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58. Effects of Culture Conditions on Gene Expression Profile of Human Bone Marrow CD34+ Cells
Hematologic - Transduction, Engraftment and Transgene Expression Hematologic – Transduction, Engraftment and Transgene Expression 57. High Level Long-Term Marking in Pigtailed Macaques Using Lentiviral Vectors without Progression to Clonal Dominance
Grant D. Trobridge,1,2 Brian C. Beard,1 Philip Olsen,1 Christina Gooch,1 Punam Malik,3 Hans-Peter Kiem.1,2 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington, Seattle, WA; 3Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
Lentiviral vectors are attractive for hematopoietic stem cell gene therapy because they do not require mitosis for transduction, and can efficiently transduce hematopoietic repopulating cells. Lentiviral vectors also do not integrate as frequently near promoter regions as gammaretroviral vectors, and self-inactivating lentiviral vectors pseudotyped with VSV-G can be produced at high-titer and concentrated by centrifugation. Experiments to evaluate HIVderived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies, due in part to host cell restriction by Trim5α. We have obtained efficient transduction of pigtailed macaques (Macaca nemestrina) using lentiviral vectors pseudotyped with VSV-G and report here long term marking in the absence of leukemia or progression towards clonal dominance. Pigtailed macaque CD34+ cells were transduced at similar frequencies to human CD34+ cells and at much higher frequencies compared to baboons (Papio cynocephalus anubis) or rhesus macaques (Macaca mulatta). In three macaques transduced using relatively low MOIs (5-10) we have observed high level marking with HIV-derived lentiviral vectors. In two monkeys that contain an EGFP reporter gene stable marking has been obtained well over one and two years respectively. In these studies a rapid 2-day transduction protocol was used to preserve the engraftment potential of the ex vivo cultured cells. In the first animal marking is stable with over 27% of granulocytes and 12% of lymphocytes expressing EGFP at 463 days post-transplantation. The second animal has 35% EGFP-positive granulocytes and 35% EGFP-positive lymphocytes 834 days after transplantation. LAM-PCR analysis of integration sites has demonstrated that repopulating cells are highly polyclonal and transgene expression has been detected in all hematopoietic lineages (B-cells, T-cells, granulocytes, red blood cells as well as platelets). These data show that lentiviral vectors are highly effective for hematopoietic stem cell gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells by using a rapid transduction protocol is critical. Importantly, these data also show that the pigtailed macaque is an excellent model to evaluate the efficacy and safety of HIV-derived lentiviral vectors proposed for clinical gene therapy protocols.
58. Effects of Culture Conditions on Gene Expression Profile of Human Bone Marrow CD34+ Cells
Amrita Ghosh,1,2 Chenwei Wang,1 Abdel G. Elkahloun,1 Fabio Candotti.1 1 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; 2Univ of Medicine & Dentistry of NJ, Newark, NJ.
Gene transfer into hematopoietic stem cells (HSCs) using gammaretrovirus-based vectors has proven beneficial as a therapeutic approach for immunodeficiencies. Such vectors have been shown to target transcriptional start sites of actively transcribing genes, indicating that the gene expression profile of target cells can be Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
predictive of the pattern of vector integrations. Therefore, different cytokines and growth factor conditions used for gene transfer into HSCs can induce expression of different genes that may be targets for integration. A recent analysis of the integration patterns in X-SCID patients treated under two trials in France and UK showed differences in the frequency of common integration sites that may have resulted from distinct gene expression profiles induced by the seemingly minor differences of in vitro culture conditions (60 U/ml IL3 and 4% FCS vs. 20 U/ml IL3 and 1% HSA, respectively). To explore this issue, we cultured human bone marrow CD34+ cells from three donors under stimulation conditions that reproduced the French (S1) and UK (S2) protocols. Cells were harvested after 48, 72, and 96 hours of stimulation, representing times of transductions performed in the trials. Total RNA was extracted, amplified, and hybridized to Affymetrix HG U133 Plus 2.0 microarrays and robust multi-array average was performed. “Activated Genes” were defined as those with ≥1.5-fold upregulated expression in each condition compared to unstimulated cells by pairwise t-test (p≤0.05). Lists of genes activated in all three time points for either S1 or S2 were generated and named “S1-All” and “S2-All”, respectively. Bioinformatic comparison of S1-All and S2-All showed significant differences in gene ontologies and functional pathways, which led us to analyze the characteristics of genes that were uniquely activated (Unique Genes) at each time point of either stimulation protocol (S1-48h, S1-72h, S1-96h and S2-48h, S2-72h, S2-96h). Unsupervised hierarchical clustering of functional annotation categories showed two main clusters, one corresponding to S1-48h, S2-48h, S2-72h, and S2-96h, the other corresponding to S1-72h and S1-96h. Pathway analysis of unique gene lists showed that enrichment of genes involved in differentiation increased in both stimulation protocols as time progressed. Furthermore, networked genes associated with cell cycle and DNA replication, recombination, and repair showed functional enrichment in S1-48h and S2-96h. Increased enrichment of annotated cancer genes was also found in S1-48h and S2-96h. These results indicate that genes uniquely upregulated at early time points by the S1 conditions share functional characteristics with genes induced at later time points by the S2 culture regimen. This analysis suggests that cells stimulated with 60 U/ml of IL3 and FCS for 48h differ from cells cultured with 20 U/ml of IL3 and HSA but achieve similar transcriptional activity only after 96h. If this is true, even small differences in culture conditions can result in differences in transcription profiles that may lead to differences in integration profiles
59. Promotor-Deprived Self-Inactivating γ-Retroviral Vectors Allow Marking of Serially Reconstituting Hematopoietic Stem Cells
Kerstin Cornils,1 Claudia Lange,2 Axel Schambach,3 Michael Lioznov,2 Christopher Baum,3,4 Boris Fehse.1 1 Experimental Pediatric Oncology and Hematology, Pediatric Clinic III, University Hospital of the J.W. Goethe-University, Frankfurt, Germany; 2Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany; 4Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Based on their ability to stably integrate in their host cell’s genome, γ-retroviral vectors have been successfully used for gene marking as well as gene therapy, mainly in the hematopoietic system. However, recently retroviral vectors were shown to influence growth and/ or survival characteristics of hematopoietic stem cells (HSC) by insertional mutagenesis. This type of genotoxicity may result in clonal dominance, but also malignant transformation of affected clones. Using our established serial bone marrow transplantation (BMT) model we investigated whether γ-retroviral self-inactivating vectors deprived of an internal promotor (pd-SIN vectors) allow stable S23
Grant D. Trobridge,1,2 Brian C. Beard,1 Philip Olsen,1 Christina Gooch,1 Punam Malik,3 Hans-Peter Kiem.1,2 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington, Seattle, WA; 3Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.
Lentiviral vectors are attractive for hematopoietic stem cell gene therapy because they do not require mitosis for transduction, and can efficiently transduce hematopoietic repopulating cells. Lentiviral vectors also do not integrate as frequently near promoter regions as gammaretroviral vectors, and self-inactivating lentiviral vectors pseudotyped with VSV-G can be produced at high-titer and concentrated by centrifugation. Experiments to evaluate HIVderived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies, due in part to host cell restriction by Trim5α. We have obtained efficient transduction of pigtailed macaques (Macaca nemestrina) using lentiviral vectors pseudotyped with VSV-G and report here long term marking in the absence of leukemia or progression towards clonal dominance. Pigtailed macaque CD34+ cells were transduced at similar frequencies to human CD34+ cells and at much higher frequencies compared to baboons (Papio cynocephalus anubis) or rhesus macaques (Macaca mulatta). In three macaques transduced using relatively low MOIs (5-10) we have observed high level marking with HIV-derived lentiviral vectors. In two monkeys that contain an EGFP reporter gene stable marking has been obtained well over one and two years respectively. In these studies a rapid 2-day transduction protocol was used to preserve the engraftment potential of the ex vivo cultured cells. In the first animal marking is stable with over 27% of granulocytes and 12% of lymphocytes expressing EGFP at 463 days post-transplantation. The second animal has 35% EGFP-positive granulocytes and 35% EGFP-positive lymphocytes 834 days after transplantation. LAM-PCR analysis of integration sites has demonstrated that repopulating cells are highly polyclonal and transgene expression has been detected in all hematopoietic lineages (B-cells, T-cells, granulocytes, red blood cells as well as platelets). These data show that lentiviral vectors are highly effective for hematopoietic stem cell gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells by using a rapid transduction protocol is critical. Importantly, these data also show that the pigtailed macaque is an excellent model to evaluate the efficacy and safety of HIV-derived lentiviral vectors proposed for clinical gene therapy protocols.
58. Effects of Culture Conditions on Gene Expression Profile of Human Bone Marrow CD34+ Cells
Amrita Ghosh,1,2 Chenwei Wang,1 Abdel G. Elkahloun,1 Fabio Candotti.1 1 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; 2Univ of Medicine & Dentistry of NJ, Newark, NJ.
Gene transfer into hematopoietic stem cells (HSCs) using gammaretrovirus-based vectors has proven beneficial as a therapeutic approach for immunodeficiencies. Such vectors have been shown to target transcriptional start sites of actively transcribing genes, indicating that the gene expression profile of target cells can be Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
predictive of the pattern of vector integrations. Therefore, different cytokines and growth factor conditions used for gene transfer into HSCs can induce expression of different genes that may be targets for integration. A recent analysis of the integration patterns in X-SCID patients treated under two trials in France and UK showed differences in the frequency of common integration sites that may have resulted from distinct gene expression profiles induced by the seemingly minor differences of in vitro culture conditions (60 U/ml IL3 and 4% FCS vs. 20 U/ml IL3 and 1% HSA, respectively). To explore this issue, we cultured human bone marrow CD34+ cells from three donors under stimulation conditions that reproduced the French (S1) and UK (S2) protocols. Cells were harvested after 48, 72, and 96 hours of stimulation, representing times of transductions performed in the trials. Total RNA was extracted, amplified, and hybridized to Affymetrix HG U133 Plus 2.0 microarrays and robust multi-array average was performed. “Activated Genes” were defined as those with ≥1.5-fold upregulated expression in each condition compared to unstimulated cells by pairwise t-test (p≤0.05). Lists of genes activated in all three time points for either S1 or S2 were generated and named “S1-All” and “S2-All”, respectively. Bioinformatic comparison of S1-All and S2-All showed significant differences in gene ontologies and functional pathways, which led us to analyze the characteristics of genes that were uniquely activated (Unique Genes) at each time point of either stimulation protocol (S1-48h, S1-72h, S1-96h and S2-48h, S2-72h, S2-96h). Unsupervised hierarchical clustering of functional annotation categories showed two main clusters, one corresponding to S1-48h, S2-48h, S2-72h, and S2-96h, the other corresponding to S1-72h and S1-96h. Pathway analysis of unique gene lists showed that enrichment of genes involved in differentiation increased in both stimulation protocols as time progressed. Furthermore, networked genes associated with cell cycle and DNA replication, recombination, and repair showed functional enrichment in S1-48h and S2-96h. Increased enrichment of annotated cancer genes was also found in S1-48h and S2-96h. These results indicate that genes uniquely upregulated at early time points by the S1 conditions share functional characteristics with genes induced at later time points by the S2 culture regimen. This analysis suggests that cells stimulated with 60 U/ml of IL3 and FCS for 48h differ from cells cultured with 20 U/ml of IL3 and HSA but achieve similar transcriptional activity only after 96h. If this is true, even small differences in culture conditions can result in differences in transcription profiles that may lead to differences in integration profiles
59. Promotor-Deprived Self-Inactivating γ-Retroviral Vectors Allow Marking of Serially Reconstituting Hematopoietic Stem Cells
Kerstin Cornils,1 Claudia Lange,2 Axel Schambach,3 Michael Lioznov,2 Christopher Baum,3,4 Boris Fehse.1 1 Experimental Pediatric Oncology and Hematology, Pediatric Clinic III, University Hospital of the J.W. Goethe-University, Frankfurt, Germany; 2Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3Experimental Hematology, Hannover Medical School, Hannover, Germany; 4Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. Based on their ability to stably integrate in their host cell’s genome, γ-retroviral vectors have been successfully used for gene marking as well as gene therapy, mainly in the hematopoietic system. However, recently retroviral vectors were shown to influence growth and/ or survival characteristics of hematopoietic stem cells (HSC) by insertional mutagenesis. This type of genotoxicity may result in clonal dominance, but also malignant transformation of affected clones. Using our established serial bone marrow transplantation (BMT) model we investigated whether γ-retroviral self-inactivating vectors deprived of an internal promotor (pd-SIN vectors) allow stable S23