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965. Integration of Retroviral Vectors into the Human Genome Is Biased by Specific Subsets of Transcription Factor Binding Sites
RNA Virus Vectors: Integration Profiles RNA Virus Vectors: Integration Profiles 965. Integration of Retroviral Vectors into the Human Genome Is Biased by Specific Subsets of Transcription Factor Binding Sites
Alessandra Recchia,1 Davide Cittaro,2 Claudia Cattoglio,3 Barbara Felice,2 Annarita Miccio,4 Giuliana Ferrari,4 Lucilla Luzi,2 Fulvio Mavilio.1 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2FIRC Institute of Medical Oncology, Milano, Italy; 3Istituto Scientifico H San Raffaele, Milano, Italy; 4HSR-TIGET, Istituto Scientifico H San Raffaele, Milano, Italy. Integration of gamma-retroviral (RV) and lentiviral (LV) vectors follows different, non-random patterns in mammalian genomes. The molecular basis of the interaction between retroviral preintegration complexes (PICs) and chromatin is, however, poorly understood. To obtain information about the genetic determinants of integration preferences, we mapped 〉2,500 integration sites of RV and LV vectors carrying wild-type or modified LTRs in human CD34+ hematopoietic cells transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for 〉20% of the RV integration events, while they are significantly less frequent in the case of LV vectors. Genes involved the control of growth, differentiation and development of the hematopoietic and immune system are targeted at high frequency and enriched in hot spots, suggesting that the cell gene expression program is instrumental in directing RV integration. To investigate the role of transcriptional regulatory networks in directing RV and LV integration, we evaluated the local abundance and arrangement of putative transcription factor binding sites (TFBSs) in the genomic regions flanking (+/-1,000 bp) the integrated proviruses. We show that RV, but not LV vectors integrate preferentially in genomic regions flanked by specific subsets of TFBSs, independently from their location (within genes, outside genes, or around transcription start sites). Hierarchical clustering and a principal component analysis of TFBSs flanking RV integration sites in CD34+, T and HeLa cells showed that TFBS subsets are vector- and cell type-specific. The same analysis shows that both protein (RV vs. LV integrase) and DNA (LTR enhancer) components of the PIC have a causal role in directing proviral integration in TFBS-rich regions of the genome. Chromatin immunoprecipitation analysis indicates that transcription factors are bound to unintegrated LTR enhancers into the nucleus, and might synergize with the viral integrase in tethering retroviral PICs to specific domains of transcriptionally active chromatins. This study indicates that the vector design and the target cell gene expression program have a significant impact in determining the integration characteristics of retroviral vectors, and predict substantial differences in the potential genotoxic risk of RV vs. LV vectors for human gene therapy.
966. Importance of Vector Content and Vector Backbone To Reduce the Risk of Insertional Mutagenesis
Ute Modlich,1 Susana Navarro,3 Anjali Mishra,2 Daniela Zychlinski,1 Tobias Maetzig,1 Martijn Brugman,1 Axel Schambach,1 Juan A. Bueren,3 Manuel Grez,4 Christopher Baum.1,2 1 Dept. of Exp. Hematology, Hannover Medical School, Hannover, Germany; 2Div. of Exp. Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 3Hematopoietic Gene Therapy Division, Ciemat, Madrid, Spain; 4Inst. for Biomedical Research, Georg-Speyer-Haus, Frankfurt/Main, Germany. Randomly integrating gene vectors may cause clonal imbalance and malignant transformation by upregulating cellular proto-oncogenes (insertional mutagenesis). In serial bone marrow transplantation S360
studies involving C57Bl6 mice observed for up to 21 months, we found that (1) a single vector insertion next to the cellular protooncogenes Evi1 or Prdm16 suffices to induce leukemia, and (2) side effects of ectopic transgene expression co-operate with insertional mutagenesis in leukemic transformation. Both LTR-driven and selfinactivating (SIN) vectors containing an internal promoter derived from spleen focus-forming virus (SFFV-IP) triggered insertional leukemia. To develop a sensitive transformation assay with shorter observation time, we introduced cell culture conditions for B6 hematopoietic cells in which insertional mutants can be detected by induction of replating ability. Testing gammaretroviral (GV) and lentiviral (LV) SIN vectors containing SFFV-IP, the average incidence of independent mutants was ~2x10-5 cells for GV-SIN and ~5x10-6 for LV-SIN (14 assays for each). A second parameter scored in this assay is the fitness of the clones, which is reflected by the frequency of positive wells in limiting dilution analysis. Interestingly, GV and LV SIN vectors showed no major difference in the replating frequency. The lower transforming capacity of LV-SIN is therefore likely related to the reduced integration bias in the promoter-proximal region while the enhancer/promoter had the same activation potential as in GVSIN vectors. Deletion versions of GV-SIN showed them to depend on the retroviral enhancer to induce replating. Importantly, GV-SIN vectors with a human cellular promoter (EF1alfa) did not induce replating (risk < 1.5x10-6, resulting in >10x increase of the maximal tolerated dose). Similarly, PGK or VAV promoters greatly reduced the transforming potential of LV-SIN vectors. The cHS4 insulator core (~250 bp) tended to reduce the replating index of GV-SIN vectors containing SFFV-IP (P=0.09). Next, we adapted this assay to a disease-specific genetic background. Using hematopoietic cells of gp91phox-/- mice, we reproduced the transforming capacity of corrective LTR-driven vectors that induced severe clonal imbalance in a clinical phase 1 trial treating patients with X-linked chronic granulomatous disease (X-CGD). Corrective GV-SIN-gp91phox vectors with a myelotropic promoter (FES) did not induce insertional transformation (incidence < 9x10 -7). We conclude that the in vitro assay offers a quick assessment of insertional side effects of integrating vectors in wt and disease-specific genetic backgrounds. Importantly, the enhancer/promoter of SIN vectors is more important than their backbone (GV or LV) to decrease the risk of insertional transformation. If vectors cannot be effectively shielded by genetic insulator elements, the LV backbone is safer.
967. A Gene Targeting Strategy Based on Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors Allowing Functional Correction of a Wide Spectrum of Mutations Causing X-Linked SCID
Angelo Lombardo,1 Pietro Genovese,1 Richard Gabriel,2 Manfred Schmidt,2 Christof von Kalle,2 Philip D. Gregory,3 Michael C. Holmes,3 Luigi Naldini.1 1 San Raffaele Telethon Institute for Gene Therapy and Vita Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 2National Center for Tumor Deseases (NCT), Heidelberg, Germany; 3Research and Development, Sangamo BioSciences, Inc., Richmond, CA.
Gene targeting by homologous recombination allows correction of inherited mutations and introduction of novel sequences into a predetermined genomic site. The development of Zinc Finger Nucleases (ZFNs) has brought these long-sought objectives within the reach of gene therapy. We have shown that integrase-defective lentiviral vectors (IDLV) can be used to express ZFNs and provide the template DNA for gene targeting in different cell types, including human primary stem cells. By this approach, we reported efficient editing of an endogenous gene and site-specific gene addition at the ZFN target site by a process that required ZFN cleavage and Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Alessandra Recchia,1 Davide Cittaro,2 Claudia Cattoglio,3 Barbara Felice,2 Annarita Miccio,4 Giuliana Ferrari,4 Lucilla Luzi,2 Fulvio Mavilio.1 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2FIRC Institute of Medical Oncology, Milano, Italy; 3Istituto Scientifico H San Raffaele, Milano, Italy; 4HSR-TIGET, Istituto Scientifico H San Raffaele, Milano, Italy. Integration of gamma-retroviral (RV) and lentiviral (LV) vectors follows different, non-random patterns in mammalian genomes. The molecular basis of the interaction between retroviral preintegration complexes (PICs) and chromatin is, however, poorly understood. To obtain information about the genetic determinants of integration preferences, we mapped 〉2,500 integration sites of RV and LV vectors carrying wild-type or modified LTRs in human CD34+ hematopoietic cells transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for 〉20% of the RV integration events, while they are significantly less frequent in the case of LV vectors. Genes involved the control of growth, differentiation and development of the hematopoietic and immune system are targeted at high frequency and enriched in hot spots, suggesting that the cell gene expression program is instrumental in directing RV integration. To investigate the role of transcriptional regulatory networks in directing RV and LV integration, we evaluated the local abundance and arrangement of putative transcription factor binding sites (TFBSs) in the genomic regions flanking (+/-1,000 bp) the integrated proviruses. We show that RV, but not LV vectors integrate preferentially in genomic regions flanked by specific subsets of TFBSs, independently from their location (within genes, outside genes, or around transcription start sites). Hierarchical clustering and a principal component analysis of TFBSs flanking RV integration sites in CD34+, T and HeLa cells showed that TFBS subsets are vector- and cell type-specific. The same analysis shows that both protein (RV vs. LV integrase) and DNA (LTR enhancer) components of the PIC have a causal role in directing proviral integration in TFBS-rich regions of the genome. Chromatin immunoprecipitation analysis indicates that transcription factors are bound to unintegrated LTR enhancers into the nucleus, and might synergize with the viral integrase in tethering retroviral PICs to specific domains of transcriptionally active chromatins. This study indicates that the vector design and the target cell gene expression program have a significant impact in determining the integration characteristics of retroviral vectors, and predict substantial differences in the potential genotoxic risk of RV vs. LV vectors for human gene therapy.
966. Importance of Vector Content and Vector Backbone To Reduce the Risk of Insertional Mutagenesis
Ute Modlich,1 Susana Navarro,3 Anjali Mishra,2 Daniela Zychlinski,1 Tobias Maetzig,1 Martijn Brugman,1 Axel Schambach,1 Juan A. Bueren,3 Manuel Grez,4 Christopher Baum.1,2 1 Dept. of Exp. Hematology, Hannover Medical School, Hannover, Germany; 2Div. of Exp. Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 3Hematopoietic Gene Therapy Division, Ciemat, Madrid, Spain; 4Inst. for Biomedical Research, Georg-Speyer-Haus, Frankfurt/Main, Germany. Randomly integrating gene vectors may cause clonal imbalance and malignant transformation by upregulating cellular proto-oncogenes (insertional mutagenesis). In serial bone marrow transplantation S360
studies involving C57Bl6 mice observed for up to 21 months, we found that (1) a single vector insertion next to the cellular protooncogenes Evi1 or Prdm16 suffices to induce leukemia, and (2) side effects of ectopic transgene expression co-operate with insertional mutagenesis in leukemic transformation. Both LTR-driven and selfinactivating (SIN) vectors containing an internal promoter derived from spleen focus-forming virus (SFFV-IP) triggered insertional leukemia. To develop a sensitive transformation assay with shorter observation time, we introduced cell culture conditions for B6 hematopoietic cells in which insertional mutants can be detected by induction of replating ability. Testing gammaretroviral (GV) and lentiviral (LV) SIN vectors containing SFFV-IP, the average incidence of independent mutants was ~2x10-5 cells for GV-SIN and ~5x10-6 for LV-SIN (14 assays for each). A second parameter scored in this assay is the fitness of the clones, which is reflected by the frequency of positive wells in limiting dilution analysis. Interestingly, GV and LV SIN vectors showed no major difference in the replating frequency. The lower transforming capacity of LV-SIN is therefore likely related to the reduced integration bias in the promoter-proximal region while the enhancer/promoter had the same activation potential as in GVSIN vectors. Deletion versions of GV-SIN showed them to depend on the retroviral enhancer to induce replating. Importantly, GV-SIN vectors with a human cellular promoter (EF1alfa) did not induce replating (risk < 1.5x10-6, resulting in >10x increase of the maximal tolerated dose). Similarly, PGK or VAV promoters greatly reduced the transforming potential of LV-SIN vectors. The cHS4 insulator core (~250 bp) tended to reduce the replating index of GV-SIN vectors containing SFFV-IP (P=0.09). Next, we adapted this assay to a disease-specific genetic background. Using hematopoietic cells of gp91phox-/- mice, we reproduced the transforming capacity of corrective LTR-driven vectors that induced severe clonal imbalance in a clinical phase 1 trial treating patients with X-linked chronic granulomatous disease (X-CGD). Corrective GV-SIN-gp91phox vectors with a myelotropic promoter (FES) did not induce insertional transformation (incidence < 9x10 -7). We conclude that the in vitro assay offers a quick assessment of insertional side effects of integrating vectors in wt and disease-specific genetic backgrounds. Importantly, the enhancer/promoter of SIN vectors is more important than their backbone (GV or LV) to decrease the risk of insertional transformation. If vectors cannot be effectively shielded by genetic insulator elements, the LV backbone is safer.
967. A Gene Targeting Strategy Based on Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors Allowing Functional Correction of a Wide Spectrum of Mutations Causing X-Linked SCID
Angelo Lombardo,1 Pietro Genovese,1 Richard Gabriel,2 Manfred Schmidt,2 Christof von Kalle,2 Philip D. Gregory,3 Michael C. Holmes,3 Luigi Naldini.1 1 San Raffaele Telethon Institute for Gene Therapy and Vita Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 2National Center for Tumor Deseases (NCT), Heidelberg, Germany; 3Research and Development, Sangamo BioSciences, Inc., Richmond, CA.
Gene targeting by homologous recombination allows correction of inherited mutations and introduction of novel sequences into a predetermined genomic site. The development of Zinc Finger Nucleases (ZFNs) has brought these long-sought objectives within the reach of gene therapy. We have shown that integrase-defective lentiviral vectors (IDLV) can be used to express ZFNs and provide the template DNA for gene targeting in different cell types, including human primary stem cells. By this approach, we reported efficient editing of an endogenous gene and site-specific gene addition at the ZFN target site by a process that required ZFN cleavage and Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy