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558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients
Targeted Genome Editing III widely used vector in gene therapy applications in human. However, lack of suitable animal models that accurately reflect the human disease phenotype often stalls the development of treatment strategies. To overcome this problem, research is focused on generating induced pluripotent stem cells (iPSCs) from affected human subjects as in vitro models to explore the potential of gene augmentation therapy. An advantage of iPSCs is that these cells can be differentiated along a retinal cell lineage. In this study, we differentiated iPSCs into Neural Progenitor Cells (NPC) and compared the in vitro transduction efficiency of AAV serotypes 1-9. Differentiated NPCs were characterized by immunocytochemistry with the expression of NPCs specific markers (Sox2, Nestin, Pax6). Experiments were carried out using different AAVs serotypes containing the same transgene cassette: an enhanced green-fluorescent protein (eGFP) reporter gene driven by the cytomegalovirus immediate-early (CMV) promoter. At 24 to 48 hours post transduction, positive GFP expressing cells are examined by laser scanning in Typhoon. Relative GFP expression was evaluated by using the software ImageJ. In addition to the laser scanning method, flow cytometry was used to evaluate GFP expression in the cell population. Live cell imaging and flow cytometry evaluation revealed that AAV2 is the most efficient vector for in vitro transduction in NPC followed by AAV1, AAV3 and AAV6 while AAV4, AAV5, AAV7, AAV8 and AAV9 showed no transduction or extremely low transduction efficiency.
Targeted Genome Editing III 557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression
Colin L. Sweeney1, Uimook Choi1, Jizhong Zou2, Randall K. Merling1, Suk See De Ravin1, Harry L. Malech1 1 Laboratory of Host Defenses, Genetic Immunotherapy Section, NIAID, NIH, Bethesda, MD, 2Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPRmediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB213 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.
558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients
Begoña Díez1, Pietro Genovesse2, Giulia Schiroli2, Lara Álvarez1, Francisco J. Román-Rodriguez1, Philip D. Gregory3, Michael Holmes3, Angelo Lombardo2, Luigi Naldini2, Paula Rio1, Juan A. Bueren1 1 Hematopoietic Innovative Therapies, CIEMAT/CIBERER/IIS. Fundación Jiménez Díaz, Madrid, Spain, 2San Raffaelle Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy, 3Sangamo BioSciences Inc., Richmond, CA
Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1-specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.
559. Induction of Fetal Hemoglobin in Adult Erythroblasts by Genome Editing of the BetaGlobin Locus
Chiara Antoniani1, Annalisa Lattanzi2, Giulia Pavani2, Vasco Meneghini1, Elisa Magrin1, Leslie Weber1, Matthew Porteus3, Marina Cavazzana1, Fulvio Mavilio2, Annarita Miccio1 1 Imagine Institute, Paris, France, 2Genethon, Evry, France, 3 Stanford University, Stanford, CA Sickle cell disease (SCD) and β-thalassemia are severe anemias characterized by abnormal or reduced production of hemoglobin β-chains. Autologous transplantation of genetically corrected hematopoietic stem cells (HSC) is an attractive therapeutic alternative for patients lacking a compatible HSC donor. Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence S223
Targeted Genome Editing III 557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression
Colin L. Sweeney1, Uimook Choi1, Jizhong Zou2, Randall K. Merling1, Suk See De Ravin1, Harry L. Malech1 1 Laboratory of Host Defenses, Genetic Immunotherapy Section, NIAID, NIH, Bethesda, MD, 2Center for Molecular Medicine, NHLBI, NIH, Bethesda, MD
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPRmediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB213 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.
558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients
Begoña Díez1, Pietro Genovesse2, Giulia Schiroli2, Lara Álvarez1, Francisco J. Román-Rodriguez1, Philip D. Gregory3, Michael Holmes3, Angelo Lombardo2, Luigi Naldini2, Paula Rio1, Juan A. Bueren1 1 Hematopoietic Innovative Therapies, CIEMAT/CIBERER/IIS. Fundación Jiménez Díaz, Madrid, Spain, 2San Raffaelle Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy, 3Sangamo BioSciences Inc., Richmond, CA
Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1-specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.
559. Induction of Fetal Hemoglobin in Adult Erythroblasts by Genome Editing of the BetaGlobin Locus
Chiara Antoniani1, Annalisa Lattanzi2, Giulia Pavani2, Vasco Meneghini1, Elisa Magrin1, Leslie Weber1, Matthew Porteus3, Marina Cavazzana1, Fulvio Mavilio2, Annarita Miccio1 1 Imagine Institute, Paris, France, 2Genethon, Evry, France, 3 Stanford University, Stanford, CA Sickle cell disease (SCD) and β-thalassemia are severe anemias characterized by abnormal or reduced production of hemoglobin β-chains. Autologous transplantation of genetically corrected hematopoietic stem cells (HSC) is an attractive therapeutic alternative for patients lacking a compatible HSC donor. Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence S223