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Definitions

• the invention relates to retroviral expression vectors and more particularly to pseudotyped retroviral vectors for gene therapy of cancer.
• Tumor cells modified to express the Herpes Simplex Virus Thymidine Kinase gene 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.
• tumors undergoing a necrotic death 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 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.
• 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.
• 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.
• VPCs murine retroviral producer cells
• Retroparticles which incorporate the Vesicular Stomatitis Virus G
• VSVG murine retroviral pseudotypes
• 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).
• 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.
• One aim of the present invention is to provide a suitable retroviral vector for gene therapy of a cancer.
• VSVG-typed retroparticles may be suitable for delivering a therapeutic gene to a tumor tissue.
• the cancer may be a brain cancer.
• therapeutic genes include suicide genes.
• 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.
• the reporter protein allows for a sorting of producer cells and facilitates the measurement of the retroviral titer.
• 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 MD1 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 RARB2; IRF3; IRF3-5d; MCP1; Rantes; MIP1 alpha; MlPlbeta; MCP1.
• Preferred JGH2 expression vector derivatives include, without limitation, the following:
• GMCSF (MD2); IRF3 (AP6); IRF3/5D (AP7).
• Figs. 1A-1C illustrate schematic representations of plasmids and retrovectors.
• Fig. 1A 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. IB 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. 1C Target cells transduced with vTKiGFP will integrate the retrovector in their genomic DNA.
• 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.
• 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).
• Fig. 5 illustrates flow cytometric analysis of 293AP3 producer cells.
• 293 GPG 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”.
• IX and 84X virus stock were diluted (as indicated on left) in a final volume of 1 ml and applied to 2.3x10 5 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 5.4x10 5 C6 glioma cells and analyzed three days later for GFP expression. Titer extrapolated from these experiments were: IX: 2.9xl0 7 cfu/ml, 84X: 2.2x10 9 cfu/ml, 1000X: 2.3x10 10 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
• MIDDLE tumor from a control rat which did not receive vTKIGFP but was treated with GCV (rat was sacrificed on day 43).
• BOTTOM Panel E, F
• GFP expression 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 2x10 4 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 9 ⁇ L of lOOOx vTKiGFP stock in the same stereotactic coordinates as the previous C6/lacZ implant.
• 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.
• 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 stable retroviral producers with the 293 GPG 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 Nhel, 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.
• MFI Mean Fluorescence Intensity
• 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 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.
• Retroviral vectors pseudotyped with the Vesicular Stomatitis Virus G protein (VSVG) may serve as a remedy to this conundrum.
• VSVG Vesicular Stomatitis Virus G protein
• retroviral particles differ 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.
• TK Herpes Simplex Virus Thymidine Kinase
• TKiGFP Green Fluorescence Protein
• the 293GPG packaging cell line was utilized to generate vTKiGFP retroparticles.
• 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 2.3xl0 10 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 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, unpublished), pJ ⁇ bleo 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.
• pMClTK plasmid was rationally 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 bp EGFP cDNA was excised by Eco47-3 and Notl digest of pEGFP-Nl (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 bp Internal Ribosomal Entry Site was excised from the BSICZSVPA plasmid (Ghattas, I.R. 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 Spel-Ascl 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.
• 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 pMClTK plasmid was cut with Bglll-BsaWl to generate a 1207 bp fragment containing the HSVTK cDNA (excluding polyadenylation signal) and was ligated into Bglll-Xmal-cut AP2 to generate pTKiGFP (Fig. IB).
• 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 pro viral genome (Fig. 1C).
• the pMSCV-DHFR (L22Y)/IRES/EGFP vector was derived by incorporating the 654 bp BamHl-Xhol DHFR (L22Y) cDNA from Ha-L22Y into Bglll-Sall 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 G418 (Mediatech, Herndon, VA) and 2 ⁇ g/ml puromycin (Sigma, Oakville, ONT), 1 ⁇ g/ml tetracycline (Fisher Scientific, Nepean, ONT) and 50 units/ml of Pen-Strep ].
• 293GPG cells were transfected with 5 ⁇ g 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 4 ⁇ g Fspl linearized retrovector plasmid and 1 :25 ratio of pJ6 ⁇ Bleo plasmid.
• Transfected cells were subsequently selected in 293GPG media supplemented with 100 ⁇ g/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 utilized to generate high titer virus.
• 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 2xl0 4 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 ⁇ g/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.
• adherent transduced cells were trypsinized and resuspended in RPMI at ⁇ 10 5 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 Nhel 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 GFP cDNA probe, washed and exposed on photographic film.
• 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, Hoffman-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 mitochondrial 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 2xl0 5 cells per well in a 6 well tissue 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
• Titer (cfu/ml) was calculated as [ (% GFP+ cells) X (cell number at initial viral exposure) / (viral volume in ml applied)].
• C6/lacZ glioma cells reproducibly generate lethal intra-cerebral tumors when injected in Sprague-Dawley rats.
• the constitutive ⁇ -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 mg/kg) and xylazine (2 mg/kg).
• C6/lacZ rat glioma cells (2xl0 4 cells in 5 ⁇ l of HBSS) were injected intracranially into the frontal lobe using a Hamilton syringe in a stereo- 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 dural surface.
• vTKiGFP concentrated stock of 2.3xl0 10 cfu/ml
• the AP2 expression vector (Fig. 1A) 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.
• MCS Multiple cloning site
• IRS Internal Ribo- somal Entry Site
• EGFP Enhanced Green Fluorescent Protein
• 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 293 GPG retroviral packaging cell line.
• the 293 GPG packaging cell line was transiently transfected with pTKiGFP (Fig. IB) 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.
• HSV TK expression will lead to the conversion of the prodrug gancyclovir to its cytotoxic metabolite gancyclovir monophosphate.
• Cells which do not express this enzyme are refractory to gancyclovir toxicity.
• vTKiGFP transduced 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.
• vTKiGFP-expressing cell lines were sensitized to gancyclovir. Comparing 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.
• viable vTKiGFP retroparticles could be concentrated by ultracentrifugation as previously described.
• As a first step we transfected 293 GPG cells with pTKiGFP and a zeocin resistance plasmid (pJ ⁇ bleo). A stably transfected, Zeocin-resistant polyclonal producer cell population (293AP3) was generated.
• Supernatant was concentrated 84 fold (20 mis 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.
• C6/lacZ glioma cells will reliably lead to the establishment of intra-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 ⁇ -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.
• Retroparticles which are pseudotyped with the NSNG protein will adopt the wide host range of the vesicular stomatitis virus.
• the putative NSNG receptor on target cells - which is believed to be membrane phospholipid - is ubiquitously found in all eukaryotic cells.
• a retroviral expression vector which incorporates HSVTK and the EGFP reporter cD ⁇ A within a bicistronic transcript (pTKiGFP).
• pTKiGFP bicistronic transcript
• co-dominant expression of the HSVTK cD ⁇ A 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.
• 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).
• vTKiGFP producer cell line (293AP3) derived from the 293GPG packaging cell line (Fig. 5).
• this retroviral producer cell line will express the VSVG envelope protein and generate pseudotyped retroviral particles.
• VSVG-pseudotyped retroparticles incorporating vTKiGFP will lead to high efficiency retrovector transfer to human glioma cell lines in vitro.
• Retroparticle conditioned media collected from 293 GPG cells transiently transfected with pTKiGFP was used to generate vTKiGFP transduced glioma cell lines.
• 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).
• 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 mis of media to a final volume of 0.1 ml (1000X concentration on volume basis). This was accompanied by a 800 fold increase in titer from 2.9 to 2300x107 cfu/ml.
• TK-expressing cells may have acquired resistance to gancyclovir via some other means.
• 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 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.
• 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.
• 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.
• Retrovector encoding for GFP & HSVTK fusion protein serves as a versatile suicide/reporter instrument for cell and gene therapy applications
• 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 ( ⁇ 3xl0e6 retroparticles/ml).
• vGFP/TKfus VSV-G pseudotyped retrovector
• ⁇ 3xl0e6 retroparticles/ml high titer stable retroviral producer generated
• 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.
• 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.
• the pMClTK plasmid containing the HSVTK cDNA was intuitively provided by Gerald Batist (Lady Davis Institute for Medical Research, Montreal, QC).
• the pJ6 ⁇ bleo 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 by Moulai Alaoui-Jamali (Lady Davis Institute for Medical Research, Montreal,
• 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 pMClTK by a HincII-Xmal digest and ligated with 4693 bp fragment generated from a Ecll36II-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.
• pGFP/Tkfus -derived retroviral particles Transduction of target cells with pGFP/Tkfus -derived retroviral particles (vGFP/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 2xl0 5 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.
• 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, Hoffman-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 mitochondrial 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 [OD 570 test - OD 570 empty well]/[OD 570 untreated cells - OD 570 empty well] x 100. All data points were measured in triplicate in at least three separate experiments.
• HSVTK protein 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-OMAT 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.
• adherent transduced cells were trypsinized and resuspended in RPMI at ⁇ 10 5 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 15mins at room temperature, and washed again several times with PBS.
• PBS phosphate buffered saline
• the cover glasses were then removed and mounted on pre- 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
• mice Female Balb/c mice were implanted subcutaneously with lxlO 6 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.
• Retrovector plasmids expressing GFP only (pGFP) and a bicistronic vector encoding for HSVTK and GFP (pTKiGFP) are also depicted in Fig. 9, panel B.
• plasmid vector constructs were utilized to generate retroviral producer cell lines derived from the 293 GPG retroviral packaging cell line as previously described. Following co-transfection of 293 GPG cells with pGFPTKfus and BLEO, and Zeocin drug selection, a mixed population of cells (293 GFPTKfus) was maintained.
• the 293GFPTKfus retroviral producer cells will generate Vesicular Stomatitis Virus-G protein (VSVG) pseudotyped retroparticles following tetracycline withdrawal.
• VSVG Vesicular Stomatitis Virus-G protein
• the retroviral titer generated from the vGFPTKfus producer was 3 x 10 6 particles/ml as measured on the human A549 lung carcinoma cell line.
• Green fluorescence in retrovirally transduced cells Green fluorescence (-530 nm) emitted from cells excited with "blue” (470 nm bandwith) light serves as a reporter of GFP transgene expression in genetically- engineered cells.
• DA3/GFPTKfus cells the mean fluorescence intensity (MFI) of DA3/GFPTKfus cells is 100 fold greater than control and 30 fold greater than DA3/TKiGFP cells.
• MFI mean fluorescence intensity
• 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). HSVTK expression and sensitization to gancyclovir
• DA3/GFPTKfus 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.
• DA3/TKiGFP express the expected 50 kd native HSVTK protein
• DA3/GFPTKfus cells bear a 75kd immunoreactive protein whose size is consistent with the predicted mass of GFP (25kd) + HSVTK (50 kd) fusion product.
• equivalent amounts of anti- HSVTK immunoreactive protein is generated by both vGFPTKfus and vTKiGFP transduced cells.
• 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.
• both DA3/TKiGFP and D A3 GFPTKfus cells are significantly sensitized to gancyclovir with an IC50 -1000 fold (Fig. 14). Doubling time (-24 hours) for all cell lines are similar.
• DA3 cells are tumorigenic in Balb/c mice.
• 7/9 rodents implanted with D A3 GFPTKfus tumors were rendered tumor-free by gancyclovir treatment.
• Mice in which D A3 GFPTKfus 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 D A3 GFP implants and mock-treated DA3GFPTKfus implants. 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.
• DLI donor lymphocyte infusion
• NGFR Nerve Growth Factor Receptor reporter
• HSVTK/Neomycin phosphotransferase II fusion gene was retrovirally introduced in donor lymphocytes, selected in G418, 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.
• 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 (-8-1 Okb) gene packing space.
• 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.
• GFP Green Fluorescent Protein
• a GFP & HSVTK fusion protein as a bifunctional reporter/marker and suicide transgene as part of a therapeutic retrovector.
• 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 inco ⁇ orated in a retroviral expression vector and VSVG-pseudotyped retroparticles were generated as a gene transfer vehicle.
• 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).
• 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.
• 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.
• 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.
• GFPTKfus inco ⁇ oration of GFPTKfus in multigenic vector constructs where other gene products of interest are included 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.

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