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Experimental Therapeutics |
Department of Medicine (Division of Hematology-Oncology), Lady Davis Institute for Medical Research, Montreal, Quebec, H3T 1E2 Canada [A. P., F. S., J. G.], and Montreal Neurological Institute, McGill University, Montreal, Quebec, H2A 3B4 Canada [J. N., H. L., G. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The "bystander" effect is dependent, in part, on cell-cell contact (5) and intercellular communications, gap junctions, through which GCV-phosphate can circulate between TK-positive and TK-negative tumor cells (6) . Phagocytosis of GCV-phosphate laden cell debris by adjacent tumor cells will also lead to cell death (5) . Blood vessel endothelial cells within or adjacent to the tumor may also acquire TK, and their destruction with GCV therapy, thus, may also contribute to tumor regression (7) . It has also been noted that "suicide" tumors release inflammatory cytokines that promote hemorrhagic necrosis in local, but noncontiguous, tumor deposits (8) . Furthermore, tumors undergoing a necrotic death, as opposed to apoptosis, will up-regulate the expression of proteins such as heat shock protein 70, IL-10, and IL-12, which may enhance immune recognition and rejection (9 , 10) . Necrotic tumors may be infiltrated with a wide assortment of immunocompetent cells such as CD4+ lymphocytes, CD8+ lymphocytes, natural killer cells and antigen-presenting cells (10 , 11) . These infiltrating cells may take part in a tumor-specific immune response that is an important component of the local (12, 13, 14, 15) as well as distant antitumor immune bystander effect (16 , 17) . Intracerebral tumors are also susceptible to immune clearance after suicide gene expression (18 , 19) , suggesting that the brain is not an immune sanctuary for cancer (20) . Therefore, tumor-targeted suicide gene delivery will lead 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, because 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 (reviewed in Ref. 21 ). This is usually achieved by direct intratumoral or i.v. injection of 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 the local injection site and to transduce contiguous normal brain (22) , including astrocytes, neurons, and ependymal cells (23) , suicide gene expression may lead to significant toxicity after GCV treatment (24, 25, 26) .
Recombinant retroviral vectors are well characterized as vehicles for tumor-targeted gene delivery (27) . Retroviruses can integrate only in cells undergoing mitosis shortly after infection (28) . Quiescent cells, such as normal brain tissue adjacent to a targeted tumor deposit, will be refractory to gene transfer and spared from subsequent toxicity (4) . 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 that do not express the retroviral receptor (29) ; and the low particle concentration in clinical-grade viral preparations. Clinical-grade retroviral particle preparations usually have titers <107 particles/ml. Assuming that a target tumor having a 1-cm diameter contains at least 108 cells, it would be necessary to inject intratumorally at least >10 ml of viral preparation to deliver an equal number of viral particles. This logistical impediment to retroparticle delivery has been addressed by directly injecting murine retroviral VPCs in to tumors in vivo. The idea is that locally produced viral particles could transduce cancer cells. Although 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 (4 , 7 , 19) . In human clinical trials, where this strategy was duplicated by injecting amphotropic VPCs with a titer of 1 x 105 cfu/ml, low-albeit detectable-TK gene transfer efficiency was noted in tumor cells. Furthermore, a specific immune response against VPCs was elicited (30) . 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 that incorporate the VSVG protein differ from traditional murine retroviral pseudotypes by their high affinity for a wide assortment of eukaryotic cells (31) . This is primarily due to the ability of VSVG to recognize membrane phospholipid as a minimal receptor (32 , 33) . Unlike standard murine retroviruses, VSVG retrovectors are also relatively resistant to deactivation by human complement (34) . Furthermore, like adenoviruses, VSVG-typed retroviruses can be concentrated to high titers by centrifugation and frozen/thawed without loss of activity (31) . The VSVG pseudotype does not alter the retroviral genome’s restricted targeting of cycling cells. Thus, VSVG-typed retroparticles may be a suitable delivery vehicle for suicide gene transfer, combining high titer, particle stability, and tumor specificity.
To test this hypothesis, we designed a HSV TK-expressing retrovector and generated VSVG-pseudotyped retroparticles. We show that human glioma cell lines can be transduced in vitro and express functionally significant amounts of HSV TK. Concentrated retroparticles were administered intratumorally in a rat model of brain cancer, and a significant survival benefit was noted after GCV therapy.
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MATERIALS AND METHODS |
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bleo plasmid (35)
, and 293GPG retroviral packaging cell line (34)
were generous gifts from Dr. Richard C. Mulligan (Children’s Hospital, Boston, MA). MSCV-Neo plasmid (36)
and BSICZSVPA plasmid (37)
were kindly provided by Dr. Robert G. Hawley (The Toronto Hospital, Toronto, Ontario, Canada). SKI-1, SKMG-4, SKMG-1, T98G, UW28, and UWR7 human glioma cell lines were generously provided by Lawence Panasci (Lady Davis Institute for Medical Research, Montreal, Quebec, Canada). C6 and C6/lacZ glioma cells originated from American Type Culture Collection. pMC1TK plasmid (38)
was graciously provided by Dr. Gerald Batist (Lady Davis Institute for Medical Research, Montreal, Quebec, Canada). HaL22Y plasmid (39)
was kindly provided by Dr. Raymond L. Blakley (St. Jude Children’s Research Hospital, Memphis, TN).
Retrovector Design and Synthesis.
We engineered a plasmid encoding for a bicistronic, nonsplicing murine retrovector that incorporates a multiple cloning site, allowing insertion of cDNA of interest, linked to the enhanced green fluorescence reporter (AP2). The synthesis of AP2 was as follows. The 805-bp EGFP cDNA was excised by Eco47-3 and NotI digest of pEGFP-N1 (Clontech, Palo Alto, CA) and ligated into the MSCV (36)
retroviral plasmid to generate MSCV-EGFP. The 555-bp IRES was excised from the BSICZSVPA plasmid (37)
by SacII-NcoI digest and cloned into SacII-NcoI cut MSCV-EGFP to generate MSCV-IRES/EGFP. MSCV-IRES/EGFP was digested with SpeI-AscI to generate a 2524-bp 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-bp fragment from SpeI-AscI cut pCMMP-LZ, an unpublished MFG-based retrovector, to generate AP2 (Fig. 1A)
. AP2 is designed to coexpress 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 nonsplicing. The pMC1TK plasmid (38)
was cut with BglII-BsaW1 to generate a 1207-bp fragment containing the HSVTK cDNA (excluding polyadenylation signal) and was ligated into BglII-XbaI-cut AP2 to generate pTKiGFP (Fig. 1B)
. The retroviral genome produced from pTKiGFP will not incorporate the cytomegalovirus 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-bp BamHI-XhoI DHFR(L22Y) cDNA from Ha-L22Y (39)
into BglII-SalI cut MSCV-IRES/EGFP.
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Bleo plasmid (35)
. Transfected cells were subsequently selected in 293GPG media supplemented with 100 µg/ml Zeocin (Invitrogen, San Diego, CA) as described (34)
. Resulting stable polyclonal producer populations were used to generate high titer virus. All viral supernatants were filtered with 0.45 µm syringe-mounted filters (Gelman Sciences, Ann Arbor, MI) and stored at -20°C. Concentration of VSVG retroparticles was performed as described previously (34)
. 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 min. Viral pellets were resuspended overnight in 100 µl of serum-free RPMI (Life Technologies, Inc.) at 4°C, pooled, and concentrated through a second centrifugation. Concentrated virus was aliquoted and stored at -80°C. Viral preparations were devoid of Replication-defective Retrovirus (RCR) by EGFP marker rescue assay using supernatant from transduced UWR7 cells.
Transduction of Glioma Cells, Flow Cytometry, and Southern Blot Analysis.
Human glioma cell lines were plated at 2 x 104 cells per well in a 24-well dish and allowed to adhere. Medium was removed and replaced with 500 µl of thawed, retrovirus-conditioned medium collected from transiently transfected 293GPG. Polybrene (Sigma) was added to a final concentration of 6 µg/ml. This procedure was repeated daily for 3 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 2 weeks after 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/ml. Analysis was performed on a Epics XL/MCL Coulter analyzer. Live cells were gated based on forward scatter/side scatter profile and analyzed for GFP fluorescence. Southern blot analysis was performed on 15 µg 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 bp of 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/well in a flat-bottomed, tissue culture-treated, 96-well plate (Costar Corp., Cambridge, MA). Clinical grade GCV (Hoffman-La Roche, Mississauga, Ontario, Canada) was added to achieve a range of concentrations from 0.01 to 5000 µg/ml in a final volume of 100 µl of RPMI/10% FBS. Cells were incubated at 37°C, and medium was replaced with fresh GCV after 3 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 mitochondrial substrate MTT into formazan, which is detected by measurement of the absorbance at 570 nm (40)
. Percent survival is calculated as follows:
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Titration of Retrovector.
Target glioma cells were plated at 2 x 105 cells/well in a six-well tissue culture dish. The next day, cells from a test well were trypsinized and enumerated to determine baseline cell count at the moment of virus exposure. Virus was serially diluted (range, 100 to 0.001 µl) in a final volume of 1 ml of RPMI/10% FBS supplemented with 6 µg/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 nonsaturating transduction conditions prevailed (i.e., transduction 2efficiency <80%). Titer (cfu/ml) was calculated as:
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Animal Model of Brain Cancer, in Vivo Retrovector Delivery, and GCV Treatment.
C6/lacZ glioma cells will reproducibly generate lethal intracerebral tumors when injected in Sprague Dawley rats. The constitutive ß-galactosidase expression facilitates delineation (by X-gal staining) of tumor cells and extent of the tumor infiltrate in postmortem brain sections. Adult Sprague Dawley rats were anesthesized with i.p. injection of ketamine (50 mg/kg) and xylazine (2 mg/kg). C6/lacZ rat glioma cells (2 x 104 cells in 5 µl of HBSS) were injected intracranially into the frontal lobe using a Hamilton syringe in a stereotactic apparatus (Kopf) over a period of 15 min. 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 dural surface. Six days after glioma cell implantation, rats were anesthesized, and vTKiGFP (concentrated stock of 2.3 x 1010 cfu/ml) was injected of into six different sites (1 mm apart) in the preestablished tumor guided by the previous stereotactic coordinates. A total volume of 9 µl was injected in each tumor (6 x 1.5-µl increment), and the needle was left in place for at least 5 min/increment (for a total of 30 min/tumor). Two days after retrovector delivery, rats are treated with 50 mg/kg GCV i.p. twice daily for 5 days, followed 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 µm) were prepared. GFP activity was observed by epifluorescence microscopy and recorded photographically. Subsequently, sections were stained histochemically for ß-galactosidase activity as described previously (41)
before counter staining with H&E.
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RESULTS |
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allows the incorporation of a cDNA sequence in a Multiple cloning site upstream of an IRES and the EGFP cDNA. The transcription initiation from a cytomegalovirus promoter will lead to the production 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 codominant expression of both protein products. Live cells expressing EGFP, which is detectable by fluorescence microscopy or flow cytometry, will coexpress the linked gene product. Gene-modified cells can be implanted or transplanted in animal models, and their localization and function can 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' LTR. Transfection of an appropriate retroviral packaging cell line can lead to production of 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 cotransfection with a drug resistance plasmid. We have generated retroparticles by both methods with good success using 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, and UWR7) were transduced with three consecutive daily applications of thawed vTKiGFP supernatant. The MOI was 4 at each viral application. Six days after transduction, polyclonal cell lines were subjected to flow cytometric analysis to determine the proportion of cells that expressed the GFP reporter protein. All polyclonal cell lines were 100% GFP-positive by fluorescence-activated cell sorting analysis, and transduced UWR7 cells served 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 (data not shown). Southern blot analysis confirmed that unrearranged vTKiGFP vector integrated in chromosomal DNA of transduced target cells (Fig. 3)
. vTKiGFP transduced cells have been passaged in excess of 30 times without loss of GFP expression.
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). Growth rates for transduced and parental cell lines in the absence of GCV were identical (data not shown).
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. 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 after tetracycline withdrawal. Supernatant was cleared of cellular debris with a 0.45 µm filter and frozen. We have noted that twice daily media collection, as opposed to once daily, doubled the yield of retroparticles from producer cells after tetracycline withdrawal (data not shown). Media were thawed, pooled, and subjected to ultracentrifugation as described in "Materials and Methods." Supernatant was concentrated 84-fold (20–0.24 ml) by ultracentrifugation. The concentration step raised the titer from 2.9 x 107 cfu/ml to 220 x 107 cfu/ml, as measured on UWR7 human glioma cells (Fig. 6)
. Concentrates (84x) 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 2.3 x 1010, 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 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).
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, including distant micrometastasis. Normal surrounding brain tissue is bereft of green fluorescence. No green fluorescence was observed in untransduced brain tumors (Fig. 7C)
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. Significantly enhanced survival was obtained; 8 of 12 GCV-treated test rats (66%) remain long-term survivors (>120 days). A supplementary control cohort of six 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, 31–63 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 versus 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 9 L glioma implants (42)
. These differences may be due different biological properties of these two experimental glioma models. |
DISCUSSION |
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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 that express a specific inorganic phosphate transporter (29) . If a target tumor does not express the retrovirus receptor, gene transfer-and therapeutic benefit–is unlikely to occur. Retroparticles that are pseudotyped with the VSVG protein will adopt the wide host range of the VSV. The putative VSVG receptor on target cells, which is believed to be membrane phospholipid (44) , is ubiquitously found in all eukaryotic cells. This has led to the use of VSVG-pseudotyped retrovectors as gene delivery vehicles in a wide assortment of mammalian, nonmammalian, and invertebrate cells (31 , 45, 46, 47, 48, 49) . A major advance in pseudotyping retrovectors with VSVG was achieved when a practical "transient" VSVG retroviral packaging cell line was designed (31) . The subsequent publication of reliable "stable" high-titer VSVG packaging cell lines (50 , 51) , including 293GPG (34) , has allowed the development and characterization of pseudotyped retrovectors for a wide variety of gene transfer applications (52) , including tumor cell-targeted gene delivery (53) .
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 that incorporates HSV TK and the EGFP reporter cDNA within a bicistronic transcript (pTKiGFP). We have found that codominant expression of the HSV TK 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 end point 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 withdrawal, this retroviral producer cell line will express the VSVG envelope protein and generate pseudotyped retroviral particles. We found that VSVG-pseudotyped 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 >90% gene transfer in targeted cells (Fig. 6)
. Gene expression was durable as assessed by persistent GFP expression (>30 passages) and by functional HSV TK expression, rendering VSVG-associated pseudotransduction (46
, 54)
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 is of some importance, especially in light of recent reports documenting rearranged "suicide" retroviral vectors as a cause of GCV resistance in transduced tumors (55)
. Virtually all glioma cell lines transduced with vTKiGFP acquired substantial and significant sensitivity to GCV 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 GCV (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 after tetracycline withdrawal from the 293AP3 producer cell line. Retroparticle-containing medium 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 ml of media to a final volume of 0.1 ml (1000x concentration on volume basis). This was accompanied by an 800-fold increase in titer from 2.9 to 2300 x 107 cfu/ml. We noted that supernatant from tetracycline-deprived 293AP3 producer cells could be toxic to target cells if applied repeatedly. We also observed this phenomena with other 293GPG-derived producer cells (data not shown). 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 constituent(s) that are readily discarded upon concentration procedure.
To test the therapeutic usefulness of this reagent, we used a rodent model of brain cancer. We established C6/lacZ glioma tumors in immunocompetent Sprague Dawley rats and subsequently administered concentrated vTKiGFP retrovector intratumorally. Intratumoral delivery of 9 µl (108 retroparticles) of concentrated vTKiGFP retrovector stock did not improve survival of animals who did not subsequently receive GCV. These control rats (tumor+, retrovector+, but no GCV) had a mean survival of 38 days (range, 20–52 days). Postmortem 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 (data not shown). 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 after tumor-targeted vTKiGFP delivery. Of these, two died shortly (within 2 weeks) following the end of GCV treatment. This "acute" death rate attributable to direct GCV toxicity (16%) is comparable with that observed by other investigators who administered GCV at equal or lesser doses (7
, 42)
. The mechanism of death is likely related to cytopenia and immunosuppression associated with severe, albeit reversible, bone marrow toxicity. Surviving test rats fully recovered from GCV toxicity 2 weeks after its completion.
All of the test rats remained alive and well more than 80 days after tumor implantation. Two rats developed symptomatic tumor recurrences and were sacrificed on day 82 after tumor implantation (Fig. 8)
. Examination of brain tissue sections on these late relapses revealed large tumors with areas of green fluorescence interspaced 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 after 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 GCV via some other means. Lastly, the "bystander" effect, especially its immune effector arm, may vary in intensity from animal to animal. This may explain the observed pattern of late relapses, suggesting that that there was an early "suicide/bystander" effect that 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 after intratumoral injection of TK retroviral producer cells (19)
and compares favorably with that obtained with suicide adenovectors (42)
, including those incorporating tumor-specific promoter elements (22)
.
In the experimental group, 2 of 12 animals died from GCV toxicity and 2 of 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 after implant), led 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 coadministering immunomodulatory genes (IL-2 and granulocyte/macrophage colony-stimulating factor) with TK such as has been described by others (56) . Furthermore, it may be useful to readminister the suicide retrovector to those animals who have residual disease after 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 antitumor 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 intratumor 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.
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ACKNOWLEDGMENTS |
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Note Added in Proof
A supplementary test animal that had received vTKiGFP and GCV died on day 125 after tumor implantation. On post mortem, it had a large intracerebral tumor which was GFP negative. All the other test animals (7 of 12) which had received treatment remain alive and well 270 days after tumor implantation.
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FOOTNOTES |
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1 Supported in part by Medical Research Council of Canada Grant MA-15017 (to J. G.) and the National Cancer Institute of Canada (Terry Fox Funds; to J. N. and G. K.). J. G. is supported by the Medical Research Council of Canada Clinician-Scientist Award, and J. N. is a Senior Research Scholar of the Fond de Recherches en Sante du Quebec. 
2 To whom requests for reprints should be addressed, at Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine Road, Montreal, Quebec, H3T 1E2 Canada. Phone: (514) 340-8260; Fax: (514) 340-7576; E-mail: jgalipea{at}lab.jgh.mcgill.ca
3 The abbreviations used are: HSV TK, herpes simplex virus thymidine kinase; GCV, ganciclovir; IL, interleukin; VPC, viral producer cell; VSVG, vesicular stomatitis virus G; EGFP, enhanced green fluorescence protein; IRES, internal ribosomal entry site; LTR, long terminal repeat; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; MOI, multiplicity of infection.
4 J-S. Lee and R. C. Mulligan, unpublished data.
Received 11/30/98. Accepted 3/19/98.
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 W. Wang, D. Mullikin-Kilpatrick, J. E. Crandall, R. M. Gronostajski, E. D. Litwack, and D. L. Kilpatrick Nuclear Factor I Coordinates Multiple Phases of Cerebellar Granule Cell Development via Regulation of Cell Adhesion Molecules J. Neurosci., June 6, 2007; 27(23): 6115 - 6127. [Abstract] [Full Text] [PDF]
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 S. R. Perri, D. Martineau, M. Francois, L. Lejeune, L. Bisson, Y. Durocher, and J. Galipeau Plasminogen Kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways Mol. Cancer Ther., February 1, 2007; 6(2): 441 - 449. [Abstract] [Full Text] [PDF]
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T. Thomas, K. Jordan, J. Simek, Q. Shao, C. Jedeszko, P. Walton, and D. W. Laird Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration J. Cell Sci., October 1, 2005; 118(19): 4451 - 4462. [Abstract] [Full Text] [PDF]
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 S. R. Perri, J. Nalbantoglu, B. Annabi, Z. Koty, L. Lejeune, M. Francois, M. R. Di Falco, R. Beliveau, and J. Galipeau Plasminogen Kringle 5-Engineered Glioma Cells Block Migration of Tumor-Associated Macrophages and Suppress Tumor Vascularization and Progression Cancer Res., September 15, 2005; 65(18): 8359 - 8365. [Abstract] [Full Text] [PDF]
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J. Stagg, J. H. Wu, N. Bouganim, and J. Galipeau Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2 Fusion cDNA for Cancer Gene Immunotherapy Cancer Res., December 15, 2004; 64(24): 8795 - 8799. [Abstract] [Full Text] [PDF]
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 M. Bani-Yaghoub, S. E. Kendall, D. P. Moore, S. Bellum, R. A. Cowling, G. N. Nikopoulos, C. J. Kubu, C. Vary, and J. M. Verdi Insulin acts as a myogenic differentiation signal for neural stem cells with multilineage differentiation potential Development, September 1, 2004; 131(17): 4287 - 4298. [Abstract] [Full Text] [PDF]
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G. I. Veitch, J. E. I. Gittens, Q. Shao, D. W. Laird, and G. M. Kidder Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface J. Cell Sci., June 1, 2004; 117(13): 2699 - 2707. [Abstract] [Full Text] [PDF]
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T. Thomas, D. Telford, and D. W. Laird Functional Domain Mapping and Selective Trans-dominant Effects Exhibited by Cx26 Disease-causing Mutations J. Biol. Chem., April 30, 2004; 279(18): 19157 - 19168. [Abstract] [Full Text] [PDF]
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 J. M. Sarjeant, A. Lawrie, C. Kinnear, S. Yablonsky, W. Leung, H. Massaeli, W. Prichett, J. P. Veinot, E. Rassart, and M. Rabinovitch Apolipoprotein D Inhibits Platelet-Derived Growth Factor-BB-Induced Vascular Smooth Muscle Cell Proliferated by Preventing Translocation of Phosphorylated Extracellular Signal Regulated Kinase 1/2 to the Nucleus Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2172 - 2177. [Abstract] [Full Text] [PDF]
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H. Qin, Q. Shao, S. A. Igdoura, M. A. Alaoui-Jamali, and D. W. Laird Lysosomal and Proteasomal Degradation Play Distinct Roles in the Life Cycle of Cx43 in Gap Junctional Intercellular Communication-deficient and -competent Breast Tumor Cells J. Biol. Chem., August 8, 2003; 278(32): 30005 - 30014. [Abstract] [Full Text] [PDF]
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L. Yen, N. Benlimame, Z.-R. Nie, D. Xiao, T. Wang, A.-E. A. Moustafa, H. Esumi, J. Milanini, N. E. Hynes, G. Pages, et al. Differential Regulation of Tumor Angiogenesis by Distinct ErbB Homo- and Heterodimers Mol. Biol. Cell, November 1, 2002; 13(11): 4029 - 4044. [Abstract] [Full Text] [PDF]
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D. Duguay, F. Mercier, J. Stagg, D. Martineau, J. Bramson, M. Servant, R. Lin, J. Galipeau, and J. Hiscott In Vivo Interferon Regulatory Factor 3 Tumor Suppressor Activity in B16 Melanoma Tumors Cancer Res., September 15, 2002; 62(18): 5148 - 5152. [Abstract] [Full Text] [PDF]
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H. Qin, Q. Shao, H. Curtis, J. Galipeau, D. J. Belliveau, T. Wang, M. A. Alaoui-Jamali, and D. W. Laird Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication J. Biol. Chem., August 2, 2002; 277(32): 29132 - 29138. [Abstract] [Full Text] [PDF]
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A. J. Mao, J. Bechberger, D. Lidington, J. Galipeau, D. W. Laird, and C. C. G. Naus Neuronal Differentiation and Growth Control of Neuro-2a Cells After Retroviral Gene Delivery of Connexin43 J. Biol. Chem., October 27, 2000; 275(44): 34407 - 34414. [Abstract] [Full Text] [PDF]
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M. M. Aarts, D. Davidson, A. Corluka, E. Petroulakis, J. Guo, F. R. Bringhurst, J. Galipeau, and J. E. Henderson Parathyroid Hormone-related Protein Promotes Quiescence and Survival of Serum-deprived Chondrocytes by Inhibiting rRNA Synthesis J. Biol. Chem., October 5, 2001; 276(41): 37934 - 37943. [Abstract] [Full Text] [PDF]
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) or the control retrovector vDHFRiGFP (
). These and untransduced controls (
) were subsequently exposed to GCV for 6 days, and cell survival was measured by the MTT assay as described in "Materials and Methods." Percent survival is plotted against GCV concentration (log scale). Data points, mean survival measured in three separate experiments; bars, SD. SD smaller than data point icon are not displayed.
