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846. Defining the Efficacy of Pharmacologically Regulated In Vivo Selection in Large Animals at the Molecular Level
GENE REGULATION: CELL THERAPIES AND ANIMAL MODELS GENE REGULATION: CELL THERAPIES AND ANIMAL MODELS
846. Defining the Efficacy of Pharmacologically Regulated In Vivo Selection in Large Animals at the Molecular Level
845. Longterm In Vivo Selection Using an MplBased Cell Growth Switch
Kerstin Schwarzwaelder,1 Manfred Schmidt,1,2 Tobias Neff,3 Anthony Blau,4 Christoph Peters,1 Hans-Peter Kiem,3 Christof von Kalle.5 1 Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; 2Department I of Internal Medicine, University of Freiburg, Freiburg, Germany; 3Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 4 Department of Medicine, University of Washington, Seattle, WA; 5 Division of Experimental Hematology, Cincinnati Childrens Research Foundation, Cincinnati, OH.
Michael A. Weinreich,1 Masayoshi Masuko,1 Sylvia Chien,1 James Yan,1 C. A. Blau.1 1 Hematology, University of Washington, Seattle, WA. From our previous work we have reported the use of a conditionally dimerizable version of the thrombopoietin receptor (F36Vmpl) to selectively expand transduced cells in vivo. In both murine and canine animal models the administration of the chemical inducer of dimerization (CID) caused a reversible expansion of transduced cells. In these studies we again turned to the mouse model to ask questions of the limitations and variability of CID response. To study the long term effects of CID treatment mice were transplanted with bone marrow cells that were transduced with a vector encoding F36Vmpl and a GFP reporter gene. The mice were given three three-day courses of CID at four-week intervals. Two weeks following the last course of CID mice were killed and bone marrow from each mouse was transplanted into a lethally irradiated secondary host. Three months after transplant these secondary recipients were given the same CID regiment. We monitored GFP expression throughout the experiment. Also we used DNA from bone marrow and spleen of primary and secondary transplant recipients to perform southern blots to look at clonality. In the primary recipients 10 of 11 mice responded to the three courses of CID in both red cells and platelets. In the responding mice GFP marked erythrocytes increased from 535 percent at baseline to greater than 80 percent after CID. The outlying mouse had a response to the initial treatment but later its GFP marking fell to undetectable levels. The secondary recipient of this mouse also did not have GFP marking and neither recipient showed any bands by southern. This shows that in some cases CID may cause the exhaustion of a stem cell or long-lived progenitor. In contrast we saw in three mice similar CID responses in both the primary and secondary mice. In these mice the same bands by southern were also detected in primary and secondary recipients following CID treatment. In these cases it is likely that the same stem cells or stem cell with multiple integration sites is responsible for the response in both recipients. These are examples of stem cells that have responded to CID multiple times and reconstituted a secondary recipient without exhausting. The remaining mice had lesser or no responses in the secondary recipients. These could be attributable to either inability for the CID responsive stem cells to engraft or exhaustion of these cells. When there was a loss of GFP marking it occurred in both the red cells and the platelets but not in the white cells. This could be explained by the long life span of some T and B cells or CID may be causing the differentiation of a common red cell/platelet progenitor that was not regenerated. To investigate the reasons for these variable responses we analyzed the correlation between GFP expression and response to CID. In most cases the clone with the highest GFP expression by flow cytometry had the best response to CID. However in a few cases a lower expressing clone was the best responder demonstrating that vector expression level alone is not the only factor that determines CID responsiveness. We are performing studies to determine whether site of integration affects the response to CID.
S326
As described previously, CD34+ cells from two dogs were engineered with a retrovirus vector (MSCV) to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Before transplantation, both dogs received myeloablative total body irradiation. Activation of the receptor through administration of a chemical inducer of dimerization, AP20187 (CID), produced reversible, drug dependent surges of genetically modified red cells, white cells and platelets in both animals, with minimal side effects (Neff et al., 2002). Until now both dogs have received three CIDtreatment cycles. In order to determine their clonal derivation and the extent of specific clones contributing to the repopulation, a highly sensitive linear amplification mediated PCR (LAM-PCR) was carried out. This method is designed to detect retrovirus integration sites at genomic level unique for each clone. Thus, it is possible to track the fate of randomly selected single clones as well as their descendents. DNA of BM, PBL and LN cells and of CD3+, DM5+-, CD21+-, and CD14+-cells before, during and between CID treatments was analyzed. Through sequencing of the retrovirus integration sites it was possible to generate primers for individual genomic flanks. With primers for the genomic flank of a integration site and primers for the LTR of the vector a specific tracking for one specific clone in samples from different time points and lineages via nested PCR. The integration site also allowed to synthesize an internal standard (IS), whose PCR-product is 26 bps shorter than the PCR-product of the wild type DNA. Quantitative-competitive PCR with the sample-DNA and the IS with a specific copy-number allowed to analyze the contribution of single clones to hematopoiesis. Clonality analysis showed that the repopulation in both dogs remained oligoclonal after one year with three CID selection cycles. Individual clones from both animals (six and three, respectively) were analyzed by quantitative-competitive PCR. Samples of the first dog were analyzed semi-quantitatively for three clones. Two out of the three clones were detected in all time points and samples analyzed, demonstrating stable long term activity and pluripotency. The third clone could only be detected in sorted B-cells. These data demonstrate pluripotency of the genetically modified cells. The sustained contribution to the hematopoietic repopulation of single clones for more than one year and three successive CID-treatment cycles show that positive selection with a dimerizing drug is highly efficient at the clonal progenitor cell level and a promising approach in gene and cell therapy.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
846. Defining the Efficacy of Pharmacologically Regulated In Vivo Selection in Large Animals at the Molecular Level
845. Longterm In Vivo Selection Using an MplBased Cell Growth Switch
Kerstin Schwarzwaelder,1 Manfred Schmidt,1,2 Tobias Neff,3 Anthony Blau,4 Christoph Peters,1 Hans-Peter Kiem,3 Christof von Kalle.5 1 Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; 2Department I of Internal Medicine, University of Freiburg, Freiburg, Germany; 3Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 4 Department of Medicine, University of Washington, Seattle, WA; 5 Division of Experimental Hematology, Cincinnati Childrens Research Foundation, Cincinnati, OH.
Michael A. Weinreich,1 Masayoshi Masuko,1 Sylvia Chien,1 James Yan,1 C. A. Blau.1 1 Hematology, University of Washington, Seattle, WA. From our previous work we have reported the use of a conditionally dimerizable version of the thrombopoietin receptor (F36Vmpl) to selectively expand transduced cells in vivo. In both murine and canine animal models the administration of the chemical inducer of dimerization (CID) caused a reversible expansion of transduced cells. In these studies we again turned to the mouse model to ask questions of the limitations and variability of CID response. To study the long term effects of CID treatment mice were transplanted with bone marrow cells that were transduced with a vector encoding F36Vmpl and a GFP reporter gene. The mice were given three three-day courses of CID at four-week intervals. Two weeks following the last course of CID mice were killed and bone marrow from each mouse was transplanted into a lethally irradiated secondary host. Three months after transplant these secondary recipients were given the same CID regiment. We monitored GFP expression throughout the experiment. Also we used DNA from bone marrow and spleen of primary and secondary transplant recipients to perform southern blots to look at clonality. In the primary recipients 10 of 11 mice responded to the three courses of CID in both red cells and platelets. In the responding mice GFP marked erythrocytes increased from 535 percent at baseline to greater than 80 percent after CID. The outlying mouse had a response to the initial treatment but later its GFP marking fell to undetectable levels. The secondary recipient of this mouse also did not have GFP marking and neither recipient showed any bands by southern. This shows that in some cases CID may cause the exhaustion of a stem cell or long-lived progenitor. In contrast we saw in three mice similar CID responses in both the primary and secondary mice. In these mice the same bands by southern were also detected in primary and secondary recipients following CID treatment. In these cases it is likely that the same stem cells or stem cell with multiple integration sites is responsible for the response in both recipients. These are examples of stem cells that have responded to CID multiple times and reconstituted a secondary recipient without exhausting. The remaining mice had lesser or no responses in the secondary recipients. These could be attributable to either inability for the CID responsive stem cells to engraft or exhaustion of these cells. When there was a loss of GFP marking it occurred in both the red cells and the platelets but not in the white cells. This could be explained by the long life span of some T and B cells or CID may be causing the differentiation of a common red cell/platelet progenitor that was not regenerated. To investigate the reasons for these variable responses we analyzed the correlation between GFP expression and response to CID. In most cases the clone with the highest GFP expression by flow cytometry had the best response to CID. However in a few cases a lower expressing clone was the best responder demonstrating that vector expression level alone is not the only factor that determines CID responsiveness. We are performing studies to determine whether site of integration affects the response to CID.
S326
As described previously, CD34+ cells from two dogs were engineered with a retrovirus vector (MSCV) to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Before transplantation, both dogs received myeloablative total body irradiation. Activation of the receptor through administration of a chemical inducer of dimerization, AP20187 (CID), produced reversible, drug dependent surges of genetically modified red cells, white cells and platelets in both animals, with minimal side effects (Neff et al., 2002). Until now both dogs have received three CIDtreatment cycles. In order to determine their clonal derivation and the extent of specific clones contributing to the repopulation, a highly sensitive linear amplification mediated PCR (LAM-PCR) was carried out. This method is designed to detect retrovirus integration sites at genomic level unique for each clone. Thus, it is possible to track the fate of randomly selected single clones as well as their descendents. DNA of BM, PBL and LN cells and of CD3+, DM5+-, CD21+-, and CD14+-cells before, during and between CID treatments was analyzed. Through sequencing of the retrovirus integration sites it was possible to generate primers for individual genomic flanks. With primers for the genomic flank of a integration site and primers for the LTR of the vector a specific tracking for one specific clone in samples from different time points and lineages via nested PCR. The integration site also allowed to synthesize an internal standard (IS), whose PCR-product is 26 bps shorter than the PCR-product of the wild type DNA. Quantitative-competitive PCR with the sample-DNA and the IS with a specific copy-number allowed to analyze the contribution of single clones to hematopoiesis. Clonality analysis showed that the repopulation in both dogs remained oligoclonal after one year with three CID selection cycles. Individual clones from both animals (six and three, respectively) were analyzed by quantitative-competitive PCR. Samples of the first dog were analyzed semi-quantitatively for three clones. Two out of the three clones were detected in all time points and samples analyzed, demonstrating stable long term activity and pluripotency. The third clone could only be detected in sorted B-cells. These data demonstrate pluripotency of the genetically modified cells. The sustained contribution to the hematopoietic repopulation of single clones for more than one year and three successive CID-treatment cycles show that positive selection with a dimerizing drug is highly efficient at the clonal progenitor cell level and a promising approach in gene and cell therapy.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy