for MLV-E targeting.
The present paper describes a strategy to achieve retrovirus targeting
by tropism restriction of MLV-A. The insertion of a PRO spacer
(35) between the scFv and the envelope protein blocked RAM-1
binding but allowed the displayed scFv to bind HMW-MAA. After virus
attachment to HMW-MAA, cell surface MMPs removed the scFv and the PRO
spacer, allowing MLV-A interaction with its receptor RAM-1 and
efficient infection. This report describes the first retroviral
targeting strategy which produces sufficiently high-titer virus for in
vivo gene delivery.
While several reports have described the use of liposomes to enhance
retroviral infection (8, 26), this is also the first to
describe enhancement of targeted infection by liposomes with no loss of
specificity. The use of DOSPA-DOPE liposomes is critical for this
maintenance of specificity. We have previously shown that complexing of
nonenveloped retroviral particles with
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfonate allows infection while
3[N-(N',N'-dimethylaminoethane)==carbamoyl]cholesterol-DOPE or DOSPA-DOPE enhancement is dependent on retroviral envelope-receptor interaction (26).
Our targeting approach has several features which make it attractive
for clinical gene delivery. Firstly, the target cell must express both
a given surface antigen and a given surface protease. This double
requirement creates an extra degree of specificity. Secondly, the
targeted viruses do not attach to cells without the surface antigen.
This prevents uptake of virus by nontarget cells, which remains a
problem with approaches such as transcriptional targeting. Furthermore,
envelopes based on MLV-A can be efficiently incorporated into
lentiviral vectors, such as those based on human immunodeficiency virus
(28). This targeting method could therefore also be used for
specific gene delivery to nondividing cells.
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ACKNOWLEDGMENTS |
This work was supported by the Cancer Research Campaign, United
Kingdom, and the Medical Research Council, United Kingdom.
We thank S. Valsesia-Wittman for the plasmid pEGFPRO4070A, N. Phillipps for technical assistance, and S. Russell and Y. Takeuchi for
helpful comments on the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology, Windeyer Institute for Medical Science, University College London, 46 Cleveland St., London W1P 6DB, United Kingdom. Phone and
Fax: 44-171-504-9301. E-mail:
mary.collins{at}ucl.ac.uk.
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Journal of Virology, August 1999, p. 6923-6929, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
