Note: Descriptions are shown in the official language in which they were submitted.
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PSEUDOTYPED RETROVIRAL VECTOR FOR
GENE THERAPY OF CANCER
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to retroviral expression vectors and more
particularly to pseudotyped retroviral vectors for gene therapy of cancer.
(b) Description of Prior Art
Tumor cells modified to express the Herpes Simplex Virus Thymidine
Kinase gene (TK) acquire the ability to convert the non-toxic nucleobase
analog
gancyclovir (GCV) to its cytotoxic metabolite gancyclovir-phosphate. Cells
genetically engineered to express this "suicide" gene are eliminated if
exposed to
gancyclovir. Experimental brain tumor implants consisting of a mixture of
unmodified tumor cells with TK-expressing cells also regress following
gancyclovir treatment without harm to adjacent normal tissue. This phenomena,
where a minority of TK-expressing cells lead to the death and elimination of
adjacent tumor cells not expressing TK, has been termed the "bystander
effect".
The "bystander" effect is dependent, in part, on cell-cell contact and on
intercellular communications - gap junctions - through which gancyclovir-
phosphate can circulate between TK-positive and TK-negative tumor cells.
Phagocytosis of gancyclovir-phosphate laden cell debris by adjacent tumor
cells
also leads to cell death. Blood vessel endothelial cells within or adjacent to
the
tumor may also acquire TK, and their destruction with gancyclovir therapy,
thus,
may also contribute to tumor regression. "Suicide" tumors release inflammatory
cytokines which promote hemorrhagic necrosis in local, but non-contiguous,
tumor deposits. Furthermore, tumors undergoing a necrotic death, as opposed to
apoptosis, will up-regulate the expression of proteins such as hsp70, IL10 and
IL12, which may enhance immune recognition and rejection. Necrotic tumors
may be infiltrated with a wide assortment of immunocompetent cells such as
CD4+ lymphocytes, CD8+ lymphocytes, NK cells and Antigen Presenting Cells.
These infiltrating cells may take part in a tumor-specific immune response
which
is an important component of the local as well as distant anti-tumor immune
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bystander effect (Moolten, F.L., Cancer Research, 46: 5276-5281, 1986).
Intracerebral tumors are also susceptible to immune clearance following
suicide
gene expression, suggesting that the brain is not an immune sanctuary for
cancer.
Therefore, tumor-targeted suicide gene delivery leads to eradication of a
defined
tumor deposit if a sufficient number of targeted cells express the suicide
gene.
Malignant brain tumors are an appealing target for suicide gene delivery,
since the
entire malignancy is confined to the brain and amenable to eradication by the
bystander effect. Key components for the success of this strategy are the
genetic
vector from which the suicide gene is expressed and its delivery vehicle.
Viral vectors remain the most efficient means to introduce genetic
material in tumor cells in vivo. This is usually achieved by direct intra-
tumoral or
intravenous injection of a viral particle suspension. Among viral vector
delivery
platforms, adenoviruses are among the most studied for tumor-targeted gene
delivery. Adenoviruses can be concentrated to high titers, which facilitates
delivery of large viral doses to tumors. However, because of their ability to
disseminate beyond local injection site and to transduce contiguous normal
brain,
including astrocytes, neurons and ependymal cells, suicide gene expression may
lead to significant toxicity following gancyclovir treatment.
Recombinant retroviral vectors are well characterized as vehicles for
tumor-targeted gene delivery. Retroviruses can integrate only in cells
undergoing
mitosis shortly after infection (Miller, D.G. et al., Molecular & Cellular
Biology,
10: 4239-4242, 1990). Quiescent cells - such as normal brain tissue adjacent
to a
targeted tumor deposit - will be refractory to gene transfer and spared from
subsequent toxicity (Culver, K.W. et al., Science, 256: 1550-1552, 1992). For
this
reason, retroviral vectors have been extensively used in human clinical trials
studying suicide gene delivery to malignant brain tumors. Limitations to the
use
of retroviruses are: their inability to infect cells which do not express the
retroviral
receptor and, the low particle concentration in clinical-grade viral
preparations.
Clinical-grade retroviral particle preparations usually have titers <10'
particles/ml.
Assuming that a target tumor having a 1 cm diameter contains at least 108
cells, it
would be necessary to inject intra-tumorally at least >10 ml of viral particle
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preparation to deliver an equal number of viral particles. This logistical
impediment to retroparticle delivery has been addressed by directly injecting
murine retroviral producer cells (VPCs) in to tumors in vivo, the idea being
that
locally produced viral particles could transduce cancer cells. Though this
gene
delivery approach led to cures in a rat model of brain cancer, this was
probably
achieved as a consequence of delivering as many VPCs as there were tumor cells
(Culver, K.W. et al., Science, 256: 1550-1552, 1992). In human clinical
trials,
where this strategy was duplicated by injecting amphotropic VPCs with a titer
of
1x105 cfu/ml, low - albeit detectable - TK gene transfer efficiency was noted
in
tumor cells. Furthermore, a specific immune response against VPCs was elicited
(Ram, Z. et al., Nature Medicine, 3: 1354-1361, 1997). Although "suicide"
retrovectors are "safe", implantation of VPCs as a means to deliver
retroparticles
is of limited efficacy. Poor suicide gene transfer to tumor cells is a major
impediment to therapeutic utility.
Retroparticles which incorporate the Vesicular Stomatitis Virus G
(VSVG) protein differ from traditional murine retroviral pseudotypes by their
high affinity for a wide assortment of eukaryotic cells. This is primarily due
to
the ability of VSVG to recognize membrane phospholipid as a minimal receptor.
Unlike standard murine retroviruses, VSVG retrovectors are also relatively
resistant to deactivation by human complement (Ory, D.S. et al., Proceedings
of
the National Academy of Sciences of the United States of America, 93: 11400
11406, 1996). Furthermore, like adenoviruses, VSVG-typed retroviruses can be
concentrated to high titers by centrifugation and frozen/thawed without loss
of
activity. The VSVG pseudotype does not alter the retroviral genome's
restricted
targeting of cycling cells.
It would be highly desirable to be provided with a suitable delivery
vehicle for suicide gene transfer, combining high titer, particle stability
and
tumor-specificity.
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SUMMARY OF THE INVENTION
One aim of the present invention is to provide a suitable retroviral
vector for gene therapy of a cancer.
For example, VSVG-typed retroparticles may be suitable for
delivering a therapeutic gene to a tumor tissue.
For example, the cancer may be a brain cancer.
Examples of therapeutic genes include suicide genes.
For example, an HSVTK-expressing retrovector and VSVG
pseudotyped retroparticles were constructed. Human glioma cell lines can be
transduced in vitro and express functionally significant amounts of HSV TK.
Concentrated retroparticles were administered intra-tumorally in a rat model
of
brain cancer and a significant survival benefit was noted following
gancyclovir
therapy.
The retrovector may incorporate the AP2 expression vector. The AP2
expression vector allows for a high level expression of a transgene and
incorporates a reporter gene for monitoring of the transgene expression in
vitro
and in vivo. Furthermore, the reporter protein allows for a sorting of
producer
cells and facilitates the measurement of the retroviral titer.
In accordance with the present invention, retrovectors which may be
used include, without limitation, AP2 expression vector, AP2 derivatives
thereof
such as its first derivative AP3 which includes the HSVRK suicide gene. Other
derivatives include MD 1 which is a AP2 derivative which incorporates human
GMCSF, JGH2 which expresses a novel GFP-HSVTK fusion protein, JGH2
derivatives thereof which incorporate immunomodulatory genes as well as the
GFP/TK fusion protein. AP2 derivatives incorporate genes of therapeutic
interest
for the treatment of cancer.
Preferred AP2 expression vector derivatives include, without
limitation, the following:
HSV thymidine kinase (AP3);
GMCSF (MD1);
RARB2;
IRF3;
IRF3-5d;
MCPl;
3 5 Rantes;
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MIP 1 alpha;
MIP 1 beta;
MCP 1.
Preferred JGH2 expression vector derivatives include, without
limitation, the following:
GMCSF (MD2);
IRF3 (AP6);
IRF3/SD (AP7).
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. lA-1C illustrate schematic representations of plasmids and
retrovectors. Fig. lA: AP2 plasmid retrovector serves as a template for the co-
expression of the EGFP reporter and of a linked cDNA in eukaryotic cells. The
cDNA of interest is inserted in the multiple cloning site upstream of the
IRES.
Fig. 1B: pTKiGFP is a derivative of AP2 which contains the HSVTK gene.
Transfection of this plasmid into retroviral packaging cells will lead to the
production of replication-defective retroparticles. Fig. 1 C: Target cells
transduced with vTKiGFP will integrate the retrovector in their genomic DNA.
The DNA structure (flanked by LTRs) and coding sequences are depicted.
Fig. 2 illustrates flow cytometric analysis of vTKiGFP transduced
glioma cells. UWR7 human glioma cells were transduced with vTKiGFP and
subsequently analyzed by flow cytometry for green fluorescence, as described
in
"Materials and Methods". GFP serves as a reporter of retrovector expression in
transduced cells.
Fig. 3 illustrates Southern Blot analysis on vTKiGFP transduced
glioma cells. Following transduction with vTKiGFP, the retrovector will
integrate into genomic DNA. Digest of genomic DNA with NheI, which cuts
once in each flanking LTR, and subsequent probing of Southern blot with a
vector
complementary sequence will allow detection of integrated proviral sequences
with a predicted size of 4kb (schematic at right). Left, Southern blot
analysis of
transduced (+) and untransduced (-) UWR7 cells with a GFP cDNA-specific
probe, as described in "Materials and Method". Molecular weights are
indicated.
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Fig. 4 illustrates growth suppression of human glioma cells with
gancyclovir. The indicated human glioma cell lines were transduced with
vTKiGFP (open squares) or the control retrovector vDHFRiGFP (open circle).
These and untransduced controls (open diamonds) were subsequently exposed to
gancyclovir for 6 days, and cell survival was measured by the MTT assay as
described in "Materials and Methods". Percent survival is plotted against
gancyclovir concentration (log scale). Data points, mean survival measured in
three separate experiments; bars, SD. SD smaller than data point icon are not
displayed.
Fig. 5 illustrates flow cytometric analysis of 293AP3 producer cells.
293GPG packaging cells were stably transfected with pTKiGFP and a Zeocin
resistance plasmid. A mixed population of Zeocin resistant 293AP3 cells was
generated and characterized for GFP expression by flow cytometry as described
in
"Materials and Methods". Percent GFP+ cells is indicated. These cells were
subsequently utilized to generate vTKiGFP stock for concentration and in vivo
delivery.
Fig. 6 illustrates transduction of glioma cells with concentrated
vTKiGFP retrovector stocks. vTKiGFP retroparticles were collected and
concentrated to 84 and 1000 fold (volume/volume) as described in "Materials
and
Methods". 1X and 84X virus stock were diluted (as indicated on left) in a
final
volume of 1 ml and applied to 2.3x105 UWR7 cells in a 24 well dish. Three days
following a single application of vector, cells were analyzed for GFP
expression
by flow cytometry. Percent GFP+ is indicated in histogram figures. Dilutions
of
1000X stock was applied to 5.4x105 C6 glioma cells and analyzed three days
later
for GFP expression. Titer extrapolated from these experiments were: 1X:
2.9x10'
cfu/ml, 84X: 2.2x109 cfu/ml, 1000X: 2.3x10'° cfu/ml.
Fig. 7 illustrates in vivo transduction of C6/lacZ tumors with
vTKiGFP. Brain tumors were harvested post-mortem as described in Materials
and Methods. TOP (panel A, B), tumor from a control rat which received
vTKiGFP without subsequent treatment with GCV (rat was sacrificed on day 30
post tumor implantation due to morbid state). MIDDLE (Panel C, D), tumor from
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a control rat which did not receive vTKIGFP but was treated with GCV (rat was
sacrificed on day 43). BOTTOM (Panel E, F), tumor from a test rat which
received vTKiGFP and subsequent treatment with GCV which suffered
symptomatic recurrent tumor (rat was sacrificed on day 82). GFP expression
(panels A, C, E) was compared to subsequent histochemical staining of C6/lacZ
tumor cells with the substrate X-gal (panels B, D, F). Magnification of 100X
for
all photomicrographs.
Fig. 8 illustrates Kaplan-Meier survival curve of rats with
experimental glioma. Sprague-Dawley rats received 2x104 C6/lacZ glioma cells
by stereotactic injection in the right brain hemisphere as described in
"Materials
and Methods". Six days later, eighteen animals were administered 9pL of 1000x
vTKiGFP stock in the same stereotactic coordinates as the previous C6/lacZ
implant. 48 hours later, test animals (n=12) received GCV SOmg/kg twice daily
for 5 days followed by SOmg/kg once daily for 5 more days. The other animals
(n=6) were administered saline only. In a separate experiment, a supplementary
cohort (n=5) received a C6/IacZ glioma implant followed 9 days later by GCV
treatment (no retrovector administered). The survival seen in the test group
(vTKiGFP + GCV) is significantly grater than that in either control groups
(p<0,001 by Log rank). There is no significant difference in survival between
the
two control groups.
Fig. 9 illustrates retrovectors. Panel A. nucleotide sequence of DNA
linker region spanning the 3'-end of GFP and start codon of HSVTK.
Nucleotides derived from GFP cDNA are in bold and underligned with their
translation product also in bold. The sequence point of fusion between the
3'end
of GFP and the 5' untranslated region of HSVTK cDNA sequence is depicted.
Predicted 24 aminoacid linker is depicted. HSV TK start codon and and coding
sequence are identified in bold text and HSVTK caption. Panel B. Schematic
representation of plasmid constructs. Left, pGFP retrovector encodes for GFP
only; Center, pTKiGFP is a bicistronic expression vector incorporating HSVTK
and GFP. Right, pGFPTKfus incorporates the coding sequences for a GFP and
HSVTK fusion protein. All three plasmid constructs were utilized to generate
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stable retroviral producers with the 293GPG packaging cell line as described
in
Materials and Methods.
Fig. 10 illustrates Southern blot analysis of vGFPTKfus transduced
cells. After transduction with vGFPTKfus, the retrovector will integrate into
genomic DNA. Below, Digest of genomic DNA with NheI, which cuts once in
each LTR, and subsequent probing of Southern blot with a vector complementary
sequence will allow detection of integrated proviral sequences with a
predicted
size of 3.7 kb. Top, Southern blot analysis of tansduced (+) and untransduced
(-)
human A549 cells with a GFP cDNA-specific probe. Arrow indicates band of
predicted size. Molecular weights are indicated on left.
Fig. 11 illustrates Flow cytometry of retrovirally-transduced DA3
cells. DA3 mouse mammary carcinoma cells were transduced with either
vTKiGFP, vGFPTKfus or vGFP in a manner which leads to 100% gene transfer
efficiency. Stably transduced polyclonal cell populations were subsequently
analyzed by flow cytometry for green fluorescence as described in Materials
and
Methods. GFP serves as a reporter of retrovector expression in transduced
cells
and the Mean Fluorescence Intensity (MFI) of the analyzed populations is
indicated in the top right of each panel.
Fig. 11 illustrates fluorescent microscopy of vGFPTKfus engineered
cells.
Fig. 12 illustrates Western blot analysis. The same transduced DA3
cells analyzed by flow cytometry (Fig. 3) were utilized for Western blot
analysis
of HSVTK protein expression. Equal amounts of total protein obtained from
whole cell lysates were seperated by gel electophoresis and immunoblotted with
an anti-HSVTK polyclonal antisera as detailed in Materials and Methods.
Molecular weight markers (kd) are depicted on the left.
Fig. 13 illustrates gancyclovir growth suppression assay. The
vTKiGFP (filled square), vGFPTKfus (filled triangle) and vGFP (filled circle)
transduced DA3 mouse mammary carcinoma cells were exposed to the prodrug
gancyclovir for 6 days and cell survival was measured with the MTT assay as
described in Materials and Methods. Percent survival is plotted against GCV
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concentration (log scale). DA3 transduced with a GFP only vector serve as
negative controls. Data points, average +/- SD of three experiments is
depicted,
error bars smaller than icons are not shown.
DETAILED DESCRIPTION OF THE INVENTION
Direct in vivo tumor-targeting with "suicide" viral vectors is limited by
either inefficient gene transfer [i.e. retroviral vectors] or indiscriminate
transfer of
a conditionally toxic gene to surrounding non-malignant tissue [i.e.
adenoviral
vectors]. Retrovectors pseudotyped with the Vesicular Stomatitis Virus G
protein
(VSVG) may serve as a remedy to this conundrum. These retroviral particles dif
fer from standard murine retroviruses by their very broad tropism and the
capacity
to be concentrated by ultracentrifugation without loss of activity. A VSVG-
typed
retrovector can be utilized for efficient and tumor specific Herpes Simplex
Virus
Thymidine Kinase (TK) gene delivery in vivo. A bicistronic retroviral vector
which expresses TK and Green Fluorescence Protein (pTKiGFP) was constructed.
The 293GPG packaging cell line was utilized to generate vTKiGFP
retroparticles.
In cytotoxicity assays, vTKiGFP-transduced human glioma cell lines were
sensitized to the cytotoxic effects of gancyclovir (GCV) 10,000 fold.
Subsequently, the virus was concentrated by ultracentrifugation to a titer of
2.3x10'° cfu/ml. The anti-tumor activity of vTKiGFP retroparticles was
tested in a
rat C6 glioma model of brain cancer. Concentrated retrovector stock (9~L
volume) was injected stereotactically in pre-established intra-cerebral tumor.
Subsequently, rats were treated with GCV for 10 days. Control rats (no GCV)
had a mean survival of 38 days (range 20-52 days). Sections performed on post-
mortem brain tissue revealed large tumors with evidence of high efficiency
retrovector transfer and expression (as assessed by GFP fluorescence).
Fluorescence was restricted to malignant tissue. In the experimental group
(GCV
treated), 8/12 remain alive and well >120 days post glioma implantation. The
vTKiGFP is very efficient at transducing human glioma cell lines in vitro and
leads to significant GCV sensitization. Recombinant retroviral particles can
be
concentrated to titers which allow in vivo intra-tumoral delivery of large
viral
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doses. The therapeutic efficiency of this reagent has been demonstrated in a
pre-
clinical model of brain cancer.
MATERIALS AND METHODS
Cell lines and plasmids
pCMMP-LZ plasmid (Jeng-Shin Lee and Richard C. Mulligan, unpub-
fished), pJ6S2bleo plasmid and 293GPG retroviral packaging cell line were
generous gifts from Richard. C. Mulligan (Children's Hospital, Boston, MA).
MSCV-Neo plasmid (Hawley, R.G. et al., Gene Therapy, l: 136-138, 1994) and
BSICZSVPA plasmid (Ghattas, LR. et al., Molecular & Cellular Biology, 11:
5848-5859, 1991) were kindly provided by Robert G. Hawley (The Toronto
Hospital, Toronto, ON). SKI-1, SKMG-4, SKMG-1, T98G, UW28 & UWR7
human glioma cell lines were generously provided by Lawrence Panasci (Lady
Davis Institute for Medical Research, Montreal, QC). C6 & C6/lacZ glioma cells
originate from ATCC. pMCITK plasmid was graciously provided by Gerald
Batist (Lady Davis Institute for Medical Research, Montreal, QC). HaL22Y
plasmid was kindly provided by Raymond L. Blakley (St. Jude Children's
Research Hospital, Memphis, TN).
Retrovector design and synthesis
A plasmid encoding for a bicistronic, non-splicing murine retrovector
which incorporates a multiple cloning site - allowing insertion of cDNA of
interest - linked to the Enhanced Green Fluorescence Reporter (AP2) was
engineered. The synthesis of AP2 is as follows. The 805 by EGFP cDNA was
excised by Eco47-3 and NotI digest of pEGFP-N1 (Clontech, Palo Alto, CA) and
ligated into the MSCV (Hawley, R.G. et al., Gene Therapy, 1: 136-138, 1994)
retroviral plasmid to generate MSCV-EGFP. The 555 by Internal Ribosomal
Entry Site (IRES) was excised from the BSICZSVPA plasmid (Ghattas, LR. et al.,
Molecular & Cellular Biology, 11: 5848-5859, 1991) by SacII-NcoI digest and
cloned in to SacII-NcoI cut MSCV-EGFP to generate MSCV-IRES/EGFP.
MSCV-IRES/EGFP was digested with SpeI-AscI to generate a 2524 by fragment
encompassing part of the 5' untranslated region of the retrovector, the IRES,
EGFP and most of the 3' LTR. This insert was ligated with a 4169 by fragment
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from SpeI-AscI cut pCMMP-LZ - an unpublished MFG-based retrovector - to
generate AP2 (Fig. lA). AP2 is designed to co-express an inserted cDNA with
the EGFP reporter within a bicistronic framework. The EGFP serves as a
reporter
of provirus transfer and expression in target cells. The viral vector
generated is
non-splicing. The pMCITK plasmid was cut with BgIII-BsaWl to generate a
1207 by fragment containing the HSVTK cDNA (excluding polyadenylation
signal) and was ligated into BgIII-XmaI-cut AP2 to generate pTKiGFP (Fig. 1B).
The retroviral genome produced from pTKiGFP will not incorporate the CMV
promoter element. Transduction of target cells with pTKiGFP-derived retroviral
particles (vTKiGFP) will lead to the stable incorporation of LTR flanked
proviral
genome (Fig. 1C). The pMSCV-DHFR (L22Y)/IRES/EGFP vector (pMSCV-
DIG) was derived by incorporating the 654 by B amH 1-Xho 1 DHFR (L22Y)
cDNA from Ha-L22Y into BgIII-SaII cut MSCV-IRES/EGFP.
Production of VSVG-pseudotyped retroviral particles and concentration
Recombinant VSVG-pseudotyped retroparticles were generated either
by transient or stable transfection of the 293GPG packaging cell line (Ory,
D.S. et
al., Proceedings of the National Academy of Sciences of the United States of
America, 93: 11400-11406, 1996). 293GPG cells are maintained in 293GPG
media [DMEM (Gibco-BRL, Gaithesburg, MD), 10% heat-inactivated FBS
(Gibco-BRL) supplemented with 0,3 mg/ml 6418 (Mediatech, Herndon, VA) and
2 pg/ml puromycin (Sigma, Oakville, ONT), 1 ~g/ml tetracycline (Fisher
Scientific, Nepean, ONT) and 50 units/ml of Pen-Strep ]. For transient
production
of retroparticles, 293GPG cells were transfected with 5 pg plasmid
retrovectors
with the use of lipofectamine (Gibco-BRL). Transient transfections were done
in
tetracycline-free media and viral supernatant collected daily for 1 week, 3
days
following transfection. Stable producer cells were generated by co-
transfection of
4pg FspI linearized retrovector plasmid and 1:25 ratio of pJ6S2Bleo plasmid.
Transfected cells were subsequently selected in 293GPG media supplemented
with 100 pg/ml Zeocin (Invitrogen, San Diego, CA) as described (Ory, D.S. et
al.,
Proceedings of the National Academy of Sciences of the United States of
America,
93: 11400-11406, 1996). Resulting stable polyclonal producer populations were
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utilized to generate high titer virus. All viral supernatants were filtered
with 0.45
micron syringe mounted filters (Gelman Sciences, Ann Arbour, MI) and stored at
-20°C. Concentration of VSVG retroparticles was performed as previously
described (Ory, D.S. et al., Proceedings of the National Academy of Sciences
of
the United States of America, 93: 11400-11406, 1996). In brief, previously
harvested supernatant was thawed and 10 ml aliquots were centrifuged at 25,000
rpm in a SW41T1 rotor (Beckman Instruments Inc.) at 4°C for 90 minutes.
Viral
pellets were resuspended overnight in 100 ~L serum-free RPMI (Gibco-BRL) at
4°C, pooled and concentrated through a second centrifugation.
Concentrated
virus was aliquoted and stored at -80°C. Viral preparations were devoid
of RCR
by EGFP marker rescue assay utilizing supernatant from transduced UWR7 cells.
Transduction of glioma cells, flow cytometry and southern blot analysis
Human glioma cell lines were plated at 2x 104 cells per well in a 24
well dish and allowed to adhere. Media was removed and replaced with 500 ~L
of thawed, retrovirus conditioned media collected from transiently transfected
293GPG. Polybrene (Sigma) was added to a final concentration of 6 pg/ml. This
procedure was repeated daily for three consecutive days. Stably transduced
cells
were subsequently expanded. No clonal selection was performed, and mixed
populations of transduced cells were used for all subsequent experiments. Flow
cytometric analysis was performed within two weeks following transduction to
ascertain retrovector expression and gene transfer efficiency as measured by
GFP
fluorescence. In brief, adherent transduced cells were trypsinized and
resuspended in RPMI at 105 cells per ml. Analysis was performed on a Epics
XL/MCL Coulter analyzer. Live cells were gated based on FSC/SSC profile and
analyzed for GFP fluorescence. Southern blot analysis was performed on 15 g of
overnight NheI digested genomic DNA extracted from stably transduced cells as
well as untransduced control cells. Blots were hybridized with a P32 labeled,
full-
length 700 by GFP cDNA probe, washed and exposed on photographic film.
Growth suppression assays
Stably transduced test and control cells were trypsinized and plated at
a density of 1000 cells per well in a flat bottomed tissue-culture treated 96
well
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plate (Costar corporation, Cambridge, MA). Clinical-grade gancyclovir (GCV,
Hoffinan-Laroche, Mississauga, ONT) was added to achieve a range of concentra-
tions from 0.01 to 5000 ~g/ml in a final volume of 100 ~L of ItPMI/10% FBS.
Cells were incubated at 37°C and media was replaced with fresh GCV
after three
days for a total exposure of 6 days. The percentage of surviving cells was
meas-
ured using a method based on the metabolism, by living cells, of the
mitochondria) substrate 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium
bromide (MTT) into formazan, which is detected by measurement of the optical
density at 570nm. Percent Survival is calculated as follows [OD570 test -
OD570
empty well]/[OD570 untreated cells - OD570 empty well] x 100. All data points
were measured in triplicate in at least three separate experiments.
Titration of retrovector
Target glioma cells were plated at 2x105 cells per well in a 6 well tis-
sue culture dish. The next day, cells from a test well were trypsinized and
enu-
merated to determine baseline cell count at moment of virus exposure. Virus
was
serially diluted (range 100 to 0.001 ~L) in a final volume of 1 ml of RPMI/10%
FBS supplemented with 6 ~g/ml polybrene (Sigma) and applied to adherent cells.
Flow cytometric analysis was performed 3 days later to determine the
percentage
of GFP+ cells. Viral titer (cfu/ml) was extrapolated from the test point in
which
non-saturating transduction conditions prevailed (i.e. transduction efficiency
<80%). Titer (cfu/ml) was calculated as [ (% GFP+ cells) X (cell number at
initial
viral exposure) / (viral volume in ml applied)].
Animal model of brain cancer, In vivo retrovector delivery and gancyclovir
treatment
C6/lacZ glioma cells reproducibly generate lethal intra-cerebral tumors
when injected in Sprague-Dawley rats. The constitutive [3-galactosidase
expression facilitates delineation (by X-gal staining) of tumor cells and
extent of
the tumor infiltrate in post-mortem brain sections. Adult Sprague Dawley rats
were anesthetized with intraperitoneal injection of ketamine (50 mglkg) and
xylazine (2 mg/kg). C6/lacZ rat glioma cells (2x104 cells in 5 ~1 of HBSS)
were
injected intracranially into the frontal lobe using a Hamilton syringe in a
stereo-
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tactic apparatus (Kopf) over a period of 15 minutes. The coordinates used were
3.5 mm lateral to the bregma, 1.0 mm posterior to the coronal plane and 4.5 mm
in depth of the ducal surface. Six days post glioma cell implantation, rats
were
anesthetized and vTKiGFP (concentrated stock of 2.3x10'° cfu/ml) was
injected of
into six different sites (1 mm apart) in the pre-established tumor guided by
the
previous stereotactic coordinates. A total volume of 9 1 was injected in each
tumor (6 x 1,5 ~1 increment) and needle was left in place for at least 5 mins
per
increment (for a total of 30 mins per tumor). Two days after retrovector
delivery,
rats are treated with GCV 50 mg/kg intraperitoneally twice daily for 5 days
fol-
lowed by 50 mg/kg once daily for another 5 days. After euthanasia, brains were
removed and quickly frozen in isopentane chilled with liquid nitrogen. Coronal
sections (10 Vim) were prepared. GFP activity was observed by epi-fluorescence
microscopy and recorded photographically. Subsequently, sections were stained
histochemically for (3-galactosidase activity as previously described before
counter staining with hematoxylin and eosin.
RESULTS
Retrovector design and synthesis
The AP2 expression vector (Fig. 1 A) allows the incorporation of a
cDNA sequence in a Multiple cloning site (MCS) upstream of an Internal Ribo
somal Entry Site (IRES) and the Enhanced Green Fluorescent Protein (EGFP)
cDNA. The transcription initiation from a CMV promoter will lead to the pro-
duction of a bicistronic mRNA incorporating both the inserted cDNA and the
EGFP coding sequence. Translation of both coding sequences will be achieved
from a single mRNA molecule, thereby ensuring co-dominant expression of both
protein products. Live cells expressing EGFP, which is detectable by
fluorescence microscopy or flow cytometry, will co-express the linked gene
product. Gene-modified cells can be implanted or transplanted in animal models
and their localization and function be traced based on the expression of the
EGFP
protein. The AP2 expression vector incorporates a replication-defective
retroviral
packaging sequence and a retroviral 3'long terminal repeat (LTR). Transfection
of an appropriate retroviral packaging cell line can lead to production of
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recombinant retroviral particles. Retroparticles can be generated either by
transient transfection of packaging cell lines or alternatively, stable
producer cell
lines can be generated by co-transfection with a drug resistance plasmid. We
have
generated retroparticles by both methods with good success utilizing the
293GPG
retroviral packaging cell line.
Retrovector transfer and expression in human glioma cell lines
The 293GPG packaging cell line was transiently transfected with
pTKiGFP (Fig. 1B) and supernatant containing VSVG-typed retroparticles
(vTKiGFP) was subsequently collected, filtered and frozen for storage. Human
glioma cell lines (SKI-1, SKMG-4, SKMG-1, T98G, UW28 & UWR7) were
transduced with three consecutive daily applications of thawed vTKiGFP
supernatant. Six days post-transduction, polyclonal cell lines were subjected
to
flow cytometric analysis to determine the proportion of cells which expressed
the
GFP reporter protein. All polyclonal cell lines were 100% GFP-positive by
FACS analysis, and transduced UWR7 cells serve as a representative example
(Fig. 2). We have also found that GFP expression could be easily detected in
live
cultured cells by direct visualization with a tissue culture microscope fitted
with
an epifluorescence light source. Southern blot analysis confirmed that
unrearranged vTKiGFP vector integrated in chromosomal DNA of transduced tar-
get cells (Fig. 3). vTKiGFP transduced cells have been passaged in excess of
30
times without loss of GFP expression.
vTKiGFP Expression and Gancyclovir sensitization
HSV TK expression will lead to the conversion of the prodrug gancy
clovir to its cytotoxic metabolite gancyclovir monophosphate. Cells which do
not
express this enzyme are refractory to gancyclovir toxicity. We compared the
gan
cyclovir sensitivity of vTKiGFP transduced cells with unmodified parental
cells
as well as cells modified with a control, GFP-containing retrovector (vMSCV-
DIG). Cells were plated in 96 well dish and exposed to gancyclovir for a
period
of 6 days. Live cell content was assessed colorimetrically by MTT assay and
cell
survival was expressed as a percentage relative to untreated cells. We have
found
that all vTKiGFP-expressing cell lines were sensitized to gancyclovir.
Comparing
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the GCV concentration which inhibits cell growth by 50% (IC50), we found that
vTKiGFP transduced cells (all 6 cell lines aggregated) were up to 10,000 fold
more sensitive to GCV than controls (IC50 tests: 0.004 ug/ml vs IC50 controls:
40
ug/ml, p<0.001 by student t test) (Fig. 4). Growth rate for transduced and
parental
cell lines in the absence of gancyclovir were identical.
Concentration of vTKiGFP retroparticles
The most direct means of transducing a tumor in vivo is to inject the
therapeutic retrovector intra-tumorally. If the aim is to transduce as many
tumor
cells as possible, it would be desirable to inject a concentrated vector stock
to
achieve a high local MOI. We determined if viable vTKiGFP retroparticles could
be concentrated by ultracentrifugation as previously described. As a first
step we
transfected 293GPG cells with pTKiGFP and a zeocin resistance plasmid
(pJ6bleo). A stably transfected, Zeocin-resistant polyclonal producer cell
popula-
tion (293AP3) was generated. Flow cytometric analysis for GFP fluorescence
revealed that 42% of this mixed population stably expressed the pTKiGFP vector
DNA (Fig. 5). Tetracycline withdrawal from the culture media will lead to the
production of VSVG-typed vTKiGFP retroparticles. We collected retroparticle-
containing media daily from the 293AP3 producer cells from days 3 to 8 follow-
ing tetracycline withdrawal. Supernatant was cleared of cellular debris with a
0.45u filter and frozen. We have noted that twice daily media collection - as
opposed to once daily - doubled the yield of retroparticles from producer
cells
following tetracycline withdrawal. Media were thawed, pooled and subjected to
ultracentrifugation as described in Materials and Methods. Supernatant was
concentrated 84 fold (20 mls to 0.24 ml) by ultracentrifugation. The
concentration step raised titer from 2.9x107 cfu/ml to 220x107 cfu/ml as
measured on UWR7 human glioma cells (Fig. 6). 84X concentrates were pooled
and subjected to a second ultracentrifugation to achieve a final 1000X (100 ml
initial volume to 0.1 ml final volume) concentration. Titer of 1000X
retrovector
was 2.3x1010 as determined on rat C6 glioma cells (Fig. 6). Concentrated
retrovector aliquots were stored at -80°C until further use. We have
observed that
unmanipulated (unconcentrated) supernatant from tetracycline-deprived 293GPG
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producer cells can be toxic to target cells if applied repeatedly. However, no
toxicity was observed on target cells if concentrated supernatant was used for
transduction purposes, even at the highest tested MOI (>100).
Retrovector expression following infra-tumoral injection of concentrated
vTKiGFP retroparticles
Implantation of C6/lacZ glioma cells will reliably lead to the estab-
lishment of infra-cerebral tumors in immunocompetent. Sprague-Dawley rats.
This cell line will generate large local tumors which are uniformly lethal
within
60 days following the initial stereotactic injection of 2x104 cells.
Furthermore,
C6/lacZ cells constitutively express (3-galactosidase which permits the
assessment
of tumor extent and local invasion in X-gal stained post-mortem brain
sections.
18 rats received 2x104 C6/lacZ cells via stereotactic injection in the right
brain
hemisphere. Six days later, 9 ~L of 1000X vTKiGFP retrovector (2x10'°
cfu/ml)
was inj ected at the tumor site using the same stereotactic coordinates. Of
these 18
rats, 6 were randomly chosen and treated with saline. Saline-treated control
rats
had an average survival of 38 days (range 20 to 52 days). Post-mortem
examination of brain revealed macroscopic infra-cerebral tumors, except for 1
rat
which died with leptomeningeal tumor spread 8 days after tumor injection
(which
was excluded from further analysis). Examination of fresh frozen brain
sections
by epifluorescence microscopy shows that in all animals, a predominant
proportion of glioma cells fluoresce green (Fig 7A), including distant
micrometastasis. Normal surrounding brain tissue is bereft of green
fluorescence.
No green fluorescence was observed in untransduced brain tumors (Fig 7C).
Gancyclovir treatment of rats with vTKiGFP-targeted gliomas
Of 18 rats having received infra-tumoral vTKiGFP retrovector, 12
were subsequently treated with gancyclovir. Two days following retrovector
injection, rats received gancyclovir 50 mg/kg infra-peritoneally twice daily
for 5
days followed by 50 mg/kg once daily for another 5 days. Significant
gancyclovir
toxicity including transient limb paresis and otorhagia, was noted in some
rats in
the week following GCV treatment. Of 12 gancyclovir treated rats, two died
within 10 days following drug treatment presumably from direct GCV toxicity
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(both animals had brain tumors <lmm in diameter on post-mortem). The other 10
rats fully recovered from GCV toxicity. Two rats developed tumor relapses at
the
initial injection site and died of progressive disease at day 82. Examination
of
brain tissue sections on these late relapses, revealed focal GFP expression in
the
S tumors (Fig 7E). Significantly enhanced survival was obtained as eight of 12
GCV-treated test rats (66%) remain long term survivors (>120 days). A
supplementary control cohort of 6 rats implanted with C6/lacZ, but without
subsequent retrovector administration, was treated with the same GCV regimen.
These controls had an average life span of 47 days (range 31 to 63 days) (Fig.
8).
With our experimental C6 glioma model , we have not observed a significant
difference in average survival between the two control groups (saline controls
vs
GCV-treated null tumors, p=0.37 (Student t test)) suggesting that GCV
treatment,
on its own, does not have a measurable impact on survival, as has been
suggested
by others using 9L glioma implants. These differences may be due different
biological properties of these two experimental glioma models.
DISCUSSION
Engineering tumor cells to express the Herpes Simplex Virus Thymi-
dine Kinase cDNA will lead to their destruction if they are subsequently
exposed
to non-toxic nucleobase analogs such as gancyclovir. This "suicide" effect is
accompanied by "bystander" toxicity on adjacent tumor cells not expressing TK,
so that a minority of engineered tumor cells - perhaps no more than 10 to 25% -
will lead to 100% tumor eradication. Clinical application of this therapeutic
strategy requires relatively high efficiency TK gene transfer to pre-
established
tumors. Furthermore, "collateral" gene transfer to normal adjacent normal
tissue
would have to be curtailed to prevent GCV toxicity to normal brain tissue.
The affinity of recombinant retroparticles for target tissue is defined by
the env protein. Murine amphotropic retroviruses, from which are derived many
of the therapeutic retrovectors in glioma targeted gene delivery, will only
bind
target cells which express a specific inorganic phosphate transporter. If a
target
tumor does not express the retrovirus receptor, gene transfer - and
therapeutic
benefit - is unlikely to occur. Retroparticles which are pseudotyped with the
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VSVG protein will adopt the wide host range of the vesicular stomatitis virus.
The putative VSVG receptor on target cells - which is believed to be membrane
phospholipid - is ubiquitously found in all eukaryotic cells. This has led the
use
of VSVG-pseudotyped retrovectors as gene delivery vehicles in wide assortment
of mammalian, non-mammalian and invertebrate cells (Yee, J.K. et al.,
Proceedings of the National Academy of Sciences of the United States of
America, 91: 9564-9568, 1994). A major advance in pseudotyping retrovectors
with VSVG was achieved when a practical "transient" VSVG retroviral packaging
cell line was designed. The subsequent publication of a reliable "stable" high-
titer
VSVG packaging cell lines - including 293GPG (Ory, D.S. et al., Proceedings of
the National Academy of Sciences of the United States of America, 93: 11400-
11406, 1996) - has allowed the development and characterization of pseudotyped
retrovectors for a wide variety of gene transfer applications (Hopkins, N.,
Proceedings of the National Academy of Sciences of the United States of
America,
90: 8759-8760, 1993), including tumor cell-targeted gene delivery.
We have examined the utility of a VSVG-pseudotyped suicide
retrovector for glioma-targeted gene delivery. To facilitate analysis of
vector
transfer efficiency and expression in target cells, we engineered a retroviral
expression vector which incorporates HSVTK and the EGFP reporter cDNA
within a bicistronic transcript (pTKiGFP). We have found that co-dominant
expression of the HSVTK cDNA and of the EGFP reporter facilitates a wide
assortment of procedures associated with synthesis and characterization of
viral
vectors. Among these, are the ability to measure endpoint titer from stable
retroviral producer cells (Fig. 6) as well as potential use for selecting GFP+
producer cells with a cell sorter device. We have also found that the EGFP
reporter can serve as a sensitive marker of retrovector expression in targeted
tissue
in vitro (Fig. 2) as well as in vivo (Fig. 7).
We generated a stable retroviral vTKiGFP producer cell line (293AP3)
derived from the 293GPG packaging cell line (Fig. 5). Upon tetracycline with
drawal, this retroviral producer cell line will express the VSVG envelope
protein
and generate pseudotyped retroviral particles. We found that VSVG-pseudotyped
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retroparticles incorporating vTKiGFP will lead to high efficiency retrovector
transfer to human glioma cell lines in vitro. In contrast with standard
transfection
techniques, or with the use of more "standard" retroviral pseudotypes, we have
not required dominant selection of subpopulations of cells to achieve 100%
transgene-positive cell populations. Retroparticle conditioned media collected
from 293GPG cells transiently transfected with pTKiGFP, was used to generate
vTKiGFP transduced glioma cell lines. We noted that transducing glioma cells
with concentrated retrovector with a single application at a MOI of ~5 led to
more
than 90% gene transfer in targeted cells (Fig. 6). Gene expression was durable
as
assessed by persistent GFP expression (>30 passages) and by functional HSVTK
expression, rendering VSVG-associated pseudotransduction unlikely. Having
generated vTKiGFP transduced cell lines, we confirmed that the proviral genome
integrated unrearranged by southern analysis, demonstrating the stability of
the
viral vector as designed (Fig. 3). This of some importance especially in light
of
recent reports documenting rearranged "suicide" retroviral vectors as a cause
of
gancyclovir resistance in transduced tumors. Virtually all glioma cell lines
transduced with vTKiGFP acquired substantial and significant sensitivity to
gancyclovir in vitro (Fig. 4). Our experimental design based on the use of
polyclonal transduced cell populations for cytotoxicity assays, supports the
hypothesis that vTKiGFP gene transfer, on the average, will express
biologically
significant levels of TK in a gene-modified cell. Neither the transduction
process
(with a control retrovector), nor expression of the GFP reporter, on their
own,
sensitizes cells to gancyclovir (Fig. 4).
Important characteristics of VSVG pseudotyped retroparticles are their
ability to sustain concentration by ultracentrifugation and repeated
freeze/thaw
without loss of activity. These properties have allowed us to collect
retroparticle
conditioned media on a daily basis following tetracycline withdrawal from the
293AP3 producer cell line. Retroparticle-containing media was frozen and
stored
until further use. Large volumes of frozen supernatant can be thawed, pooled
and
subjected to at least two cycles of centrifugation with efficient retrovector
recovery. We concentrated 100 mls of media to a final volume of 0.1 ml (1000X
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concentration on volume basis). This was accompanied by a 800 fold increase in
titer from 2.9 to 2300x107 cfu/ml. We noted that supernatant from tetracycline-
deprived 293AP3 producer cells could be toxic to target cells if applied
repeat-
edly. We also observed this phenomena with other 293GPG-derived producer
cells. Interestingly, we observed that concentrated retroparticles, which had
been
resuspended in serum-free media did not have this property although they would
be delivered at a MOI higher than that achievable with the unconcentrated
supernatant. This suggests that supernatant from tetracycline-deprived 293GPG
cells contains toxic constituents) which are readily discarded upon
concentration
procedure.
To test the therapeutic usefulness of this reagent, we utilized a rodent
model of brain cancer. We established C6/lacZ glioma tumors in
immunocompetent Sprague-Dawley rats and subsequently administered
concentrated vTKiGFP retrovector intra-tumorally. Intra-tumoral delivery of
9~1
0108 retroparticles) of concentrated vTKiGFP retrovector stock did not improve
survival of animals who did not subsequently receive gancyclovir. These
control
rats (tumor+, retrovector+, but no GCV) had a mean survival of 38 days (range
20-52 days). Post-mortem examination of whole brain tissue sections, revealed
that efficient and stable tumor-specific gene transfer had occurred (Fig. 7).
Transgene expression persisted in the growing tumor as long as rats survived
after
retrovector administration. Examination of surrounding normal brain tissue
failed
to reveal GFP fluorescence (Fig. 7) suggesting that retrovector integration
and
expression occurred in tumor cells only and not in mitotically quiescent
neurons,
as would be expected from a retroviral vector.
Twelve test rats received GCV following tumor-targeted vTKiGFP
delivery. Of these, two died shortly (within two weeks) following the end of
GCV treatment. This "acute" death rate attributable to direct GCV toxicity
(~16%), is comparable to that observed by other investigators who administered
GCV at equal or lesser doses. The mechanism of death is likely related to
cytopenia and immunosuppression associated with severe, albeit reversible,
bone
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marrow toxicity. Surviving test rats fully recovered from GCV toxicity
approximately two weeks following its completion.
All of the test rats remained alive and well more than 80 days post
tumor implantation. Two rats developed symptomatic tumor recurrences and
S were sacrificed on day 82 post tumor implantation (Fig. 8). Examination of
brain
tissue sections on these late relapses, revealed large tumors with areas of
green
fluorescence inter-spaced with GFP-negative tumor cells (Fig. 7). This
suggests
that recurrence was due in part to growth of untransduced tumor cells, or of
tumor
cells in which the retrovector was silenced following integration. The
presence of
GFP+ tumor cells suggests that the GCV regimen was not intensive and/or
durable enough to eliminate all transduced tumor cells in these rats.
Alternatively, a subset of transduced, TK-expressing cells may have acquired
resistance to gancyclovir via some other means. Lastly, the "bystander" effect
-
especially its immune effector arm - may vary in intensity from animal to
animal.
1 S This may explain the observed pattern of late relapses, suggesting that
that there
was a early "suicide/bystander" effect which led to increased survival but
that
some tumor cells - transduced or not - "escaped" from the bystander effect and
eventually led to a recurrence. However, the sum of the suicide and bystander
effect was clearly sufficient to enhance survival of a majority of animals
(66%)
who received vTKiGFP and GCV. Our observed long-term survival rate (>120
days) is substantially greater than that observed following intra-tumoral
injection
of TK retroviral producer cells and compares favorably with that obtained with
suicide adenovectors, including those incorporating tumor-specific promoter
elements.
In the experimental group, 2/12 animals died from GCV toxicity and
2/12 succumbed to late tumor recurrences. These data suggest that GCV dose
reduction would be desirable to lessen toxicity, however the duration of
treatment
may need to be extended to allow elimination of all gene modified cells. The
relatively late recurrences (day 82 post implant), lead us to speculate that
the
"immune" bystander effect may have been mitigated in these two animals. It may
be possible to increase the immune response by co-administering immunomodu-
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latory genes (IL2, GMCSF) with TK such as has been described by others.
Furthermore, it may be useful to re-administer the suicide retrovector to
those
animals who have residual disease following a cycle of therapy, and to repeat
this
until maximal response has been achieved. However, it is unknown if a specific
and neutralizing - immune response against VSVG-typed retroparticles will be
elicited.
This constitutes the first report of in vivo delivery of a cell-free
retrovector concentrate with tumor-specific, high efficiency gene transfer and
expression, with evident biologically significant anti-tumor activity. We
propose
that concentrated vTKiGFP retrovector may be of therapeutic value for humans
with brain cancer. The high-titer of the concentrated reagent would allow
intra-
tumor delivery of a useful retrovector dose without the risks of injecting
relatively
large volumes in a confined space (such as brain). vTKiGFP targeting of a
tumor
mass in vivo should subsequently lead to its regression, and the bystander
effect
may have a significant impact on the biology of local and distant
micrometastatic
glioma deposits within the neuropil. This and related therapeutic reagents may
also be useful in the treatment of other locally advanced and metastatic
malignancies.
The present invention will be more readily understood by referring to
the following examples which are given to illustrate the invention rather than
to
limit its scope.
EXAMPLE I
Retrovector encoding for GFP & HSVTK fusion protein serves as a versatile
suicide/reporter instrument for cell and gene therapy applications
In accordance with a preferred embodiment of the present invention, a
pseudotyped retrovector encoding for a chimeric GFP/HSVTK fusion protein that
serves as a bifunctional suicide transgene and reporter was designed. The
fusion
gene was incorporated in a VSV-G pseudotyped retrovector (vGFP/TKfus) and
high titer stable retroviral producer generated (~3x10e6 retroparticles/ml).
Rodent
tumor cell lines transduced at an MOI of 15 for three days led to 100% gene
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transfer efficiency and southern blot analysis confirmed that unrearranged
proviral
genomes integrated in chromosomal DNA. Protein extract immunoblot with
HSVTK anti-sera revealed the presence of a 70kd protein consistent with the
predicted size of a HSVTK+GFP fusion protein. Cell growth of cell lines
expressing vGFP/TKfus was significantly suppressed in the presence of
gancyclovir thereby confirming functionality of the HSVTK C-terminal
component of the fusion protein. Fluorescence microscopy and FACS analysis
revealed that GFPTKfus-mediated fluorescence was 30 fold greater than that
observed in an equivalent bicistronic HSVTK&GFP vector. Interestingly, the
fusion protein was consistently and preferentially localized in the nucleus.
Normal human peripheral blood T-lymphocytes were phytohemagglutinin-
activated and expanded in IL2-containing media. Co-culture with vGFPTKfus
producer cells for three days led to 100% gene transfer and expression with
nucleus-restricted green fluorescence as observed in transduced tumor cell
lines.
The DA3 mouse mammary carcinoma cell line was transduced with vGFP/TKfus
and implanted in syngenic Balb/c mice. Pre-established tumors completely
regressed in 7/9 mice treated with gancyclovir. In conclusion, we demonstrate
the
utility of vGFPTKfus as a reporter/suicide transgene in tumor cells in vitro
and in
vivo. Furthermore, its potential use as an analytical and therapeutic tool
targeting
human T-lymphocytes in adoptive cell therapy applications are shown.
Materials and Methods
Cell likes and plasmids
The pMCITK plasmid containing the HSVTK cDNA was graciously
provided by Gerald Batist (Lady Davis Institute for Medical Research,
Montreal,
QC). The pJ6Ebleo plasmid and 293GPG retroviral packaging cell line were
generous gifts from Richard. C. Mulligan (Children's Hospital, Boston, MA).
MSCV-Neo plasmid was kindly provided by Robert G. Hawley (The Toronto
Hospital, Toronto, ON). The pGFPCl plasmid was purchased from Clontech. A
bicistronic retroviral expression vector encoding for HSVTK and GFP (pTKiGFP)
has been previously described by us. The DA3 mouse mammary adenocarcinoma
cell line and the A549 human lung carcinoma cell line were generously provided
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by Moulai Alaoui-Jamali (Lady Davis Institute for Medical Research, Montreal,
QC).
Retroviral vectors
The synthesis of the GFPTK fusion retrovector was as follows. A
1177 base pair fragment containing the entire HSVTK cDNA was excised from
the plasmid pMC 1 TK by a HincII Xmal digest and ligated with 4693 by fragment
generated from a Ec1136I1 Xmal digest of pEGFP-Cl (Clonetech). This fused
HSVTK sequence at the 3' end of GFP sequence whilst maintaining coding
sequences in frame (Fig. 9, panel A). The fused gene product was then imported
into our previously described retroviral expression vector. This product was
labeled pGFP/TKfus. Transduction of target cells with pGFP/Tkfus -derived
retrov_iral particles (v_GFP/TKfus) will lead to the stable incorporation of
LTR
flanked proviral genome. pGFP was generated by replacing the Neo coding
sequence from MSCV-Neo plasmid with the cDNA of GFP (Fig. 9, panel B).
Retroviral gene transfer
DA3 mouse mammary cells were plated at 2x105 cells per well in a 24
well dish and allowed to adhere. Media was removed and replaced with 500 ~,L
of thawed, retrovirus conditioned media collected from stably transfected
293GPG retoviral producers was added (MOI of ~8). Polybrene (Sigma) was
added to a final concentration of 6 ~g/ml. This procedure was repeated daily
for
three consecutive days. Stably transduced DA3 cells were subsequently
expanded. No clonal selection was performed, and mixed populations of
transduced cells were used for all subsequent experiments. As shown in Fig.
10,
Southern blot analysis was performed on 15 pg of overnight NheI digested
genomic DNA extracted from stably transduced cells as well as untransduced
control cells. Blots were hybridized with a P32 labeled, full-length 700 by
GFP
cDNA probe, washed and exposed on photographic film.
Growtlz suppression assay and Western blot analysis
Stably transduced test and control cells were trypsinized and plated at
a density of 1000 cells per well in a flat bottomed tissue-culture treated 96
well
plate (Costar corporation, Cambridge, MA). Clinical-grade gancyclovir (GCV,
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Hoffinan-Laroche, Mississauga, ONT) was added to achieve a range of
concentrations from 0.01 to 5000 ~.g/ml in a final volume of 100 ~,L of
RPMI/10%FBS. Cells were incubated at 37°C and media was replaced
with fresh
GCV after three days for a total exposure of 6 days. The percentage of
surviving
cells was measured using a method based on the metabolism, by living cells, of
the mitochondria) substrate 3-(4,5-deimethylthiazol-2-yl)-2,5-
diphenyltertrazolium bromide (MTT) into formazan, which is detected by
measurement of the optical density at 570nm(40). Percent Survival is
calculated
as follows [ODS,° test - ODS,° empty well]/[ODS,°
untreated cells - ODS,° empty
well] x 100. All data points were measured in triplicate in at least three
separate
experiments.
For the detection of the HSVTK protein in transduced cells, 20ug of
total protein from either untransduced DA3 cells or DA3 cells transduced with
vGFP/Tkfus or vTKiGFP was separated by SDS-PAGE transferred to
nitrocellulose (BioRad). Detection of HSVTK-containing proteins was done
using polyclonal rabbit anti-HSVTK (Yale University) and detection by an
Enhanced Chemilumenescence Detection kit (Amersham). Blots were exposed to
Kodak X-GMAT film for 5 min.
Flow cytometry and fluorescence microscopy
Flow cytometric analysis was performed within two weeks following
transduction to ascertain retrovector expression and gene transfer efficiency
as
measured by GFP fluorescence. In brief, adherent transduced cells were
trypsinized and resuspended in RPMI at 105 cells per ml. Analysis was
performed on a Epics XL/MCL Coulter analyzer. Live cells were gated based on
FSC/SSC profile and analyzed for GFP fluorescence. Fluorescence microscopy
was performed as follows. Transduced cells were plated over 22mm square
microscope cover glasses previously placed in wells of 6-well flat bottom
tissue
culture plates. Once cells reached subconfluency, they were washed with
phosphate buffered saline (PBS) three times, fixed by exposing to 3%
paraformaldehyde for l5mins at room temperature, and washed again several
times with PBS. The cover glasses were then removed and mounted on pre-
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cleaned frosted end microscope slides (Fisher Scientific) using gelvatol.
Photographs of cells under fluorescence microscopy (excitation of 470 nm) were
taken utilizing a Olympus BX60 microscope attached to a Compaq Deskpro
computer. Pro-Series Capture 128 Image-Pro Plus Software was used with an
integration time of 10 seconds.
Animal model of cancer
Female Balb/c mice were implanted subcutaneously with 1x106 DA3
mouse mammary cells transduced invitro with vGFP/TKfus or vGFP. Five days
post-implant the mice were given 150mg/kg of GCV twice a day i.p. for five
consecutive days. From day 10 post-implant, tumour volume at implantation site
was assessed by palpation. Discernible tumors were measured every three days
by caliper measurements.
Results
GFP and HSVTK fusion protein design and synthesis
A fusion cDNA was synthesized where the entire HSVTK coding
sequence, including 71 nucleotides from the 5' untranslated (5'UTR) HSVTK
sequence were fused to the truncated 3' end of GFP cDNA (see Materials and
methods for details). The resulting linker region between GFP and HSVTK
coding sequence and predicted translation product are shown in Fig. 9, panel
A.
The resulting fusion protein is constituted of GFP to which is fused a 24 AA
peptide linker derived from translation of the HSVTK 5'UTR. The endogenous
HSVTK start codon and subsequent entire coding sequence extending past the
STOP codon leads to a full-length HSVTK protein as the C-terminal half of this
chimera. The coding sequence for the eGFP and HSVTK fusion protein
(GFPTKfus) was incorporated in a plasmid retroviral expression vector we have
previously described (REF) to generate pGFPTKfus (Fig. 9, panel B).
Retrovector plasmids expressing GFP only (pGFP) and a bicistronic vector
encoding for HSVTK and GFP (pTKiGFP) are also depicted in Fig. 9, panel B.
These plasmid vector constructs were utilized to generate retroviral producer
cell
lines derived from the 293GPG retroviral packaging cell line as previously
described. Following co-transfection of 293GPG cells with pGFPTKfus and
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BLED, and Zeocin drug selection, a mixed population of cells (293GFPTKfus)
was maintained. The 293GFPTKfus retroviral producer cells will generate
Vesicular Stomatitis Virus-G protein (VSVG) pseudotyped retroparticles
following tetracycline withdrawal. The retroviral titer generated from the
vGFPTKfus producer was 3 x 106 particles/ml as measured on the human A549
lung carcinoma cell line. We performed DNA analysis of vGFPTKfus transduced
A549 cells to determine if the replication-defective pro-viral genome
integrated in
chromosomal DNA unrearranged. NheI digest of genomic DNA from
vGFPTKfus-transduced cells will generate a 3.6kb DNA fragment encompassing
LTR-flanked vector sequences. Southern blot of NheI digested DNA was
hybridized with a GFP sequence-specific probe and a DNA fragment of predicted
size is detected (Arrow, Fig. 10).
Green fluorescence in retrovirally transduced cells
Green fluorescence 0530 nm) emitted from cells excited with "blue" (470 nm
bandwith) light serves as a reporter of GFP transgene expression in
genetically
engineered cells. We sought to compare green fluorescence emission in DA3
mouse mammary carcinoma cells transduced with VSVG-pseudotyped
retrovectors encoding GFP alone (vGFP), HSVTK and GFP as part of a
bicistronic retrovector (vTKiGFP) or the GFPTKfus protein (vGFPTKfus). All
cells were transduced at a MOI of ~8 for three consecutive days to generate
three
polyclonal populations of cells. No clonal selection was performed and green
fluorescence was measured by flow cytometry. As shown in Fig. 11, the mean
fluorescence intensity (MFI) of DA3/GFPTKfus cells is 100 fold greater than
control and 30 fold greater than DA3/TKiGFP cells. The MFI of cells transduced
with GFP reporter only (DA3/GFP cells) is ~20 fold greater than that of
DA3/GFPTKfus. Fluorescent microscopic examination of DA3/GFPTKfus cells
revealed that green fluorescence was dominantly and sharply localized in
nucleus,
excluded from nucleoli, and that faint cytoplasmic fluorescence was observed
in
"high expressors" only. In contrast, DA3/GFP cells green fluorescence is
distributed evenly between cytoplasm and nucleus (Fig. 12).
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HSVTK expression and sensitization to gancyclovir
The expression of immunoreactive HSVTK moiety was directly
compared between DA3/GFPTKfus and DA3/TKiGFP cells. Western analysis of
whole cell lysates immunblotted with anti-HSVTK antibody was performed. As
shown in Fig. 13, DA3/TKiGFP express the expected SO kd native HSVTK
protein, whilst DA3/GFPTKfus cells bear a 75kd immunoreactive protein whose
size is consistent with the predicted mass of GFP (25kd) + HSVTK (50 kd)
fusion
product. Though of different molecular weight, equivalent amounts of anti
HSVTK immunoreactive protein is generated by both vGFPTKfus and vTKiGFP
transduced cells.
We determined if the HSVTK component of GFPTKfus remained
functional. Cells genetically-engineered to express the HSVTK suicide gene
will
convert the prodrug gancyclovir to its cytotoxic phosphorylated metabolite.
Sensitization to gancyclovir of retrovirally transduced cells was measured in
a
growth suppression assay as described in materials and methods. When compared
to DA3/GFP cells, both DA3/TKiGFP and DA3GFPTKfus cells are significantly
sensitized to gancyclovir with an IC50 1000 fold (Fig. 14). Doubling time (~24
hours) for all cell lines are similar.
In Vivo sensitzation of transduced tumor cells to gancyclovir
DA3 cells are tumorigenic in Balb/c mice. We determined if pre-
established DA3/GFPTKfus subcutaneous tumor implants would regress
following gancyclovir treatment in vivo. Five days after subcutaneous
implantation of DA3 cells, palpable tumors arise. Mice were treated with
gancyclovir 150mg/kg intraperitoneally twice daily for five days and presence
of
palpable tumor assessed over time. 7/9 rodents implanted with DA3GFPTKfus
tumors were rendered tumor-free by gancyclovir treatment. Mice in which
DA3GFPTKfus tumors regressed completely subsequent to GCV treatment
remained tumor-free for at least two months. Tumor volume growth rate over
time was identical between GCV-treated DA3GFP implants and mock-treated
DA3GFPTKfus implants.
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Discussion
The Herpes Simplex Virus TK gene product is a potent conditional
suicide gene. Conditional, since its overexpression is innocuous to engineered
cells, yet renders these same cells extremely sensitive to the cytotoxic
effects of
the nucleobase analog gancyclovir. The HSVTK transgene has been a widely
adopted tool in the field of gene therapy for adoptive cell therapy and for
cancer
gene therapy applications.
In its use as a conditional "self destruct" mechanism, its role is to
eliminate engineered cells if their in vivo behaviour is undesirable. The best
example of this approach is in the treatment of graft versus host disease
(GVHD)
that may arise following donor lymphocyte infusion (DLI). DLI are commonly
administered to patients who suffer a relapse of a hematological malignancy
following allogeneic bone marrow transplantation. In the pioneering work of
Bonini et al, a bicistronic vector containing expressing the truncated Nerve
Growth Factor Receptor reporter (NGFR) and a HSVTK/Neomycin
phosphotransferase II fusion gene was retrovirally introduced in donor
lymphocytes, selected in 6418, and subsequently administered to patients. In
vivo tracking of engineered lymphocytes was achieved by tracking cell surface
expression of NGFR which can be readily detected by flow cytometry and
immunohistochemistry. Patients in whom GVHD arose as a complication of DLI
were treated with GCV and offending engineered lymphocytes were eliminated.
In some patients GVHD reversed. For this and related strategies, pre-infusion
dominant selection of HSVTK expressing lymphocytes is mandatory. Further,
post-infusion tracking of cells in blood, lymphoid organs and diseased tissue
serves well the purpose of deciphering the role of gene-marked donor
lymphocytes in GVHD and the effect of GCV treatment on its control.
HSVTK/GCV tandem serves a potent and effective "self destruct" switch,
however the requirement of dominant selection and traceable marker, make for
bulky multigenic vector constructs, limiting the ability to introduce other
useful
therapeutic transgenes within retroviral constructs of limited (~8-lOkb) gene
packing space. Genetic engineering of autologous lymphocytes and other
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immunocompetent cells are also attracting a significant amount of attention as
tools for therapy of acquired diseases, such as AIDS and cancer. Many of these
cell therapy reagents will also require a dominant selectable marker and a
conditional "self destruct" switch, especially if an unforeseen undesirable
phenotype arises from their use in clinical studies.
Similar requirements are also relevant to the use of HSVTK as an anti-
cancer "killer" gene. HSVTK gene transfer to pre-established cancer will lead
to
tumor regression. Effectiveness is wholly dependent on gene transfer
efficiency
in vivo, and issues related to in vivo transgene tracking are critical to
ascertain
cause/effect relationship. Consequently, a dominant selectable
marker/reporter/suicide multivalent transgene would greatly enhance the
utility of
a "self destruct" or "suicide" switch in gene and cell therapy applications.
Fluorescent reporter proteins such as the Green Fluorescent Protein
(GFP) have properties well suited as a combined selectable marker/reporter.
First,
their intra-cellular expression can be readily detected in live cells without
need of
fixation, antibodies or affinity columns, allowing rapid and specific cell
sorting -
based on green fluorescence - with standard flow cytometry equipment as
described by others. Secondly, in vivo tracking of live engineered cells is
facilitated. We have also shown that tumor-targeted GFP reporter gene transfer
can also be directly visualized by fluorescence microscopy. Combining HSVTK
and fluorescent protein expression would address the objective of combining
reporter/marker with a potent "self destruct" gene. We have addressed this
previously with the use of a HSVTK and GFP reporter bicistronic vector
construct. However, we have found that to obtain optimal retrovirus-driven
HSVTK protein production, that the GFP cDNA had to be incorporated in the less
favorable IRES-dependent translation position. Though GFP reporter expression
is readily detectable in some tumor and tissue type, its expression level
often falls
below satisfactory levels, especially when utilizing fluorescent microscopy.
Further, engineering of multigenic retroviral constructs incorporating HSVTK
and
GFP with other synergistic anti-cancer immunomodulatory genes, such as IL2,
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GMCSF and others, is limited by the inadequacies of multicistronic constructs
where multiple IRES will inevitably yield poor transgene protein production.
We examined the utility of a GFP & HSVTK fusion protein as a
bifunctional reporter/marker and suicide transgene as part of a therapeutic
retrovector. As depicted in Fig. 9, we generated a fusion gene where HSVTK is
fused to the C-terminus of GFP with a 24 amino acid linker. This construct was
incorporated in a retroviral expression vector and VSVG-pseudotyped
retroparticles were generated as a gene transfer vehicle. We show that the
GFPTKfus coding sequence was permissive for high-titer virus production and
that retroviral constructs bearing this sequence were genetically stable upon
integration in target cells as shown by southern blot analysis (Fig. 10).
Target
cells transduced at an equivalent MOI reveal that GFPTKfus expression leads to
a
degree of green fluorescence that is markedly superior to that seen with
either
negative control (100 fold) or a bicistronic TKiresGFP construct (30 fold),
yet is
less "bright" than a monocistronic GFP expression vector (Fig. 11).
Interestingly,
we observed that the GFPTKfus protein localized predominantly to the nucleus
of
transduced cells as opposed to the diffuse pancellular distribution of native
GFP
protein (Fig. 12). This data strongly suggests that HSVTK protein contains a
nuclear localizing signal that may play a role in the normal physiological
role of
this viral protein. Immunoblot analysis of whole cell extracts with anti-HSVTK
antibody revealed that mass of GFPTKfus protein was 75kd, consistent with the
predicted combined mass of GFP (25kd) and HSVTK (SOkd) (Fig. 13). Further, it
is noted that equivalent amounts of TK-immunoractive protein was generated
from the GFPTKfus and TKiGFP retroviral constructs, consistent with the
prediction that the HSVTK protein translation levels obtained from the 5'
cistron
of a bicistronic construct (TKiGFP) are equivalent to a related monocistronic
construct (GFPTKfus). We found that acquired GCV sensitivity was identical
between cells transduced with either vTKiGFP or vGFPTKfus retrovectors
(Fig. 14). These biochemical observations were confirmed in a mouse tumor
implant model where pre-established GFPTKfus-expressing tumors could be
eradicated in a majority of mice by GCV treatment.
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The sum of these data suggest that the GFP fusion protein bearing a C-
terminus fused HSVTK looses some of its intrinsic "brightness" when compared
with GFP only. Yet, the fusion GFPTKfus protein conserves the ability to
sensitize cells to GCV as well as native HSVTK. Though native GFP protein is
S "brightest", its expression levels - derived from a bicistronic construct
designed to
optimize HSVTK expression (TKiGFP, Fig. 9) - are substantially lower than that
obtained from the GFPTKfus retroviral construct. Therefore, the GFPTKfus gene
product wholly preserves its vital "self destruct" and "suicide" feature and
exhibits desirable fluorescent properties superior to that achieved in
fluorescent
bicistronic constructs designed to optimize HSVTK expression. In conclusion,
GFPTKfus can serve as an improved substitute to bicistronic HSVTK and GFP
constructs, where "suicide" characteristics are preserved and green
fluorescent
reporter expression levels are superior. Further, incorporation of GFPTKfus in
multigenic vector constructs where other gene products of interest are
included
1 S will greatly facilitate their characterisation in cell and gene therapy
applications,
including dominant selection by cell sorting, analysis of vector expression in
live
cells in vitro and in vivo, and biologically relevant expression of a potent
"self
destruct" and "suicide" transgene.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations, uses,
or
adaptations of the invention following, in general, the principles of the
invention
and including such departures from the present disclosure as come within known
or customary practice within the art to which the invention pertains and as
may be
applied to the essential features hereinbefore set forth, and as follows in
the scope
of the appended claims.
