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1126. Optical Imaging of Tissue-Specific Expression of Firefly Luciferase Reporter Gene in Living Mice Using a Lentiviral Vector Carrying the Two-Step Transcriptional Amplification (TSTA) System
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN inhibiting the VEGF pathway using a lentivirus vector encoding a soluble VEGF receptor (Sin-sFLK-1). We found that in co-culture assays, the combination of Sin-End and Sin-sFLK-1 significantly inhibited the growth of HUVEC cells compared to treatment with either vector alone. These results suggest that combination antiangiogenic therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy compared to single-agent regimens.
1126. Optical Imaging of Tissue-Specific Expression of Firefly Luciferase Reporter Gene in Living Mice Using a Lentiviral Vector Carrying the Two-Step Transcriptional Amplification (TSTA) System Meera Iyer, Bergara Felix, Xiaoman Lewis, Abhijit De, Michael Carey, Lily Wu, Sanjiv S. Gambhir. Objective: The aim of this study was to non-invasively monitor long-term tissue-specific expression of firefly luciferase (fl) reporter gene in living animals using a lentiviral based gene transfer vector carrying a Two-Step Transcriptional Amplification (TSTA) system (Iyer et al, PNAS, 2001). Methods: The TSTA construct, PSEBCVP2-G5-fl was cloned into the third generation self-inactivating (SIN) lentivector, PTK 134. The SIN lentiviral vectors (LV-TSTA) were prepared by cotransfection into 293T cells. Virus from culture supernatant was concentrated by ultracentrifugation at 50,000g for 2 hours. For cell culture experiments, LNCaP cells were transiently transduced with LV-TSTA at 37 °C for 48h.The virus was also used to infect nonprostate cell lines (C6, HeLa and N2a). All cells were harvested and assayed for FL activity. For in vivo studies, the concentrated virus was injected directly into the prostate of male, nude mice (20 μl). Optical imaging was performed using a cooled Charge Coupled Device (CCD) camera using D-Luciferin as the substrate (3 mg per animal injected i.p.). The mice (n=3) were imaged at intervals of 3-4 days for 23 days and thereafter, once every week for 58 days. In a second set of mice, LV-TSTA was injected in male, nude mice carrying LAPC-4 xenografts (5-6 mm), and the animals were imaged as described earlier. The resulting bioluminescent signal was expressed as photons/sec/cm²/steridian (sr). Results: In cell culture, LV-TSTA demonstrates significantly high levels of FL activity in the presence of androgen (5-fold). Basal levels of FL activities observed in all non-prostate cell lines indicates that the system is highly cell-type specific. CCD camera imaging of mice injected with the lentivirus in the prostate reveal enhanced fl gene expression primarily in the prostate on days 7, 14 and 30 (2 x 105±1.4x104 2.1x105±1.5x104 and 3.1x105±3.9x104 p/sec/cm²/sr respectively) (Fig.1). Repetitive imaging shows persistence of gene expression in the prostate with time. Direct injection of LV-TSTA into LAPC-4 xenografts demonstrates enhanced levels of fl gene expression in the tumor for up to 18 days (4 x 105±1.0x104 p/sec/ cm²/sr). Conclusion: This study demonstrates the ability to monitor prostate-specific gene expression in living mice using a lentiviral based gene transfer vector carrying the TSTA system. These results support the use of LV-TSTA for efficient and long-term monitoring of transgene expression in prostate cancer gene therapy applications.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN 1127. Targeting Transgene Expression in Canine Megakaryocytes David A. Wilcox.1,2,3 1 Department of Pediatrics, Medical College of Wisconsin; 2 Children’s Hospital of Wisconsin; 3Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI. Megakaryocyte-specific expression of the platelet adhesion receptor, integrin αIIbb3, is mediated by regulatory elements of the αIIb gene promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop a large animal model for lineage-specific gene therapy of disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a selfinactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human αIIb gene promoter (nucleotides -889 to +35). This construct, αIIb-GFPWPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to track transgene expression in the progeny of transduced-cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, EF-1, which drives constitutively-active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from G-CSF mobilized PBC collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and Peg-rhMGDF), transduced for 2 days on plates coated with RetroNectin and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP were megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP were megakaryocytes when using the αIIb-promoter. This outcome with canine cells shows a striking correlation to results obtained using the human αIIb promoter to selectively target transgene expression of ecoli. β -galactosidase in human megakaryocyte progeny of retrovirus-transduced human CD34+ PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1promoter’s ability to drive lineage-specific transgene expression. This work suggests that there is potential feasibility for using the human αIIb gene promoter in canine models to develop methods for gene therapy for disorders affecting platelets.
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1126. Optical Imaging of Tissue-Specific Expression of Firefly Luciferase Reporter Gene in Living Mice Using a Lentiviral Vector Carrying the Two-Step Transcriptional Amplification (TSTA) System Meera Iyer, Bergara Felix, Xiaoman Lewis, Abhijit De, Michael Carey, Lily Wu, Sanjiv S. Gambhir. Objective: The aim of this study was to non-invasively monitor long-term tissue-specific expression of firefly luciferase (fl) reporter gene in living animals using a lentiviral based gene transfer vector carrying a Two-Step Transcriptional Amplification (TSTA) system (Iyer et al, PNAS, 2001). Methods: The TSTA construct, PSEBCVP2-G5-fl was cloned into the third generation self-inactivating (SIN) lentivector, PTK 134. The SIN lentiviral vectors (LV-TSTA) were prepared by cotransfection into 293T cells. Virus from culture supernatant was concentrated by ultracentrifugation at 50,000g for 2 hours. For cell culture experiments, LNCaP cells were transiently transduced with LV-TSTA at 37 °C for 48h.The virus was also used to infect nonprostate cell lines (C6, HeLa and N2a). All cells were harvested and assayed for FL activity. For in vivo studies, the concentrated virus was injected directly into the prostate of male, nude mice (20 μl). Optical imaging was performed using a cooled Charge Coupled Device (CCD) camera using D-Luciferin as the substrate (3 mg per animal injected i.p.). The mice (n=3) were imaged at intervals of 3-4 days for 23 days and thereafter, once every week for 58 days. In a second set of mice, LV-TSTA was injected in male, nude mice carrying LAPC-4 xenografts (5-6 mm), and the animals were imaged as described earlier. The resulting bioluminescent signal was expressed as photons/sec/cm²/steridian (sr). Results: In cell culture, LV-TSTA demonstrates significantly high levels of FL activity in the presence of androgen (5-fold). Basal levels of FL activities observed in all non-prostate cell lines indicates that the system is highly cell-type specific. CCD camera imaging of mice injected with the lentivirus in the prostate reveal enhanced fl gene expression primarily in the prostate on days 7, 14 and 30 (2 x 105±1.4x104 2.1x105±1.5x104 and 3.1x105±3.9x104 p/sec/cm²/sr respectively) (Fig.1). Repetitive imaging shows persistence of gene expression in the prostate with time. Direct injection of LV-TSTA into LAPC-4 xenografts demonstrates enhanced levels of fl gene expression in the tumor for up to 18 days (4 x 105±1.0x104 p/sec/ cm²/sr). Conclusion: This study demonstrates the ability to monitor prostate-specific gene expression in living mice using a lentiviral based gene transfer vector carrying the TSTA system. These results support the use of LV-TSTA for efficient and long-term monitoring of transgene expression in prostate cancer gene therapy applications.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN 1127. Targeting Transgene Expression in Canine Megakaryocytes David A. Wilcox.1,2,3 1 Department of Pediatrics, Medical College of Wisconsin; 2 Children’s Hospital of Wisconsin; 3Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI. Megakaryocyte-specific expression of the platelet adhesion receptor, integrin αIIbb3, is mediated by regulatory elements of the αIIb gene promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop a large animal model for lineage-specific gene therapy of disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a selfinactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human αIIb gene promoter (nucleotides -889 to +35). This construct, αIIb-GFPWPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to track transgene expression in the progeny of transduced-cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, EF-1, which drives constitutively-active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from G-CSF mobilized PBC collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and Peg-rhMGDF), transduced for 2 days on plates coated with RetroNectin and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP were megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP were megakaryocytes when using the αIIb-promoter. This outcome with canine cells shows a striking correlation to results obtained using the human αIIb promoter to selectively target transgene expression of ecoli. β -galactosidase in human megakaryocyte progeny of retrovirus-transduced human CD34+ PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1promoter’s ability to drive lineage-specific transgene expression. This work suggests that there is potential feasibility for using the human αIIb gene promoter in canine models to develop methods for gene therapy for disorders affecting platelets.
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