# Mouse Mammary Tumor Virus Promoter-Containing  Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional In Vitro and In Vivo

Authors: Reinhard Klein; Bärbel Ruttkowski; Sonja Schwab; Thomas Peterbauer; Brian Salmons; Walter H. Günzburg; Christine Hohenadl
Journal: Journal of Biomedicine and Biotechnology
 (2008-01-20)

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## Abstract

Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respectiveIC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.


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## Body

## 1. Introduction

Conventional cancer chemotherapy including chemotherapy of breast cancer often results in
severe systemic toxicity at drug concentrations necessary for effective killing
of tumor cells. This
obstacle can be overcome with the concept of gene-directed enzyme prodrug
therapy (GDEPT) that implies selective delivery into tumor cells and expression
of drug-metabolizing transgenes within them [1].The oxazaphosphorine cyclophosphamide (CPA) and
its structural isomer ifosfamide (IFA) are DNA-alkylating agents commonly used
in breast cancer chemotherapy [2]. These anticancer
agents are administered as prodrugs that are primarily activated by the hepatic
enzyme cytochrome P450 (CYP). Among the P450 enzymes, the subfamily 2B enzymes CYP2B1 (from rat) and
CYP2B6 (from human) have been shown to be the most active catalysts for this
enzymatic reaction [3, 4]. The generated anticancer
metabolites phosphoramide mustard (from CPA) or isophosphoramide mustard (from
IFA) as well as acrolein are systematically distributed throughout the body
eventually reaching the tumor but also causing undesired toxic side effects.
Local activation of cyclophosphamide or ifosfamide at the site of the tumor
would allow to use lower concentrations of the prodrug resulting in lower
systemic toxicity with a still effective or, if using conventional prodrug
dosages, a much more potent cell killing effect on the tumor cells. In addition,
cyclophosphamide and ifosfamide suicide gene therapy has the advantage of also
exerting a bystander effect as it causes the death of not only the therapeutic
transgene-carrying cells but also of neighboring nontransgenic cells via passive diffusion of the
cytotoxic metabolites [5, 6].Gene therapy requires strong and, if possible, selective expression of the transgene
in the requested tissue or organ. For mammary gland-specific expression of
transgenes in mammals, a number of promoters from various sources have been
evaluated. Among those are the promoters of milk protein-encoding genes such as the whey acidic
protein (WAP), β-lactoglobulin,α-s1-casein, β-casein, or the C3(1) promoter [7]. However, for mouse models of human
breast cancer, the long terminal repeat (LTR) of the mouse mammary tumor virus
(MMTV) has emerged as the most potent and frequently used promoter to drive
transgene expression [8–12]. It, therefore, also
represents one of the major candidate promoters for human breast cancer gene
therapy. The MMTV promoter is most active during lactation due to induction by lactogenic
hormones such as prolactin [13, 14]. However, the most potent inducing effects
are due to the presence of hormone response elements (HREs) within the U3
region of the viral LTR that respond to androgens, progestins,
mineralocorticoids, and glucocorticoids [15].For in vitro and in vivo
transgene delivery, a number of techniques have been elaborated. Among those,
infection with retroviral vectors represents a very efficient method. Due to their capacity to integrate
into the host genome, retroviral vectors are one choice if long-term gene
expression of a transgene is desired and thus they have been used in a variety
of gene therapy studies [16]. However, to date, several rounds of vector
delivery are necessary to achieve satisfactory transfer of a therapeutic gene in vivo. This is mainly due to
unsatisfactory virus titers, rapid clearance of the vector by the liver and the
spleen [17], and, in the case of MLV-based retroviral vectors, the fact that
only dividing cells can be reached, thereby a priori limiting the number of accessible cells [18]. In addition, repeated delivery may cause the risk of the development
of an immune response against the vector, thereby impairing gene transfer. The establishment of an in situ cell depot, constantly
producing therapeutic retroviral vectors where required, may overcome those
hurdles and may allow much more efficient delivery of the transgene. In previous experiments, for proof of principle, we have
encapsulated virus packaging cells that not only expressed a reporter gene from
a retroviral vector  but also produced virus particles. Upon
insertion into the mammary glands of mice, the virus particles were liberated
from the capsules and transferred the reporter gene to surrounding cells [19].
This type of delivery has also been shown by others to be functional using a
potentially therapeutic suicide gene for the treatment of glioblastoma [20],
and similar long-term in vivo gene delivery could also be achieved using TheraCyte immuno-isolation devices
containing spleen necrosis retrovirus packaging cells [21]. We have also shown
previously that, when combined with ifosfamide-based chemotherapy, intratumoral
injection as well as instillation of capsules containing CYP2B1-expressing (but
not virus-producing) cells into tumor-supplying blood vessels leads to a
significantly increased tumoricidal effect on experimentally generated tumors
in mice [22, 23] or on inoperable
pancreatic tumors in humans [24, 25].In this study, we have constructed
MLV-based replication-deficient retroviral promoter conversion (ProCon) vectors
for the use in breast cancer gene therapy in combination with the in situ vector supply and delivery
system described above. The vectors are designed to express the CYP2B1 gene
within the packaging cells to convert the prodrugs ifosfamide or
cyclophosphamide into their active forms. In addition, the packaging cells
produce virus particles that are liberated from the cells and, by infection,
can transfer the therapeutic gene into the surrounding target cells. In the vectors described in this
study, the original U3 region of the3′long terminal repeat (LTR) is replaced
with the heterologous MMTV promoter. In virus-packaging
cell lines, transcription of the eGFP-encoding reporter gene or of the actual
therapeutic CYP2B1-encoding gene is driven by the MLV promoter/enhancer. After infection, in the course of
reverse transcription, the heterologous MMTV promoter is duplicated and one
copy is translocated to the U3-region of the 5′LTR. This
rearrangement finally brings the expression of the transduced gene under
control of the MMTV promoter. We show that the genes incorporated into the
vectors are efficiently expressed in virus packaging cells as well as in infected
cells. Virus titers obtained with the generated vectors are sufficiently high
to allow efficient infection of target cells both in vitro and in a murine tumor model in vivo. We further show
that the generated therapeutic CYP2B1 protein is enzymatically active and exerts
a strong cell killing effect on infected breast cancer cells upon treatment
with ifosfamide or cyclophosphamide in vitro.


## 2. Materials and Methods

### 2.1. Plasmids

Plasmids pPCCMm1 and pPCEMm1 (see Figure1) are
identical in construction except for the fact that pPCCMm1 carries the Cyp2B1
gene and pPCEMm1 carries the eGFP gene. pPCCMm1 was constructed by replacing a
cytomegalovirus (CMV) promoter-harboring MluI–SacII
fragment of plasmid pPCCmCMV.WPRE (Harry Holzmüller, Austrianova Biotechnology
GmbH) with an MluI–SacII
fragment from plasmid pPCEMa [26] harboring the MMTV promoter. Plasmid pPCEMm1
was constructed as described elsewhere [27]. pCMV-dsRed-Express was purchased
from BD Clontech.Figure 1
MLV-based retroviral ProCon vectors.5′LTRs consist of unique 3′(U3), repeated (R), and
unique 5′(U5) regions. 3′LTRs consist of repeated (R) and unique 5′(U5) regions.
The 3′LTR U3 region has been replaced by the MMTV promoter that drives
expression of the transgene (eGFP or CYP2B1) in infected cells. All vectors
contain the CMV enhancer region (CMV enh) upstream of the 5′LTR, a packaging
region (ψ), an internal SV40 promoter (SV40) driving the expression of a neomycin resistance
gene (neo), the Woodchuck hepatitis virus posttranscriptional regulatory
element (WPRE), and an origin of replication (ori) for replication in E. coli.


### 2.2. Nucleic Acid Extraction

Plasmid DNA was isolated using a QIAprep spin miniprep kit (Qiagen, Calif, USA) or a Qiagen-tip 100
plasmid midi kit (Qiagen). Linear DNA fragments were purified using a QIAquick
gel extraction kit (Qiagen).


### 2.3. Cell Lines

Human 2GP19Talf amphotropic retroviral
packaging cells [28], human 293 embryonic kidney cells (ATCC CRL-1573) [29],
and feline CRFK kidney cells (ATCC CCL-94) [30] were grown in Dulbecco’smodified
Eagle’s medium (DMEM)/Glutamax (Invitrogen Life Technologies, Calif, USA)supplemented
with 10% fetal bovine serum (FBS; Invitrogen Life Technologies). Human T-47D
(mammary gland; ductal carcinoma) cells (ATCC HTB-133) [31] were cultivated in
DMEM/Glutamax supplemented
with 10% FBS and 6.5 μg/mL insulin (Sigma-Aldrich, Miss, USA).


### 2.4. Transfection

Transfections of 2GP19Talf cells were performed by calcium phosphate coprecipitation according
to the instructions of the manufacturer (Amersham Biosciences, NJ, USA). For
stable transfections, the transfected cells were selected in medium containing
0.4 mg/mL Geneticin(G418; Invitrogen Life Technologies) until mock-transfected
cells had died. Stably transfected cells were maintained as populations in the
presence of 0.4 mg/mL G418.


### 2.5. In Vitro Infection Experiments

Culture supernatants from2×106 virus-producing cells were usedto infect 4×105 target cells as described elsewhere [28]. For titer calculation, dilutions of infected CRFK cells were
trypsinized and replated 24 hours afterinfection in triplicates and
selected in medium containing 0.4 mg of G418/mL.After 10 to 14 days
of culture, drug-resistant colonies werecounted, and the number of colony-forming
units (CFU) per milliliter of vector supernatant wascalculated.
Populations of stably infected T-47D and CRFK cells were maintained in the presence
of 0.4 mg/mL G418. For stimulation of expression, cells were treated with 1 μM
dexamethasone (Sigma-Aldrich) every 48 hours.


### 2.6. In Vivo Infection Experiments

For establishment of mixed tumors
2GP19Talf/pPCEMm1 cells and T-47D/DsRed or CRFK/DsRed cells were trypsinized
out of the culture flasks, washed three times with PBS, and mixed in a ratio of
1 : 5 or 1 : 10. A total number of6×105 mixed cells in a total volume
of 100 μL RPMI medium containing 100 U/mL penicillin/streptomycin (Invitrogen Life
Technologies) and 50% matrigel (Becton Dickinson Biosciences, NJ, USA) was
injected into the mammary fat pads of Hsd: Athymic Nude-nu mice (Harlan
Winkelmann, Borchen, Germany). For stimulation of tumor growth, slow-release estrogen
pellets (1.13 mg/pellet, 60 days release; Innovative Research of America, Fla,
USA) were implanted. At an average tumor size of 100–200 mm3, mice were treated
with 500 μg dexamethasone/mouse (Voren, 1 mg/mL, Boehringer Ingelheim, Ingelheim, Germany)
intraperitoneally on 3 consecutive days. On day 4, tumors were explanted,
digested to single-cell suspension by incubation with 2 mg/mL collagenase
(Sigma-Aldrich) in PBS at 37°C for two hours, washed twice with DMEM/10% FBS,
and finally analyzed by fluorescence-activatedcell sorting (FACS)
and confocal laser-scanning microscopy. In vivo experiments were carried out
according to Austrian law regulating animal experimentation (GZ 68.205/109-BrGT/2003).


### 2.7. Detection of Fluorescent Cells

For detection of DsRed- and EGFP-expressing
tissue culture cells, those were trypsinized, washed twice with PBS/10% FBS, and then 50,000 cells per sample were analyzed for fluorescencewith
an FACS analyzer (FACScalibur; Becton Dickinson). Cells from explanted tumors were
treated as described above. The numbers and meanfluorescence
intensities (MFIs) of positive cells were determined using the CellQuest
software (Becton Dickinson). For confocal laser-scanning microscopy, a Zeiss
LSM 510 inverted microscope equipped with a 40 x, 1.3 numerical aperture, oil
immersion objective (Plan Neofluar, Zeiss, Göttingen, Germany) was used. Cells
analyzed by laser-scanning microscopy were prepared in the same way as for FACS
analysis.


### 2.8. FACS Sorting

Populations of cells expressing the fluorescent proteins EGFP or DsRed were FACS sorted to exclude nonfluorescent
cells before their use in in vivo experiments. Therefore, cells to be sorted were expanded to approximately3×107, harvested by trypsinization, resuspended in normal cell culture
medium, and pelleted by cetrifugation at 410× g for 5 minutes. Thereafter,
cells were washed twice in PBS and resuspended in PBS containing 5% FBS. Then,
the cell suspension was filtered through a sterile nylon gauze into an FACS tube
and stored on ice until sorting. FACS sorting was performed using the
FACSVantage SE device (Becton Dickinson) which is controlled by the Cell Quest
Pro software (Becton Dickinson).


### 2.9. Detection of Protein and Enzymatic Activity

CYP2B1 was detected by Western blotting using an anti-CYP2B1 antibody (Daiichi Pure Chemicals Co., LTD, NJ,
USA). The enzymatic activity of CYP2B1 was determined in a 96-well format
resorufin assay. Briefly, cells were washed twice with PBS, trypsinized out of
the culture flasks, and resuspended  in DMEM/Glutamax medium lacking phenol red (Invitrogen Life Technologies)
supplemented with 10% FBS. 100 μL of cell suspension containing a total of4×105 cells was pipetted
into a black clear bottom 96-well plate in quadruplicates. DMEM/Glutamax medium
containing 10% FBS but lacking phenol red was used as a blank. 5 μL of substrate (0.3 mM benzylresorufin in DMSO; Sigma-Aldrich) was
added to each well. Plates were incubated for 1 hour at 37°C/5% CO2 protected from light. Samples were fluorometrically analyzed for the presence
of generated resorufin in a plate reader (Tecan Systems) using an extinction
wavelength of 520 nm and an emission wavelength of 590 nm. The CYP2B1 enzymatic activities of a
selected single cell clone (22P1G) [32] served as a reference.


### 2.10. Cytotoxicity Assays

1×104 T-47D or
T-47D/pPCCMm1 cells were seeded into 96 well plates in quadruplicates and
cultivated in DMEM/Glutamax medium lacking phenol red (Invitrogen Life
Technologies) supplemented with 10% FBS for six days without changing the
medium. 24 hours after seeding of the cells, the medium was supplemented with
0, 0.03, 0.1, 0.3, 1, or 3 mM ifosfamide (Baxter, Ill, USA) or cyclophosphamide
(Baxter). The supplementation with dexamethasone was repeated every 48 hours.
Five days after incubation with ifosfamide or cyclophosphamide, the viabilities
of the cells were determined with an XTT assay according to the instructions of
the manufacturer (Promega, Wiss, USA). The plates were analyzed with a plate
reader (Tecan Systems Inc., Calif, USA) by reading the absorbance at 490 nm.


## 2.1. Plasmids

Plasmids pPCCMm1 and pPCEMm1 (see Figure1) are
identical in construction except for the fact that pPCCMm1 carries the Cyp2B1
gene and pPCEMm1 carries the eGFP gene. pPCCMm1 was constructed by replacing a
cytomegalovirus (CMV) promoter-harboring MluI–SacII
fragment of plasmid pPCCmCMV.WPRE (Harry Holzmüller, Austrianova Biotechnology
GmbH) with an MluI–SacII
fragment from plasmid pPCEMa [26] harboring the MMTV promoter. Plasmid pPCEMm1
was constructed as described elsewhere [27]. pCMV-dsRed-Express was purchased
from BD Clontech.Figure 1
MLV-based retroviral ProCon vectors.5′LTRs consist of unique 3′(U3), repeated (R), and
unique 5′(U5) regions. 3′LTRs consist of repeated (R) and unique 5′(U5) regions.
The 3′LTR U3 region has been replaced by the MMTV promoter that drives
expression of the transgene (eGFP or CYP2B1) in infected cells. All vectors
contain the CMV enhancer region (CMV enh) upstream of the 5′LTR, a packaging
region (ψ), an internal SV40 promoter (SV40) driving the expression of a neomycin resistance
gene (neo), the Woodchuck hepatitis virus posttranscriptional regulatory
element (WPRE), and an origin of replication (ori) for replication in E. coli.


## 2.2. Nucleic Acid Extraction

Plasmid DNA was isolated using a QIAprep spin miniprep kit (Qiagen, Calif, USA) or a Qiagen-tip 100
plasmid midi kit (Qiagen). Linear DNA fragments were purified using a QIAquick
gel extraction kit (Qiagen).


## 2.3. Cell Lines

Human 2GP19Talf amphotropic retroviral
packaging cells [28], human 293 embryonic kidney cells (ATCC CRL-1573) [29],
and feline CRFK kidney cells (ATCC CCL-94) [30] were grown in Dulbecco’smodified
Eagle’s medium (DMEM)/Glutamax (Invitrogen Life Technologies, Calif, USA)supplemented
with 10% fetal bovine serum (FBS; Invitrogen Life Technologies). Human T-47D
(mammary gland; ductal carcinoma) cells (ATCC HTB-133) [31] were cultivated in
DMEM/Glutamax supplemented
with 10% FBS and 6.5 μg/mL insulin (Sigma-Aldrich, Miss, USA).


## 2.4. Transfection

Transfections of 2GP19Talf cells were performed by calcium phosphate coprecipitation according
to the instructions of the manufacturer (Amersham Biosciences, NJ, USA). For
stable transfections, the transfected cells were selected in medium containing
0.4 mg/mL Geneticin(G418; Invitrogen Life Technologies) until mock-transfected
cells had died. Stably transfected cells were maintained as populations in the
presence of 0.4 mg/mL G418.


## 2.5. In Vitro Infection Experiments

Culture supernatants from2×106 virus-producing cells were usedto infect 4×105 target cells as described elsewhere [28]. For titer calculation, dilutions of infected CRFK cells were
trypsinized and replated 24 hours afterinfection in triplicates and
selected in medium containing 0.4 mg of G418/mL.After 10 to 14 days
of culture, drug-resistant colonies werecounted, and the number of colony-forming
units (CFU) per milliliter of vector supernatant wascalculated.
Populations of stably infected T-47D and CRFK cells were maintained in the presence
of 0.4 mg/mL G418. For stimulation of expression, cells were treated with 1 μM
dexamethasone (Sigma-Aldrich) every 48 hours.


## 2.6. In Vivo Infection Experiments

For establishment of mixed tumors
2GP19Talf/pPCEMm1 cells and T-47D/DsRed or CRFK/DsRed cells were trypsinized
out of the culture flasks, washed three times with PBS, and mixed in a ratio of
1 : 5 or 1 : 10. A total number of6×105 mixed cells in a total volume
of 100 μL RPMI medium containing 100 U/mL penicillin/streptomycin (Invitrogen Life
Technologies) and 50% matrigel (Becton Dickinson Biosciences, NJ, USA) was
injected into the mammary fat pads of Hsd: Athymic Nude-nu mice (Harlan
Winkelmann, Borchen, Germany). For stimulation of tumor growth, slow-release estrogen
pellets (1.13 mg/pellet, 60 days release; Innovative Research of America, Fla,
USA) were implanted. At an average tumor size of 100–200 mm3, mice were treated
with 500 μg dexamethasone/mouse (Voren, 1 mg/mL, Boehringer Ingelheim, Ingelheim, Germany)
intraperitoneally on 3 consecutive days. On day 4, tumors were explanted,
digested to single-cell suspension by incubation with 2 mg/mL collagenase
(Sigma-Aldrich) in PBS at 37°C for two hours, washed twice with DMEM/10% FBS,
and finally analyzed by fluorescence-activatedcell sorting (FACS)
and confocal laser-scanning microscopy. In vivo experiments were carried out
according to Austrian law regulating animal experimentation (GZ 68.205/109-BrGT/2003).


## 2.7. Detection of Fluorescent Cells

For detection of DsRed- and EGFP-expressing
tissue culture cells, those were trypsinized, washed twice with PBS/10% FBS, and then 50,000 cells per sample were analyzed for fluorescencewith
an FACS analyzer (FACScalibur; Becton Dickinson). Cells from explanted tumors were
treated as described above. The numbers and meanfluorescence
intensities (MFIs) of positive cells were determined using the CellQuest
software (Becton Dickinson). For confocal laser-scanning microscopy, a Zeiss
LSM 510 inverted microscope equipped with a 40 x, 1.3 numerical aperture, oil
immersion objective (Plan Neofluar, Zeiss, Göttingen, Germany) was used. Cells
analyzed by laser-scanning microscopy were prepared in the same way as for FACS
analysis.


## 2.8. FACS Sorting

Populations of cells expressing the fluorescent proteins EGFP or DsRed were FACS sorted to exclude nonfluorescent
cells before their use in in vivo experiments. Therefore, cells to be sorted were expanded to approximately3×107, harvested by trypsinization, resuspended in normal cell culture
medium, and pelleted by cetrifugation at 410× g for 5 minutes. Thereafter,
cells were washed twice in PBS and resuspended in PBS containing 5% FBS. Then,
the cell suspension was filtered through a sterile nylon gauze into an FACS tube
and stored on ice until sorting. FACS sorting was performed using the
FACSVantage SE device (Becton Dickinson) which is controlled by the Cell Quest
Pro software (Becton Dickinson).


## 2.9. Detection of Protein and Enzymatic Activity

CYP2B1 was detected by Western blotting using an anti-CYP2B1 antibody (Daiichi Pure Chemicals Co., LTD, NJ,
USA). The enzymatic activity of CYP2B1 was determined in a 96-well format
resorufin assay. Briefly, cells were washed twice with PBS, trypsinized out of
the culture flasks, and resuspended  in DMEM/Glutamax medium lacking phenol red (Invitrogen Life Technologies)
supplemented with 10% FBS. 100 μL of cell suspension containing a total of4×105 cells was pipetted
into a black clear bottom 96-well plate in quadruplicates. DMEM/Glutamax medium
containing 10% FBS but lacking phenol red was used as a blank. 5 μL of substrate (0.3 mM benzylresorufin in DMSO; Sigma-Aldrich) was
added to each well. Plates were incubated for 1 hour at 37°C/5% CO2 protected from light. Samples were fluorometrically analyzed for the presence
of generated resorufin in a plate reader (Tecan Systems) using an extinction
wavelength of 520 nm and an emission wavelength of 590 nm. The CYP2B1 enzymatic activities of a
selected single cell clone (22P1G) [32] served as a reference.


## 2.10. Cytotoxicity Assays

1×104 T-47D or
T-47D/pPCCMm1 cells were seeded into 96 well plates in quadruplicates and
cultivated in DMEM/Glutamax medium lacking phenol red (Invitrogen Life
Technologies) supplemented with 10% FBS for six days without changing the
medium. 24 hours after seeding of the cells, the medium was supplemented with
0, 0.03, 0.1, 0.3, 1, or 3 mM ifosfamide (Baxter, Ill, USA) or cyclophosphamide
(Baxter). The supplementation with dexamethasone was repeated every 48 hours.
Five days after incubation with ifosfamide or cyclophosphamide, the viabilities
of the cells were determined with an XTT assay according to the instructions of
the manufacturer (Promega, Wiss, USA). The plates were analyzed with a plate
reader (Tecan Systems Inc., Calif, USA) by reading the absorbance at 490 nm.


## 3. Results

### 3.1. The Generated ProCon Vector System Allows Efficient Infection of Target Cells

We have previously evaluated MLV-based ProCon
vectors (pPCEM and pPCEMm1) containing the mammary gland-specific MMTV promoter
and the eGFP gene as a reporter for their gene expression and infection capacities
in vitro [27]. Here, for in vivo
investigations of the efficacy of the therapeutic vectors, we chose the
following experimental setup: we wanted to determine if efficient infection of
target cells is (i) also achievable by cocultivation of virus-producing cells
and target cells in vitro instead of using
high-titer virus suspensions for in vitro infections and (ii) in a mixed tumor
model in vivo, thus mimicking a future therapeutic situation in which
viruses would be released from capsules. In 293-based virus-producing 2GP19Talf
cells, vector pPCEMm1 (see Figure 1) [27] that was used in this study allows
the expression of the eGFP reporter gene from an MLV promoter/CMV enhancer
cassette while in infected cells after promoter conversion, expression of the
eGFP gene is driven by the heterologous MMTV promoter.For in vitro infection studies, 2GP19Talf virus-producing cells that had stably been transfected with pPCEMm1
were mixed in ratios of 1 : 10 or 1 : 5 with red fluorescent human T-47D breast
cancer cells and as a control with red fluorescent feline CRFK kidney cells
that are known to effectively being infected by retroviruses. Red fluorescence of target cells was
achieved by stably transfecting the cells with the DsRed protein-encoding
vector pCMV-dsRed-Express and enrichment of red fluorescent
cells was performed by cell sorting. Mixed cells were
cocultivated for five weeks and the numbers of green, red, and green/red cells
were monitored by FACS (see Figure2(a)). After five weeks, about 74% of red
fluorescent T-47-D cells were also green fluorescent, indicating that they had
been infected with pPCEMm1. The initial ratio between virus-producing cells and target cells did not
play a role in this setting. The red fluorescent CRFK
cells were infected to an extent of about 19% (ratio 1 : 10 between virus-producing
cells and target cells) and 40% (ratio 1 : 5 between virus-producing cells and
target cells). In contrast, when green fluorescent nonvirus-producing  293 cells stably infected with pPCEMm1 where
mixed with red fluorescent T-47D or CRFK cells, only a very low number (<1%) of false-positive red/green cells were detected by FACS after six weeks of
cocultivation (data not shown). The obtained results were also verified by confocal laser-scanning
microscopy: no red/green double fluorescent cells could be detected as
exemplified in Figure 2(b) for a mix of 293 cells stably infected with pPCEMm1
and CRFK/DsRed cells. In contrast, when the cell mixes
of cocultivated red fluorescent T-47D or CRFK cells and virus-producing green
fluorescent cells were analyzed, clearly a number of truly red/green double
fluorescent cells could be detected as demonstrated for a mix of CRFK/DsRed and
2GP19Talf/pPCEMm1cells (see Figure 2(c)). Double fluorescence was not due to
red autofluorescence of 2GP19Talf/pPCEMm1 or green autofluorescence of
CRFK/pPCEMm1 and T-47D/pPCEMm1 cells since no red fluorescent cells could be
detected in samples only consisting of green fluorescent 2GP19Talf/pPCEMm1
cells and no green fluorescence could be detected in samples of red fluorescent
CRFK/DsRed and T-47D/DsRed cells (data not shown).In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)
FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfected
with pPCEMm1 over a time period of five weeks. The numbers of red, green, and
red/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. As
positive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cells
were used. (b) Confocal laser-scanning microscopy of CRFK/DsRed
cells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.
Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), red
fluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanning
microscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over a
time period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;
lower right (LR), UL + UR. Magnification 400 x.
(a)(b)(c)To investigate the in vivo infection capability of the MMTV promoter containing
ProCon vector, mixed tumors consisting of DsRed-expressing T-47D or CRFK cells
on the one hand and virus-producing 2GP19Talf/pPCEMm1 cells on the other hand
were established in the mammary fat pads of nude mice. Again,
the ratios between virus-producing cells and target cells at the time point of
injection were 1 : 10 and 1 : 5. After 11 weeks of tumor growth, the tumors were
explanted and analyzed by FACS (see Figure3(a)). Of the red fluorescent T-47D
cells of six mixed tumors, 22% to 55% were also green fluorescent, that is, had
been infected with pPCEMm1. A range of 13%–33% of red
fluorescent CRFK cells isolated from mixed tumors also showed green
fluorescence, indicating infection by pPCEMm1. In addition, cells were analyzed by confocal laser-scanning
microscopy (see Figures 3(b) and 3(c)). Clearly, cells
fluorescing red as well as green were visible in the samples derived from mixed
T-47D  (see Figure 3(b)) as well as from
mixed CRFK tumors (see Figure 3(c)). As expected, cells originating from
control tumors solely consisting of 2GP19Talf/pPCEMm1 cells showed only green
but no red fluorescence while cells stemming from control tumors exclusively
consisting of T-47D/DsRed or CRFK/DsRed cells showed only red but no green
fluorescence. Cells derived from control tumors only consisting of 2GP19Talf,
T-47D, or CRFK cells exhibited no fluorescence at all (data not shown).In vivo infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a) FACS analysis of cells
from explanted mixed tumors consisting of 2GP19Talf/pPCEMm1
cells mixed with either T-47D/DsRed or CRFK/DsRed cells. The numbers of red, green, and red/green fluorescent cells were
determined. Percentages of red/green doublefluorescent cells for individual tumors are shown. Individual
tumors are designated with alphabetical characters below each bar. (b)-(c) Confocal laser-scanning microscopy of cells from explanted mixed tumors consisting of 2GP19Talf/pPCEMm1
and either T-47D/DsRed (b) or CRFK/DsRed cells (c). Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;
lower right (LR), UL + UR. Magnification 400 x.
(a)(b)(c)


### 3.2. The ProCon Vector/Virus Packaging Cell  System is Functional

The eGFP reporter gene was replaced with the therapeutic gene coding for
cytochrome P450 2B1 (CYP2B1). The resulting vector pPCCMm1 (see Figure1) was
used to transfect 2GP19Talf cells and vector harboring cells were selected with
geneticin in order to obtain a population of stably transfected cells. Western
blot analysis was used to prove that CYP2B1 was produced in 2GP19Talf/pPCCMm1
cells (see Figure 4(a)). To not only test for the presence of the protein but
also for its activity, resorufin formation as a consequence of CYP2B1 activity
was monitored using a 96-well format assay. Generation of resorufin in
2GP19Talf/pPCCMm1 cells in relation to 2GP19Talf negative control cells after
incubation with pentoxyresorufin is shown in Figure 4(b). The presence of
resorufin indicated that CYP2B1 was active.CYP2B1 expression, activity, and virus titers of 2GP19Talf amphotropic
retroviral packaging cells stably transfected with pPCCMm1. (a) Expression of CYP2B1 in 2GP19Talf/pPCCMm1
cells. Cell lysates (equal amounts of protein)
were analyzed by Western blotting for the presence of CYP2B1 using an
anti-CYP2B1 antibody. The CYP2B1 band is indicated with an arrow. The molecular weights of the protein
standard are given on the left side. Neg. cntrl.: 2GP19Talf cells not transfected with pPCCMm1.
(b) Enzymatic activity of CYP2B1 in 2GP19Talf/pPCCMm1 cells. The enzymatic activity as determined
by a resorufin assay is shown as the relative emission intensity of the created
product resorufin at 590 nm in comparison to nontransfected cells (neg. cntrl.). (c) Virus titers of 2GP19Talf/pPCCMm1 cells as
determined by counting the numbers of colonies of infected G418-resistant CRFK
cells (colony-forming units per milliliter; CFU/mL). Two independent infection
experiments were performed. Plating of dilutions of cells after infection was
performed in triplicates, each. Neg. cntrl.: 2GP19Talf cells not transfected with pPCCMm1.
(a)(b)(c)For infection of target tumor cells, sufficiently high numbers of
virus particles have to be generated by the virus-producing cells. Virus titers were determined by
infection of CRFK cells with virus particles from the supernatant of
2GP19Talf/pPCCMm1 cells and followed by counting the numbers of G418 resistant
colonies. Titers were about1.3×106 colony-forming units (CFU)/mL of
cell supernatant (see Figure 4(c)) which is comparable to titers obtained with
the respective eGFP gene-containing vector pPCEMm1 [27].


### 3.3. The MMTV Promoter ProCon System Generates a Pronounced Cell Killing Effect in Vitro

As a model breast cancer cell line to study the efficacy of our vector system, the
T-47D cell line was chosen. T-47D cells were infected with pPCCMm1 and stable
populations were created by selection of infected cells with G418. For
induction of gene expression from the MMTV promoter cells were stimulated with dexamethasone
for 48 hours and analyzed by Western blotting for the presence of CYP2B1. While an anti-CYP2B1 antibody did
not detect any CYP2B1 in noninfected T-47D cells (negative control), CYP2B1 was
clearly present in T-47D cells infected with pPCCMm1 (see Figure5(a)). Resorufin assays revealed that the protein was also active in that
cell line (Figure 5(b)). Enzyme activities were 2.5-fold higher in cells that
had been treated with dexamethasone compared to those in nontreated cells (data
not shown).CYP2B1 expression and activity in T-47D cells stably infected with pPCCMm1. (a)
Expression of CYP2B1 in a population of T-47D/pPCCMm1
cells. Cell lysates (equal amounts of protein) were analyzed by Western
blotting for the presence of CYP2B1 using an anti-CYP2B1 antibody. The CYP2B1
band is indicated with an arrow. The molecular weights of the protein standard are given on the left
side. Neg. cntrl.: T-47D cells not infected with pPCCMm1. (b) Enzymatic
activity of CYP2B1 in a population of T-47D/pPCCMm1 cells. The enzymatic
activity as determined by a resorufin assay is shown as the relative emission
intensity of the created product resorufin at 590 nm in comparison to noninfected
T-47D cells (neg. cntrl.) and a reference clone of virus-packing cells (22P1G).
(a)(b)To evaluate the sensitizing effect of CYP2B1 on T-47D cells upon treatment with the anticancer prodrug ifosfamide,
T-47D cells infected with pPCCMm1 and noninfected T-47D control cells were
treated with increasing amounts of ifosfamide (ranging from 0 to 3 mM) for five
consecutive days after which cell viabilities were determined using an XTT
assay. While noninfected T-47D cells remained largely unaffected up to an
ifosfamide concentration of 0.3 mM, T-47D/pPCCMm1 cells expressing the CYP2B1
gene showed a marked decrease of 40% already at a concentration of 0.1 mM. This
viability decreased further at higher concentrations (see Figure6(a)). 0.3 mM
ifosfamide was determined as the most effective concentration at which noninfected
T-47 cells were still largely unaffected but CYP2B1-producing T-47D/pPCCMm1
cells showed a dramatic decrease in viability by 88%. IC50 values
were determined as 1.6 mM and 0.1 mM for T-47D and T-47D/pPCCMm1, respectively.
This represents a decrease of the IC50 value by sixteen folds,
underlining the potent cumulative cytotoxic effect of CYP2B1 and ifosfamide.
Treatment of the cells with cyclophosphamide instead of ifosfamide led to
similar results (see Figure 6(b)).Enhanced
cytotoxicities of ifosfamide and cyclophosphamide in T-47D/pPCCMm1 cells
expressing CYP2B1. Cells were cultivated at increasing amounts of ifosfamide
for five days as described in materials and methods. Cell viabilities were
determined using an XTT assay and survival rates of CYP2B1 expressing cells in
relation to noninfected control cells were calculated. (a) Incubation of cells
with ifosfamide. (b) Incubation of cells with cyclophosphamide.
(a)(b)


## 3.1. The Generated ProCon Vector System Allows Efficient Infection of Target Cells

We have previously evaluated MLV-based ProCon
vectors (pPCEM and pPCEMm1) containing the mammary gland-specific MMTV promoter
and the eGFP gene as a reporter for their gene expression and infection capacities
in vitro [27]. Here, for in vivo
investigations of the efficacy of the therapeutic vectors, we chose the
following experimental setup: we wanted to determine if efficient infection of
target cells is (i) also achievable by cocultivation of virus-producing cells
and target cells in vitro instead of using
high-titer virus suspensions for in vitro infections and (ii) in a mixed tumor
model in vivo, thus mimicking a future therapeutic situation in which
viruses would be released from capsules. In 293-based virus-producing 2GP19Talf
cells, vector pPCEMm1 (see Figure 1) [27] that was used in this study allows
the expression of the eGFP reporter gene from an MLV promoter/CMV enhancer
cassette while in infected cells after promoter conversion, expression of the
eGFP gene is driven by the heterologous MMTV promoter.For in vitro infection studies, 2GP19Talf virus-producing cells that had stably been transfected with pPCEMm1
were mixed in ratios of 1 : 10 or 1 : 5 with red fluorescent human T-47D breast
cancer cells and as a control with red fluorescent feline CRFK kidney cells
that are known to effectively being infected by retroviruses. Red fluorescence of target cells was
achieved by stably transfecting the cells with the DsRed protein-encoding
vector pCMV-dsRed-Express and enrichment of red fluorescent
cells was performed by cell sorting. Mixed cells were
cocultivated for five weeks and the numbers of green, red, and green/red cells
were monitored by FACS (see Figure2(a)). After five weeks, about 74% of red
fluorescent T-47-D cells were also green fluorescent, indicating that they had
been infected with pPCEMm1. The initial ratio between virus-producing cells and target cells did not
play a role in this setting. The red fluorescent CRFK
cells were infected to an extent of about 19% (ratio 1 : 10 between virus-producing
cells and target cells) and 40% (ratio 1 : 5 between virus-producing cells and
target cells). In contrast, when green fluorescent nonvirus-producing  293 cells stably infected with pPCEMm1 where
mixed with red fluorescent T-47D or CRFK cells, only a very low number (<1%) of false-positive red/green cells were detected by FACS after six weeks of
cocultivation (data not shown). The obtained results were also verified by confocal laser-scanning
microscopy: no red/green double fluorescent cells could be detected as
exemplified in Figure 2(b) for a mix of 293 cells stably infected with pPCEMm1
and CRFK/DsRed cells. In contrast, when the cell mixes
of cocultivated red fluorescent T-47D or CRFK cells and virus-producing green
fluorescent cells were analyzed, clearly a number of truly red/green double
fluorescent cells could be detected as demonstrated for a mix of CRFK/DsRed and
2GP19Talf/pPCEMm1cells (see Figure 2(c)). Double fluorescence was not due to
red autofluorescence of 2GP19Talf/pPCEMm1 or green autofluorescence of
CRFK/pPCEMm1 and T-47D/pPCEMm1 cells since no red fluorescent cells could be
detected in samples only consisting of green fluorescent 2GP19Talf/pPCEMm1
cells and no green fluorescence could be detected in samples of red fluorescent
CRFK/DsRed and T-47D/DsRed cells (data not shown).In vitro infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a)
FACS analysis of CRFK/DsRed and T-47D/DsRed cells cocultivated with 2GP19Talf cells stably transfected
with pPCEMm1 over a time period of five weeks. The numbers of red, green, and
red/green fluorescent cells of a representative experiment are shown. As negative controls, 2GP19Talf, T-47D, and CRFK cells were used. As
positive controls, pure 2GP19Talf/pPCEMm1, T-47D/DsRed, and CRFK/DsRed cells
were used. (b) Confocal laser-scanning microscopy of CRFK/DsRed
cells cocultivated with 293/pPCEMm1 cells over a time period of five weeks.
Upper left (UL), green fluorescence; upper right (UR), transmission; lower left (LL), red
fluorescence; lower right (LR), UL + LL. Magnification 400 x. (c) Confocal laser-scanning
microscopy of CRFK/DsRed cells cocultivated with 2GP19Talf/pPCEMm1cells over a
time period of five weeks. Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;
lower right (LR), UL + UR. Magnification 400 x.
(a)(b)(c)To investigate the in vivo infection capability of the MMTV promoter containing
ProCon vector, mixed tumors consisting of DsRed-expressing T-47D or CRFK cells
on the one hand and virus-producing 2GP19Talf/pPCEMm1 cells on the other hand
were established in the mammary fat pads of nude mice. Again,
the ratios between virus-producing cells and target cells at the time point of
injection were 1 : 10 and 1 : 5. After 11 weeks of tumor growth, the tumors were
explanted and analyzed by FACS (see Figure3(a)). Of the red fluorescent T-47D
cells of six mixed tumors, 22% to 55% were also green fluorescent, that is, had
been infected with pPCEMm1. A range of 13%–33% of red
fluorescent CRFK cells isolated from mixed tumors also showed green
fluorescence, indicating infection by pPCEMm1. In addition, cells were analyzed by confocal laser-scanning
microscopy (see Figures 3(b) and 3(c)). Clearly, cells
fluorescing red as well as green were visible in the samples derived from mixed
T-47D  (see Figure 3(b)) as well as from
mixed CRFK tumors (see Figure 3(c)). As expected, cells originating from
control tumors solely consisting of 2GP19Talf/pPCEMm1 cells showed only green
but no red fluorescence while cells stemming from control tumors exclusively
consisting of T-47D/DsRed or CRFK/DsRed cells showed only red but no green
fluorescence. Cells derived from control tumors only consisting of 2GP19Talf,
T-47D, or CRFK cells exhibited no fluorescence at all (data not shown).In vivo infection of T-47D/DsRed and CRFK/DsRed cells with pPCEMm1. (a) FACS analysis of cells
from explanted mixed tumors consisting of 2GP19Talf/pPCEMm1
cells mixed with either T-47D/DsRed or CRFK/DsRed cells. The numbers of red, green, and red/green fluorescent cells were
determined. Percentages of red/green doublefluorescent cells for individual tumors are shown. Individual
tumors are designated with alphabetical characters below each bar. (b)-(c) Confocal laser-scanning microscopy of cells from explanted mixed tumors consisting of 2GP19Talf/pPCEMm1
and either T-47D/DsRed (b) or CRFK/DsRed cells (c). Upper left (UL), green fluorescence; upper right (UR), red fluorescence; lower left (LL), transmission;
lower right (LR), UL + UR. Magnification 400 x.
(a)(b)(c)


## 3.2. The ProCon Vector/Virus Packaging Cell  System is Functional

The eGFP reporter gene was replaced with the therapeutic gene coding for
cytochrome P450 2B1 (CYP2B1). The resulting vector pPCCMm1 (see Figure1) was
used to transfect 2GP19Talf cells and vector harboring cells were selected with
geneticin in order to obtain a population of stably transfected cells. Western
blot analysis was used to prove that CYP2B1 was produced in 2GP19Talf/pPCCMm1
cells (see Figure 4(a)). To not only test for the presence of the protein but
also for its activity, resorufin formation as a consequence of CYP2B1 activity
was monitored using a 96-well format assay. Generation of resorufin in
2GP19Talf/pPCCMm1 cells in relation to 2GP19Talf negative control cells after
incubation with pentoxyresorufin is shown in Figure 4(b). The presence of
resorufin indicated that CYP2B1 was active.CYP2B1 expression, activity, and virus titers of 2GP19Talf amphotropic
retroviral packaging cells stably transfected with pPCCMm1. (a) Expression of CYP2B1 in 2GP19Talf/pPCCMm1
cells. Cell lysates (equal amounts of protein)
were analyzed by Western blotting for the presence of CYP2B1 using an
anti-CYP2B1 antibody. The CYP2B1 band is indicated with an arrow. The molecular weights of the protein
standard are given on the left side. Neg. cntrl.: 2GP19Talf cells not transfected with pPCCMm1.
(b) Enzymatic activity of CYP2B1 in 2GP19Talf/pPCCMm1 cells. The enzymatic activity as determined
by a resorufin assay is shown as the relative emission intensity of the created
product resorufin at 590 nm in comparison to nontransfected cells (neg. cntrl.). (c) Virus titers of 2GP19Talf/pPCCMm1 cells as
determined by counting the numbers of colonies of infected G418-resistant CRFK
cells (colony-forming units per milliliter; CFU/mL). Two independent infection
experiments were performed. Plating of dilutions of cells after infection was
performed in triplicates, each. Neg. cntrl.: 2GP19Talf cells not transfected with pPCCMm1.
(a)(b)(c)For infection of target tumor cells, sufficiently high numbers of
virus particles have to be generated by the virus-producing cells. Virus titers were determined by
infection of CRFK cells with virus particles from the supernatant of
2GP19Talf/pPCCMm1 cells and followed by counting the numbers of G418 resistant
colonies. Titers were about1.3×106 colony-forming units (CFU)/mL of
cell supernatant (see Figure 4(c)) which is comparable to titers obtained with
the respective eGFP gene-containing vector pPCEMm1 [27].


## 3.3. The MMTV Promoter ProCon System Generates a Pronounced Cell Killing Effect in Vitro

As a model breast cancer cell line to study the efficacy of our vector system, the
T-47D cell line was chosen. T-47D cells were infected with pPCCMm1 and stable
populations were created by selection of infected cells with G418. For
induction of gene expression from the MMTV promoter cells were stimulated with dexamethasone
for 48 hours and analyzed by Western blotting for the presence of CYP2B1. While an anti-CYP2B1 antibody did
not detect any CYP2B1 in noninfected T-47D cells (negative control), CYP2B1 was
clearly present in T-47D cells infected with pPCCMm1 (see Figure5(a)). Resorufin assays revealed that the protein was also active in that
cell line (Figure 5(b)). Enzyme activities were 2.5-fold higher in cells that
had been treated with dexamethasone compared to those in nontreated cells (data
not shown).CYP2B1 expression and activity in T-47D cells stably infected with pPCCMm1. (a)
Expression of CYP2B1 in a population of T-47D/pPCCMm1
cells. Cell lysates (equal amounts of protein) were analyzed by Western
blotting for the presence of CYP2B1 using an anti-CYP2B1 antibody. The CYP2B1
band is indicated with an arrow. The molecular weights of the protein standard are given on the left
side. Neg. cntrl.: T-47D cells not infected with pPCCMm1. (b) Enzymatic
activity of CYP2B1 in a population of T-47D/pPCCMm1 cells. The enzymatic
activity as determined by a resorufin assay is shown as the relative emission
intensity of the created product resorufin at 590 nm in comparison to noninfected
T-47D cells (neg. cntrl.) and a reference clone of virus-packing cells (22P1G).
(a)(b)To evaluate the sensitizing effect of CYP2B1 on T-47D cells upon treatment with the anticancer prodrug ifosfamide,
T-47D cells infected with pPCCMm1 and noninfected T-47D control cells were
treated with increasing amounts of ifosfamide (ranging from 0 to 3 mM) for five
consecutive days after which cell viabilities were determined using an XTT
assay. While noninfected T-47D cells remained largely unaffected up to an
ifosfamide concentration of 0.3 mM, T-47D/pPCCMm1 cells expressing the CYP2B1
gene showed a marked decrease of 40% already at a concentration of 0.1 mM. This
viability decreased further at higher concentrations (see Figure6(a)). 0.3 mM
ifosfamide was determined as the most effective concentration at which noninfected
T-47 cells were still largely unaffected but CYP2B1-producing T-47D/pPCCMm1
cells showed a dramatic decrease in viability by 88%. IC50 values
were determined as 1.6 mM and 0.1 mM for T-47D and T-47D/pPCCMm1, respectively.
This represents a decrease of the IC50 value by sixteen folds,
underlining the potent cumulative cytotoxic effect of CYP2B1 and ifosfamide.
Treatment of the cells with cyclophosphamide instead of ifosfamide led to
similar results (see Figure 6(b)).Enhanced
cytotoxicities of ifosfamide and cyclophosphamide in T-47D/pPCCMm1 cells
expressing CYP2B1. Cells were cultivated at increasing amounts of ifosfamide
for five days as described in materials and methods. Cell viabilities were
determined using an XTT assay and survival rates of CYP2B1 expressing cells in
relation to noninfected control cells were calculated. (a) Incubation of cells
with ifosfamide. (b) Incubation of cells with cyclophosphamide.
(a)(b)


## 4. Discussion

The ProCon vectors we have generated in this
study are a further development of an earlier, aβ-galactosidase reporter
gene-containing vector that was used for the generation of virus-producing
cells for encapsulation experiments [19]. The new vectors contain modifications
that have recently been shown by our laboratory to improve their overall
performance (i.e., a CMV enhancer to increase viral RNA production in virus-producing cells, a strong
polyadenylation signal in the modified 3′LTR to prevent read-through of viral
RNA and to stabilize the mRNA, and an elongated attachment site to increase the
integration efficiency of the provirus [26]). When transferred
into virus packaging cells, our newly generated vectors allow the production of
enzymatically active CYP2B1 that converts ifosfamide or cyclophosphamide into
their tumoricidal metabolites. As demonstrated by Western blotting and with
resorufin assays, a high concentration of CYP2B1 in virus-producing cells is
ascertained by the strong MLV promoter that drives the expression of the
therapeutic gene. Similarly, when incorporating the eGFP reporter gene into the
vectors, high MFIs in FACS analyses can be achieved in virus-producing cells. In
contrast to what was seen with similar vectors containing the WAP promoter
[27], when incorporated into our new vectors, the woodchuck hepatitis virus
posttranscriptional regulatory element (WPRE) enhanced the expression rate of
both the eGFP and the CYP2B1 genes in virus packaging cells as well as in
infected cells (data not shown). Therefore, vectors
pPCCMm1 and pPCEMm1 harboring the WPRE were chosen for further experiments over
versions of those vectors lacking the WPRE (data not shown).pPCCMm1 and the reporter gene-harboring vector
pPCEMm1 allow the generation of high virus titers in virus-packaging cells and
productive infection of target cells in
vitro. In cocultivation experiments employing
virus packaging cells and target cells, the infection rates of T-47D and CRFK
target cells reached 74% and 40%, respectively. The lower infection rate of the CRFK cells was
due to the higher growth rate of the CRFK cells compared to that of the
2GP19Talf/pPCEMm1 virus-packaging cells, quickly leading to the displacement of
the latter ones. As a consequence, virus particle numbers in the cell
supernatants decreased over time. The infection rates
were also reflected by similar results obtained with confocal laser-scanning
microscopy, demonstrating true infection of target cells. The small numbers of double
fluorescent cells (<1%) obtained after six weeks of cocultivation of red
fluorescent target cells and green fluorescent nonvirus-producing cells
represented false-positive cells since no such double fluorescent cells were
observed in laser-scanning microscopy. Most likely, the
small numbers of false-positive red/green cells observed in FACS experiments
were rather due to small numbers of red and green cells sticking together,
thus, giving the impression of double-fluorescent cells. This is also
underlined by the fact that small numbers of red/green fluorescent cells
were even detected when red fluorescent cells were mixed with green fluorescent
cells immediately prior to FACS (data not shown).Since in vivo infection may be less efficient compared to invitro
infection, we tested our vectors in a mixed tumor model in nude mice in vivo.
Analysis of explanted tumors revealed that 22% to 55% of T-47D target cells and
13% to 33% of CRFK target cells had been infected with vector pPCEMm1. The
somewhat lower infection rate for CRFK cells was probably due to their
excellent growth rate in vivo compared to the poor growth rate of the
293-based virus-producing cells and a subsequent shift in the ratio between
virus-producing cells and target cells in favor of the target cells. This
probably led to a substantial decrease of virus titer within the tumor over
time. However, this will not be an issue if vector-producing cells will be encapsulated. In
addition, an infection rate of 100% is not necessarily required during a
therapy employing encapsulated viral vector-producing cells because of the
bystander effect of cyclophosphamide or ifosfamide on neighboring noninfected cells
in GDEPT [6, 33].Incorporation of the MMTV promoter into the retroviral vectors allowed high transgene
expression of the eGFP reporter gene or the therapeutic CYP2B1 gene in infected
cells. This was not only seen for T-47D and CRFK cells but also for a number of
other human or nonhuman cell lines (mainly of mammary origin; data not shown).
In most of the cell lines, transgene expression could be further boosted by
stimulation with the glucocorticoid dexamethasone. Transgene expression was
also increased in most of the cell lines when cells were treated with
progesterone, although the effect was less pronounced (data not shown). Dexamethasone is frequently used in
cancer chemotherapy, either as a tumoricidal therapeutic [34, 35] or to prevent
or diminish nausea and other side effects in patients (including breast cancer
patients) undergoing chemotherapy [36, 37]. Thus, in a
GDEPT setting, dexamethasone would decrease the symptoms of the side effects of
the chemotherapy and at the same time increase the expression of the
therapeutic gene from the MMTV promoter.In this study, we also showed that the level of
enzymatically active CYP2B1 generated in infected cells with the newly
developed vectors is pronounced enough to create a highly increased cell
killing effect on T-47D/pPCCMm1 cells upon ifosfamide or cyclophosphamide treatment
in vitro. The enhanced cytotoxic effect due to
infection with pPCCMm1 became manifest in a sixteen fold-lowered IC50 value for ifosfamide in T-47D/pPCCMm1 compared to noninfected T-47D cells. This
value was similar for cyclophosphamide. Effective concentrations of 0.1–0.5 mM
ifosfamide have been measured in the plasma of patients after administration of
typical dosages of ifosfamide during conventional chemotherapy [38]. These pharmacologically
active concentrations are identical to those we determined, which resulted in
efficient killing of CYP2B1-expressing T-47D cells while leaving T-47D cells
not expressing CYP2B1 largely unaffected.Taken together, the newly generated vectors
constitute the basis for the development of a novel breast cancer GDEPT system:
the system is anticipated to employ the encapsulation of cells that (i)
generate the therapeutic enzyme CYP2B1 and, (ii) in addition, produce viral
vector particles that transfer the therapeutic CYP2B1 gene directly into tumor
cells.



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*Source: 683505-2008-04-08.xml*