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Journal of Virology, December 1998, p. 9706-9713, Vol. 72, No. 12
Gene Therapy Program, Comprehensive Cancer
Center, University of Alabama at Birmingham, Birmingham, Alabama
35294-3300
Received 11 June 1998/Accepted 2 September 1998
Recombinant adenoviruses (Ad) have become the vector system of
choice for a variety of gene therapy applications. However, the utility
of Ad vectors is limited due to the low efficiency of Ad-mediated gene
transfer to cells expressing marginal levels of the coxsackievirus and
adenovirus receptor (CAR). In order to achieve CAR-independent gene
transfer by Ad vectors in clinically important contexts, we proposed
modification of viral tropism via genetic alterations to the viral
fiber protein. We have shown that incorporation of an Arg-Gly-Asp
(RGD)-containing peptide in the HI loop of the fiber knob domain
results in the ability of the virus to utilize an alternative receptor
during the cell entry process. We have also demonstrated that due to
its expanded tissue tropism, this novel vector is capable of efficient
transduction of primary tumor cells. An increase in gene transfer to
ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an
incorporated RGD peptide may be of great utility for treatment of
neoplasms characterized by deficiency of the primary Ad type 5 receptor.
Adenovirus (Ad) vectors are useful
in a wide variety of gene therapy applications. One of the principal
attributes recommending the employment of these vectors is their
unparalleled efficacy in accomplishing gene transfer in vivo. This
property has been noted in a variety of different organs.
There are, however, some limitations associated with the use of
recombinant Ad for gene therapy. One such disadvantage is related to
the reliance of the virus on the presence of the coxsackievirus and
adenovirus receptor (CAR) to achieve high levels of gene transfer. In
certain settings, this may result in sequestration of recombinant virions by nontarget, yet high-CAR-expressing cells, whereas the true
target cells, if low in CAR, are poorly transduced. In order to
compensate for this sequestration, significant escalation in the dose
of administered vector is needed, which increases the risk of inducing
both direct toxicity and immune responses against the vector, thus
further compromising the overall efficacy of the therapy.
Therefore, the utility of the present generation of Ad vectors
for gene therapy may be significantly improved by achieving targeted
transduction of specific cell types by the virus.
In this regard, the initial steps of Ad infection involve at least two
sequential virus-cell interactions, each being mediated by a specific
protein component of Ad capsid. The primary binding of the virus to the
cell surface receptor, CAR (9, 10, 38), is mediated by the
knob domain of the fiber protein (23). This is followed by
the internalization of the virus within a clathrin-coated endosome
(39). The virus then escapes from the endosome by triggering its acidification via a secondary interaction of the
argininine-glycine-aspartic acid (RGD) motif of Ad penton base protein
with cellular integrins As the fiber and the penton base are key mediators of the cell entry
mechanism developed by Ad, targeting of recombinant Ad vectors may be
achieved via genetic modifications of these capsid proteins. In order
to overcome the limitations imposed by the CAR dependence of Ad
infection, Michael et al. (27) proposed the incorporation of
small peptide motifs possessing receptor binding specificities into the
carboxy terminus of Ad fiber protein, thus enabling the virus to attach
and infect via a novel cell surface receptor. This concept has been
further developed by Wickham et al. (43, 44), who have
proved the feasibility of this approach by generating several
recombinant Ad containing fibers with targeting ligands positioned at
the carboxy terminus of the fiber molecule.
Although in some cases genetic modification of the carboxy terminus of
Ad fiber has proved its utility with respect to vector retargeting, it
failed in others (44), thereby suggesting that this locale
in the fiber molecule is not the optimal site for incorporation of
targeting protein moieties. In this regard, published findings
(15, 44) strongly suggest that the addition of more than 25 to 30 amino acid residues of heterologous protein sequence to the
carboxy terminus of the fiber molecule has dramatic negative effect on
the stability of the fiber trimer and, therefore, is incompatible with
the fiber functions. In addition, the three-dimensional structure of
the fiber knob (45) clearly indicates that the carboxy
terminus of the fiber points toward the virion, that is, away from the
cell surface, thereby providing a suboptimal environment for the
incorporation of targeting ligands.
With these findings in mind, we recently reported that another locale
within the fiber molecule, the HI loop of the fiber knob domain, could
be used as a convenient site for incorporation of heterologous ligands
(21). As the next logical step, we explored the utility of
the HI loop for incorporation of targeting ligands to allow
modification of Ad tropism. Specifically, we sought to capitalize on
the recently published reports on phage biopanning (3, 31)
by choosing an RGD motif proven to have in vivo targeting capabilities.
We have shown that incorporation of this peptide into the fiber knob
allowed the virus to utilize the RGD-integrin interactions as an
alternative infection pathway, thereby dramatically improving the
ability of the virus to transduce several types of cells, which
normally are refractory to Ad infection. In order to show the utility
of the modified virion for application which may have immediate
clinical translation, we employed this viral vector as a means for
efficient gene transfer to primary ovarian cancer cells. Specifically,
we have shown that recombinant Ad vector containing fibers with RGD
motif in the HI loop is capable of dramatically augmenting gene
delivery to target cells via a CAR-independent cell entry mechanism.
Cells and tissues.
The 293 human kidney cell line
transformed with Ad type 5 (Ad5) DNA was purchased from Microbix
(Toronto, Ontario, Canada). Human ovarian carcinoma cell lines
SKOV3.ip1 and OV-4 were obtained from Janet Price (M. D. Anderson
Cancer Center, Houston, Tex.) and Timothy J. Eberlein (Brigham and
Women's Hospital, Harvard Medical School, Boston, Mass.),
respectively. Human umbilical vein endothelial cells (HUVEC) and human
embryonal rhabdomyosarcoma (RD) cells were from American Type Culture
Collection (Rockville, Md.). All cell lines were grown at 37°C in
media recommended by the suppliers in a humidified atmosphere of 5%
CO2.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
An Adenovirus Vector with Genetically Modified Fibers
Demonstrates Expanded Tropism via Utilization of a
Coxsackievirus and Adenovirus Receptor-Independent Cell
Entry Mechanism
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
v
3 and
v5 (4, 5, 41, 42). Following
the endosome escape, partially dismantled virus translocates to the
nuclear pore complex and releases its genome into the nucleoplasm where subsequent steps of viral replication take place.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until needed.
Enzymes. Restriction endonucleases, Klenow enzyme, T4 DNA ligase, T4 polynucleotide kinase, and proteinase K were from either New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.).
Monoclonal antibodies.
Anti-v3
integrin monoclonal antibody LM609 and
anti-v5 integrin monoclonal antibody P1F6
were purchased from Chemicon International, Inc. (Temecula, Calif.) and
Gibco-BRL (Gaithersburg, Md.), respectively. Anti-CAR monoclonal
antibody RmcB (9) was obtained from R. W. Finberg
(Dana-Farber Cancer Institute, Harvard Medical School, Boston, Mass.).
Viruses. A recombinant Ad5 vector, AdCMVLuc, containing a firefly luciferase-expressing cassette in place of the E1 region of the Ad genome, was obtained from R. D. Gerard (University of Texas Southwestern Medical Center, Dallas). Ad vector, Ad5lucRGD, containing recombinant fiber-RGD protein and expressing the firefly luciferase was generated by transfection of 293 cells with PacI-digested pVK703 by the method described previously (12). Ad were propagated on 293 cells and purified by centrifugation in CsCl gradients by a standard protocol. Determination of virus particle titer was accomplished spectrophotometrically by the method of Maizel et al. (24), using a conversion factor of 1.1 × 1012 viral particles per absorbance unit at 260 nm. To determine the titer of infectious viral particles, the plaque assay on 293 cells was performed by the method of Mittereder et al. (28) was used.
Construction of recombinant plasmids.
In order to generate a
recombinant Ad5 fiber gene encoding the fiber with the RGD-4C peptide
within the HI loop of the knob domain, a duplex made of
oligonucleotides CAC ACT AAA CGG TAC ACA GGA AAC AGG AGA CAC AAC TTG
TGA CTG CCG CGG AGA CTG TTT CTG CCC and GGG CAG AAA CAG TCT CCG CGG CAG
TCA CAA GTT GTG TCT CCT GTT TCC TGT GTA CCG TTT AGT GTG was cloned into
the EcoRV site of previously designed plasmid pQE.KNOB
HI
(21), thereby generating pQE.KNOB.RGDHI.
PC. This plasmid was then used for homologous DNA
recombination with ClaI-linearized pVK503 in order to
generate pVK703, containing the genome of Ad5lucRGD.
To derive a recombinant baculovirus expressing fiber-RGD, previously
made transfer vector pFB.F5HIFLAG (21) was
modified in the following way. First, EcoRI linker, CGG CGA
ATT CGC, was incorporated into the ClaI site of
pFB.F5HIFLAG, resulting in pFB.F5.RI. Then, the
NcoI-MunI fragment of pNEB.PK.FHIRGD
containing the 3' portion of the fiber-RGD gene was used to replace an
NcoI-MunI fragment in pFB.F5.RI, generating
pFB.F5HIRGD. This plasmid was then used to generate
recombinant baculovirus genome via site-specific transposition by
utilizing a Bac-to-Bac kit (Gibco-BRL) according to the manufacturer's recommendations.
Flow cytometry. Cells grown in T75 flasks were released from the flasks by the addition of EDTA and resuspended in SM buffer (HEPES-buffered saline, 0.1% sodium azide, 1% bovine serum albumin [BSA]) at 2 × 106 cells/ml. Two hundred thousand cells were incubated with 5 µg of LM609, P1F6, RmcB, or no primary monoclonal antibody (negative control) per ml in 200 µl of SM for 2 h at 4°C. Cells were then washed with SM and incubated with secondary fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G serum (Jackson Labs, West Grove, Pa.) (5 µg/ml) for 1 h at 4°C. After the cells were washed with SM, 104 cells per sample were analyzed by flow cytometry at the UAB FACS Core Facility.
Recombinant proteins. Recombinant Ad5 fiber knob protein was expressed in E. coli and purified by immobilized metal ion affinity chromatography (IMAC) on Ni-nitrilotriacetic acid (NTA)-Sepharose (Qiagen, Valencia, Calif.) as recommended by the manufacturer.
Human Ad2 penton base protein was expressed in Spodoptera frugiperda Sf9 cells by recombinant baculovirus AcNPV-PbWT (18) provided by P. Boulanger (Institute of Biology, Montpellier, France). The penton base protein was purified from baculovirus-infected cells by two-step ion-exchange chromatography utilizing a DEAE-Sepharose FF column (Pharmacia, Piscataway, N.J.) followed by purification on POROS HQ column (PerSeptive Biosystems, Framingham, Mass.). Recombinant fiber proteins expressed in baculovirus-infected Sf9 cells were purified by chromatography on Ni-NTA-Sepharose as previously described (21). The protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, Calif.) with bovine gamma globulin as the standard.ELISA.
Solid-phase binding assay was performed by a method
previously described by Sharma et al. (36). Briefly,
purified fiber proteins or Ad virions were diluted in 50 mM
carbonate-bicarbonate buffer, pH 9.6, to a concentration of 10 µg of
protein per ml, and 100-µl aliquots were added to the wells of a
96-well Nunc-Maxisorp enzyme-linked immunosorbent assay (ELISA) plate.
Plates were incubated overnight at 4°C and then blocked for 2 h
at room temperature by the addition of 200 µl of blocking buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% casein) to each well. Wells
were then washed three times with the washing buffer (20 mM Tris-HCl
[pH 7.5], 150 mM NaCl). Purified integrin
v3 (Chemicon) diluted in binding buffer
(20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM CaCl2, 1 mM
MgCl2, 1 mM MnCl2, 0.5% casein) to
concentrations ranging from 0.04 to 0.5 µg/ml was added to the wells
in 100-µl aliquots. After overnight incubation at 4°C, the wells
were washed three times with washing buffer containing 2 mM
CaCl2, 1 mM MgCl2, and 1 mM MnCl2.
Bound integrin was detected with mouse monoclonal anti-human integrin
v subunit antibody VNR139 (Gibco-BRL). VNR139 antibody
diluted 1:3,000 in binding buffer was added to the wells in 100-µl
aliquots, incubation was continued for 1 h at room temperature, and then the wells were washed again. The ELISA plate was then developed with VECTASTAIN kit (Vector Laboratories, Burlingame, Calif.)
as recommended by the manufacturer. Color development was stopped by
the addition of 1 N H2SO4, and plates were read in a microtiter plate reader set at 490 nm.
Ad-mediated gene transfer assay. Ad-mediated transduction experiments utilizing cell lines were performed as described previously (22).
Primary cells from ascitic fluid samples obtained from ovarian cancer patients were prepared for this analysis as follows. First, the erythrocytes present in the samples were lysed by the addition of buffer containing 150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA. Then, the cell debris and dead cells were separated from the live cells by slow-speed centrifugation on a step gradient of Ficoll-Hypaque (Media Preparation Shared Facility, UAB Comprehensive Cancer Center, Birmingham, Ala.). The cells were washed twice with Dulbecco's modified Eagle's medium/F12 (DMEM/F12) (Cellgro, Herndon, Va.) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, Utah), 100 U of penicillin per ml, and 100 µg of streptomycin per ml.Ad binding assay. Binding of 125I-labeled Ad to 293, HUVEC, or RD cells was assayed in a procedure described previously (21).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fiber-RGD protein efficiently interacts with integrins via the RGD tripeptide. Recently we demonstrated that the FLAG octapeptide incorporated in the HI loop of Ad5 fiber does not interfere with correct folding of the cell-binding site localized in the knob and is available for binding to FLAG-specific antibody in immunoprecipitation assay (21). To utilize these findings for the purposes of Ad retargeting, we chose to introduce in the HI loop of the fiber knob an RGD-4C peptide, CDCGRDCFC, which is known to bind with high affinities to several types of integrins present on the surfaces of mammalian cells. This effort was undertaken in an attempt to generate an Ad vector, which would be able to bind to cells by utilizing fiber-RGD-integrin interaction. Therefore, the infection by such virus would not be dependent on the presence of CAR on a cell membrane.
We first chose to express an RGD-4C-containing fiber protein,
fiber-RGD, in a baculovirus expression system in order to characterize the protein with respect to its ability to perform the targeting functions. The sequence encoding the amino-terminal six-His tag was
incorporated in the fiber-RGD gene in order to facilitate downstream
purification of the product. As anticipated from our previous
experiments done with fiber-FLAG protein, electrophoresis of
IMAC-purified fiber-RGD protein showed that the fiber retains its
native trimeric structure (data not shown), which is known to be
crucial for association of the fiber with the penton base during virion
assembly. In order to assess the ability of the fiber-RGD protein to
bind to integrins, we employed this fiber protein for an ELISA
utilizing purified integrin v3. This
assay showed that, in contrast to the wild-type fiber protein used as a
negative control, the fiber-RGD protein binds
v3 integrin very efficiently (Fig.
1). Therefore, these experiments
confirmed the functional utility of the modified fiber and provided a
rationale for generation of recombinant Ad containing such fibers.
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The virus was derived by the method described by Chartier et al.
(12). To simplify the downstream gene transfer assays, an
expression cassette containing the firefly luciferase gene driven by
cytomegalovirus promoter was introduced in place of the E1 region of
the Ad genome. The genome of the new virus designated Ad5lucRGD was
generated in E. coli via a two-step protocol utilizing homologous DNA recombination between the previously designed plasmid pVK50 (21) and fragments of DNA isolated from two shuttle
vectors, pNEB.PK.FHIRGD and pACCMV.LucPC, which contain
the fiber gene and the luciferase expression cassette flanked by Ad DNA
sequences, respectively. Utilization of this method requires the
digestion of the resultant recombinant plasmid containing the newly
generated Ad genome with restriction endonuclease PacI to
release inverted terminal repeats of Ad5 DNA from the plasmid backbone.
In order to be able to use the firefly luciferase gene, which contains an internal PacI site, in the context of this method, we
eliminated this site by introducing a silent mutation into the gene.
The plasmid obtained as a result of aforementioned DNA recombinations, pVK703, was then utilized for transfection of 293 cells to rescue Ad5lucRGD. The identity of the virus was confirmed by PCR as well as by
cycle sequencing of viral DNA isolated from CsCl-purified virions of Ad5lucRGD.
To demonstrate the accessibility of the RGD tripeptide incorporated in
the fiber of Ad5lucRGD, we utilized this virus in an ELISA analogous to
the one used previously for purified fiber protein. This analysis
clearly showed efficient binding of the v3 integrin to immobilized particles of
Ad5lucRGD, while binding of v3 to a
control virus was at the background level at all concentrations of
integrin used (Fig. 2). Based on these
results, we hypothesized that Ad5lucRGD is able to interact in vitro
and in vivo with various types of RGD-binding integrins, thereby
utilizing this interaction at early steps of infection in order to
attach to target cells.
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Ad5lucRGD is capable of mediating a CAR-independent gene
delivery.
Our next goal was to examine whether introduction of the
RGD motif in the fiber of Ad5lucRGD resulted in any changes with respect to the ability of this virus to infect cells. In order to
investigate the infection pathway utilized by Ad5lucRGD, we sought to
employ this virus for gene transfer to several cell lines, expressing
various levels of CAR as well as integrins
v3 and v5.
To achieve this goal, a panel of the cell lines, including the 293 human kidney cells, human umbilical cord endothelial cells, HUVECs, and
human embryonal RD cells, was employed for a series of flow cytometry
assays. While 293 cells readily support Ad infection, HUVECs have been
shown to bind Ad poorly (43), whereas CAR expression in RD
cells was reported to be passage dependent (35). Our flow cytometry assay showed that 293 cells express high levels of CAR (Fig.
3A) and v5
integrin, while expression of v3 is
moderate (Fig. 3B). HUVECs demonstrated moderate levels of CAR
expression (Fig. 3C), whereas both integrins were present at the cell
surface in rather large amounts (Fig. 3D). RD cells were CAR negative (Fig. 3E), while being high v5 and
moderate v3 expressors (Fig. 3F).
Therefore, for our subsequent gene transfer experiments, we established
a set of cell lines covering a full range of CAR expression profiles
and with moderate to high levels of integrins v3 and v5
present on their cytoplasmic membranes.
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v3 and
v5 integrins detected in these cells
suggested that HUVECs should be readily transduced with Ad5lucRGD.
Although the levels of luciferase activity in HUVECs mediated by either
virus were considerably lower than those in 293 cells, our experiment
revealed striking differences between the transduction profiles
demonstrated by these two viruses (Fig. 4B). First,
luciferase expression in the Ad5lucRGD-transduced cells was
about 30-fold higher than in the cells transduced with AdCMVLuc.
Second, the effect of the Ad5 fiber knob on AdCMVLuc-mediated transduction was less dramatic than in our experiments with 293 cells,
consistent with a relative lack of CAR in the HUVECs. Most importantly,
recombinant knob protein had no inhibition effect on the levels of
luciferase expression directed by Ad5lucRGD.
Very similar results were then generated on RD cells, which do not
express CAR. The luciferase activity detected in the lysates of
AdCMVLuc-transduced RD cells was extremely low: at an MOI of 1 PFU/cell, it was almost equal to background readings obtained in
mock-infected cells (Fig. 4C). Once again, Ad5lucRGD was capable of
directing the levels of transgene expression 16- to 47-fold higher than
those mediated by AdCMVLuc. This expression was not responsive to
inhibition by the fiber knob.
These experiments clearly showed that incorporation of the RGD-4C
peptide into the fiber of Ad5lucRGD resulted in dramatic changes in the
initial steps of virus-cell interaction, presumably by creating an
alternative cell attachment pathway.
Ad5lucRGD demonstrates increased efficiencies of cell binding due to utilization of RGD-integrin interaction. Having established that AdCMVLuc and Ad5lucRGD demonstrate different efficiencies of gene delivery as well as different profiles of fiber knob-mediated inhibition of transduction, our next task was to compare the cell binding profiles of these two viruses. To address this issue, we labeled both viruses with 125I and employed them in the virus binding assay on 293, HUVEC, and RD cells. This assay was performed under conditions (4°C) allowing the viruses to bind the cells, but preventing virus internalization.
As shown in Fig. 5, binding efficiencies demonstrated by Ad5lucRGD and AdCMVLuc on CAR-positive 293 cells were similar, while the percentages of labeled Ad5lucRGD virions bound to HUVEC and RD cells were significantly higher than those of AdCMVLuc virions.
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v3 and v5
integrins and therefore competes for binding to these cellular
receptors with other molecules or macromolecular complexes containing
an RGD motif.
When binding of the viruses to 293 cells was assayed (Fig.
6A), the penton base protein failed to
inhibit cell binding of either virus, whereas the fiber knob protein,
alone as well as together with the penton base, blocked 94% of
AdCMVLuc and 75% of Ad5lucRGD binding.
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Ad5lucRGD mediates enhanced gene transfer to human ovarian cancer cells. Since a number of clinical trials utilizing Ad vectors to treat cancer patients via direct in vivo gene delivery are currently under way, we chose to investigate whether the expanded tropism of Ad5lucRGD would render it useful for this type of clinical application.
First, we looked into the ability of this recombinant vector to deliver genes to cultured human ovarian cancer cells. Characterization of two cell lines, SKOV3.ip1 and OV-4, by flow cytometry showed that they both express moderate to high levels of integrinsv3 and v5
(Fig. 7B and D), SKOV3.ip1 expresses a
high level of CAR (Fig. 7A), whereas OV-4 is modest CAR
expressor (Fig. 7C).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this report, we describe the generation and characterization of recombinant Ad vector containing fibers with an RGD-4C sequence genetically incorporated within the HI loop of the carboxy-terminal knob domain. An effort to create such a virus was undertaken in order to demonstrate the utility of the HI loop of the fiber knob as an optimal site for incorporation of short peptide ligands, which would allow the virus to bind to ligand-specific cellular receptors, thereby resulting in altered or expanded tropism of the vector.
The interaction between cellular integrins and various proteins containing an RGD tripeptide is one of the best characterized interactions between macromolecules. This interaction plays an important role in a variety of fundamental biological processes, including cell adhesion and viral infection. In this regard, it has been shown that the RGD motif contained in adhesive proteins, such as fibrinectin, vitronectin, collagen, osteopontin, thrombospondin, fibrinogen, laminin, and von Willebrand factor (16, 29), allows efficient and specific interaction between these proteins and integrin molecules. It is also known that an RGD motif is present in some viral proteins including the VP1 proteins of coxsackievirus (32-34) and foot-and-mouth disease virus (13), the penton base protein of the majority of known Ad (25), the VP7 proteins of the African horsesickness virus and bluetongue virus (7), the Tat protein of human immunodeficiency virus (6), and the glycoprotein H of herpes simplex virus (14). In some of these instances, this tripeptide has been shown to play an important role in the process of viral infection by mediating primary or secondary interactions between the virion and cell surface-localized integrins. Furthermore, recent studies showed that genetic incorporation of the RGD-containing sequences into chimeric hepatitis B cores (11, 36), poliovirus particles (30), bacteriophage fd (19, 20), and human Ad virions (44) allows specific interaction of these viral particles with cellular integrins, thereby resulting in binding of aforementioned structures to cell surface.
We utilized a similar genetic strategy in order to expand the tropism
of recombinant Ad vector with respect to cell types which normally are
refractory to Ad infection. Based on our previous findings on
accessibility of the HI loop-localized FLAG peptide (21), we
hypothesized that positioning of the RGD-4C peptide in close proximity
to the putative cell binding domain localized within the knob of Ad5
fiber protein (45) should make this ligand available for
efficient interaction with integrins on the cell membrane. By using an
ELISA-based binding assay, we have been able to show direct interaction
between the RGD motif of the fiber-RGD protein with purified integrin
v3. This key finding provided a rationale
for the generation of recombinant Ad vector, Ad5lucRGD, containing
such fiber-RGD proteins. The data generated with Ad5lucRGD on
several cell lines showed that this virus demonstrates profiles of gene
transfer significantly different from those by the virus with
unmodified fibers. This difference was especially dramatic when
CAR-negative cells were utilized for the gene delivery experiments. Investigation of radiolabeled virus binding to the cells in vitro paralleled our gene transfer experiments, thereby supporting the concept of augmented efficiency of transgene expression as a result of
more-efficient primary interaction between the virus and the target cell.
In order to demonstrate the utility of the newly generated viral vector for clinical applications in the context of gene therapy, we employed Ad5lucRGD for gene delivery to cells isolated from ascitic fluid samples obtained from ovarian cancer patients. Our experiments showed that in this model Ad5lucRGD was able to direct levels of transgene expression 2 to 3 orders of magnitude higher that those mediated by control virion containing unmodified fibers. These results strongly suggest that recombinant Ad vectors containing fibers with genetically incorporated RGD peptides may be of great utility in the context of cancer gene therapy approaches based on in vivo gene delivery. In addition, well-documented overexpression of several types of integrins in tumor vasculature (26) suggests that derivatives of Ad5lucRGD expressing therapeutic genes may be utilized for eradication of tumors via abrogation of their blood supply.
Successful utilization of the RGD tripeptide incorporated into HI loop of Ad fiber protein for the purposes of vector retargeting suggests that other peptide ligands may work just as well in a context of the fiber molecule. In this regard, the rapidly emerging technology of phage display libraries has proved its utility as a means to identify peptides, which demonstrate the ability to specifically bind to certain molecules on a cell surface in vivo. This high-throughput method is based on the capability of small peptide ligands to target a bacteriophage particle to previously characterized as well as to unknown structures on a cell membrane. Recent successes in phage biopanning in an in vivo context (3) strongly suggest that this novel technology may provide an ideal source of targeting peptides to be used for modification of endogenous tropism of recombinant Ad vectors.
Whereas we have demonstrated the utility of small peptides to be
incorporated into the HI loop of the fiber knob, the size restrictions
of this locale have not been fully defined. In this regard, the
compatibility of the HI loop structure with protein ligands of a larger
size, such as, for example, single-chain antibodies (scFv), would
significantly expand the range of potential targeting approaches.
Furthermore, incorporation of large polypeptide ligands into the HI
loop, which connects -strands H and I involved in the formation of
the cell-binding site, may create a steric hindrance, thereby
preventing direct interaction of the fiber knob with CAR and resulting
in elimination of endogenous tropism of the virus. This, in turn, would
result in a new generation of truly retargeted Ad vectors, capable of
cell-specific gene delivery exclusively via CAR-independent mechanisms.
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ACKNOWLEDGMENTS |
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This work was supported by the following grants: NIH grants RO1 CA-74242, CA-68245, and RO1 HL-50255; a grant from the American Lung Association; and a grant from the U.S. Department of Defense, DAMD 17-94-J4398.
We are grateful to Pierre Boulanger for making the recombinant baculovirus AcNPV-PbWT available and Robert Gerard for providing the plasmid pACCMVpLpA. We are indebted to Robert Finberg for anti-CAR monoclonal antibody RmcB. We thank Paul Reynolds and Alex Pereboev for fruitful discussions and proofreading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Gene Therapy Program, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294-3300. Phone: (205) 934-8627. Fax: (205) 975-7476. E-mail: david.curiel{at}ccc.uab.edu.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Virology, December 1998, p. 9706-9713, Vol. 72, No. 12
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