- Email: [email protected]
292. Application of a Human Multidrug Transporter (ABCG2) Variant as Selectable Marker in Gene Transfer to Heamatopoietic Progenitor Cells
NOVEL APPROACHES FOR HEMATOPOIETIC GENE THERAPY transduced than G-CSF-alone or G-CSF + Flt3L mobilized cells (Hematti et al, Blood, 2002 online) and our results showing more efficient mobilization using G-CSF + SCF, we conclude that this combination may be optimal in the context of haemopoietic stem cell gene transfer protocols.
292. Application of a Human Multidrug Transporter (ABCG2) Variant as Selectable Marker in Gene Transfer to Heamatopoietic Progenitor Cells Olga Ujhelly,1 Csilla Özvegy,2 György Várady,1 Balázs Sarkadi,1 Katalin Német.1 1 National Medical Center, Institute of Haematology, Budapest, Hungary; 2Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary. Stem cell based gene therapy is often unsuccessful because of the relatively low number of genetically modified cells with repopulating capabilities. In order to provide a selective advantage for the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells as a selectable marker. This protein is active as a homodimer, and its relatively small cDNA is an advantage in gene therapy applications. In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate-specificity was co-expressed with a therapeutic gene by using a bicistronic vector and an efficient retroviral transduction protocol. Expression of the gp91phox protein in human gp91phoxknock-out hematopoietic progenitor cells corrected the loss-offunction mutation responsible for human chronic granulomatous disease, while the mutant ABCG2 protein selectively protected the transduced cells against clinically applicable cytotoxic agents. Overexpression of ABCG2 did not affect hematopoietic cell maturation or the restoration of granulocyte function by gp91phox. We suggest that the mutant ABCG2 protein is an ideal candidate for human stem cell protection and for use as a selectable marker in gene therapy.
293. Protection of the Hematopoietic Stem Cell Compartment by Retroviral Gene Transfer of Drug Resistance Genes Thomas D. Southgate,1 Lorna B. Woolford,1 Claire F. Stevens,1 Leslie J. Fairbairn.1 1 Gene Therapy, Paterson Institute for Cancer Research, Manchester, United Kingdom. Even after high-dose chemotherapy followed by autologous transplantation with peripheral blood-derived progenitor cells, relapse of metastatic tumours still constitutes an important problem. Elimination of residual malignant cells will require the practice of a consolidation treatment but with many chemotherapeutic agents conferring acute bone marrow toxicity there is a need to minimise side effects whilst allowing effective tumour management. Retrovirally transduced hematopoietic stem cells overexpressing the ATP-dependent drug efflux pumps, P-glycoprotein or multidrug resistance associated protein (MRP) may confer chemotherapeutic protection. This could avoid the associated acute bone marrow toxicity in these patients, and in doing so widen the therapeutic window for an earlier onset of post-transplantation chemotherapy, whilst eliminating the risk of leukopenia, anemia and/or thrombocytopenia which so far jeopardises treatment and can compromise the patient’s quality of life. Here we present data from a mouse model, that mimics clinically achievable gene transfer rates, to demonstrate the efficacy of protection of a minor proportion of the repopulating cells with
S116
ATP-dependent efflux pumps. We have utilised novel retroviral vectors expressing P-glycoprotein or MRP in the SFβ91 vector to facilitate a direct in vivo competition assay of these two transgenes. Transplantation of bone marrow expressing both MRP-IRESGFP and MDR-IRES-YFP within the same animal allows us to determine the relative protection conferred to these two hematopoietic populations in response to etoposide and paciltaxel both in the presence and the absence of the ATP-dependent drug efflux pump inhibitor verapamil. Here we compare this protection to determine whether P-glycoprotein or MRP confers greater chemoprotection against clinically approved chemotherapeutic agents.
CANCER IMMUNOTHERAPY 294. Regression of Murine Bladder Tumors by AdCD40L Therapy Angelica S. I. Loskog,1 Thomas H. Totterman.1 Oncology, Radiology and Clinical Immunology, Clinical Immunology Division, Uppsala, Sweden. 1
Aim In previous studies we have shown that s.c.vaccinations with CD40L expressing MB49 tumor cells protect mice from challenge with parental tumor (J.Urol.166:1093, 2001). In this preclinical study we investigated the immunological mechanisms and evaluated the capacity of treating established tumors with CD40L immunogene therapy. Background Many tumors exhibit immune escape properties that enable their survival. Murine bladder cancer cells (MB49) induce local IL10 production. IL10 is known to drive the potent anti-tumor Th1 response towards a less favorable Th2 response. We have studied the immunological effects (cytokine levels) of introducing the CD40L, a potent dendritic cell activation molecule, into the tumor micro milieu by use of ELISA and quantitative PCR. Further, we studied induction of MB49 specific CTLs by 51Cr release assays. Finally, human biopsy material was screened for immune escape properties by PCR. Results AdCD40L injections induced regression of MB49 tumors and further, stabilized and suppressed growth of large solid tumors. Fibroblasts or DCs can also be used as carrier cells of the CD40L gene in order to suppress tumor growth. The cytokine pattern of untreated tumors were of Th2 type with high IL10 levels. Expression of CD40L in the tumor environment suppressed IL10 production and favored Th1 cytokine production. Further, AdCD40L immunogene therapy stimulated induction of MB49 specific CTLs. MB49 cells and human biopsies exhibited immune escape mechanisms such as deteriorated FAS expression and high levels of the granzyme B inhibitor SPI-6/PI-9 or apoptosis inhibitor cFLIP. Conclusion In conclusion, despite powerful escape mechanisms such as IL10 induction, poor FAS and high SPI-6 expression, AdCD40L immunogene therapy can effect the regression of s.c. tumors. This study further argues for using CD40L immunogene therapy in the treatment of bladder carcinomas.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
292. Application of a Human Multidrug Transporter (ABCG2) Variant as Selectable Marker in Gene Transfer to Heamatopoietic Progenitor Cells Olga Ujhelly,1 Csilla Özvegy,2 György Várady,1 Balázs Sarkadi,1 Katalin Német.1 1 National Medical Center, Institute of Haematology, Budapest, Hungary; 2Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary. Stem cell based gene therapy is often unsuccessful because of the relatively low number of genetically modified cells with repopulating capabilities. In order to provide a selective advantage for the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells as a selectable marker. This protein is active as a homodimer, and its relatively small cDNA is an advantage in gene therapy applications. In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate-specificity was co-expressed with a therapeutic gene by using a bicistronic vector and an efficient retroviral transduction protocol. Expression of the gp91phox protein in human gp91phoxknock-out hematopoietic progenitor cells corrected the loss-offunction mutation responsible for human chronic granulomatous disease, while the mutant ABCG2 protein selectively protected the transduced cells against clinically applicable cytotoxic agents. Overexpression of ABCG2 did not affect hematopoietic cell maturation or the restoration of granulocyte function by gp91phox. We suggest that the mutant ABCG2 protein is an ideal candidate for human stem cell protection and for use as a selectable marker in gene therapy.
293. Protection of the Hematopoietic Stem Cell Compartment by Retroviral Gene Transfer of Drug Resistance Genes Thomas D. Southgate,1 Lorna B. Woolford,1 Claire F. Stevens,1 Leslie J. Fairbairn.1 1 Gene Therapy, Paterson Institute for Cancer Research, Manchester, United Kingdom. Even after high-dose chemotherapy followed by autologous transplantation with peripheral blood-derived progenitor cells, relapse of metastatic tumours still constitutes an important problem. Elimination of residual malignant cells will require the practice of a consolidation treatment but with many chemotherapeutic agents conferring acute bone marrow toxicity there is a need to minimise side effects whilst allowing effective tumour management. Retrovirally transduced hematopoietic stem cells overexpressing the ATP-dependent drug efflux pumps, P-glycoprotein or multidrug resistance associated protein (MRP) may confer chemotherapeutic protection. This could avoid the associated acute bone marrow toxicity in these patients, and in doing so widen the therapeutic window for an earlier onset of post-transplantation chemotherapy, whilst eliminating the risk of leukopenia, anemia and/or thrombocytopenia which so far jeopardises treatment and can compromise the patient’s quality of life. Here we present data from a mouse model, that mimics clinically achievable gene transfer rates, to demonstrate the efficacy of protection of a minor proportion of the repopulating cells with
S116
ATP-dependent efflux pumps. We have utilised novel retroviral vectors expressing P-glycoprotein or MRP in the SFβ91 vector to facilitate a direct in vivo competition assay of these two transgenes. Transplantation of bone marrow expressing both MRP-IRESGFP and MDR-IRES-YFP within the same animal allows us to determine the relative protection conferred to these two hematopoietic populations in response to etoposide and paciltaxel both in the presence and the absence of the ATP-dependent drug efflux pump inhibitor verapamil. Here we compare this protection to determine whether P-glycoprotein or MRP confers greater chemoprotection against clinically approved chemotherapeutic agents.
CANCER IMMUNOTHERAPY 294. Regression of Murine Bladder Tumors by AdCD40L Therapy Angelica S. I. Loskog,1 Thomas H. Totterman.1 Oncology, Radiology and Clinical Immunology, Clinical Immunology Division, Uppsala, Sweden. 1
Aim In previous studies we have shown that s.c.vaccinations with CD40L expressing MB49 tumor cells protect mice from challenge with parental tumor (J.Urol.166:1093, 2001). In this preclinical study we investigated the immunological mechanisms and evaluated the capacity of treating established tumors with CD40L immunogene therapy. Background Many tumors exhibit immune escape properties that enable their survival. Murine bladder cancer cells (MB49) induce local IL10 production. IL10 is known to drive the potent anti-tumor Th1 response towards a less favorable Th2 response. We have studied the immunological effects (cytokine levels) of introducing the CD40L, a potent dendritic cell activation molecule, into the tumor micro milieu by use of ELISA and quantitative PCR. Further, we studied induction of MB49 specific CTLs by 51Cr release assays. Finally, human biopsy material was screened for immune escape properties by PCR. Results AdCD40L injections induced regression of MB49 tumors and further, stabilized and suppressed growth of large solid tumors. Fibroblasts or DCs can also be used as carrier cells of the CD40L gene in order to suppress tumor growth. The cytokine pattern of untreated tumors were of Th2 type with high IL10 levels. Expression of CD40L in the tumor environment suppressed IL10 production and favored Th1 cytokine production. Further, AdCD40L immunogene therapy stimulated induction of MB49 specific CTLs. MB49 cells and human biopsies exhibited immune escape mechanisms such as deteriorated FAS expression and high levels of the granzyme B inhibitor SPI-6/PI-9 or apoptosis inhibitor cFLIP. Conclusion In conclusion, despite powerful escape mechanisms such as IL10 induction, poor FAS and high SPI-6 expression, AdCD40L immunogene therapy can effect the regression of s.c. tumors. This study further argues for using CD40L immunogene therapy in the treatment of bladder carcinomas.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy