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572. Antibiotic-Free Nonviral pFAR4 Vector Displays Efficient Transgene Delivery in Mouse and Human Cells
Gene Targeting and Gene Correction III 572. Antibiotic-Free Nonviral pFAR4 Vector Displays Efficient Transgene Delivery in Mouse and Human Cells
Marie Pastor,1 Sandra Johnen,2 Mickael Quiviger,1 Zsuzsanna Izsvak,3 Daniel Scherman,1 Gabriele Thumann,4 Corinne Marie.1 1 UTCBS, INSERM U1022, CNRS UMR8258, Paris, France; 2 Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany; 3Max Delbruck Center for Molecular Medicine, Berlin, Germany; 4Departement des Neurosciences Cliniques, Hopitaux Universitaires de Geneve, Geneva, Switzerland. New generations of plasmid vectors used for non-viral gene therapy have a reduced size, thus allowing an increase in transfection efficiency and expression levels. We designed a novel vector, called pFAR4, which is Free of Antibiotic Resistance markers. In bacteria, the production of pFAR4 derivatives relies on a plasmid-borne function that suppresses a nonsense mutation introduced into an essential Escherichia coli gene. Thanks to its reduced size, pFAR4 appears to be an efficient gene vector as it displays a superior transgene expression level either after transfection of cultured B16 melanoma cells or electrotransfer into skin or tumors. Furthermore, hydrodynamic delivery into liver revealed that pFAR4 allowed a prolonged expression level, in comparison with kanamycin resistant plasmids that promote transgene silencing. We took advantage of the pFAR4 superiority in liver to assess a novel therapeutic approach for the Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome. MPS-IIIA is a lysosomal storage genetic disease which results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and patients’ death. In MPS-IIIA mice, hydrodynamic delivery of a pFAR4 derivative expressing the deficient enzyme from a liver-specific promoter allowed high and prolonged serum levels of sulfamidase protein that was efficiently taken-up by neighboring organs after its endocytosis by mannose 6-phosphate receptors-expressing cells. This led to the correction of GAG accumulation in all peripheral organs as well as in the brain, albeit at early stages of the disease. Improvement of our approach is provided by fusing the SGSH protein with peptides mediating transcytocis across the blood-brain barrier. Having established that pFAR4 is a potent expression vector in a non-integrative approach, our next step consisted in combining the pFAR4 technology and the Sleeping Beauty (SB) transposon system, which is composed of a transposase that excises the transgenecontaining transposon from a donor plasmid and mediates its integration into the host cells’ genome. The reduced size of pFAR4 allowed an efficient delivery of SB components into human cells. Our current goal is to take the pFAR4 and Sleeping Beauty technologies to the clinic, in the context of an ex vivo gene therapy approach involving the transfection of autologous primary pigment epithelial cells for the treatment of neovascular age-related macular degeneration disease, in two phase I/II clinical trials.
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573. Towards Gene Correction in a Large Animal Model: Gene Correction of Canine Coagulation Factor IX Deficiency Via Homologous Recombination Mediated By Designer Nucleases
Thorsten Bergmann,1 Eric Schulz,2 Verena Schildgen,1 Maren Gebbing,1 Stephan David,1 Oliver Schildgen,2 Anja Ehrhardt.1 1 Institute of Virology and Microbiology, Center for Biological Education and Research, Department of Human Medicine, University Witten/Herdecke, Witten, Germany; 2Institute of Pathology, Kliniken der Stadt Köln gGmbH, Hospital of the University Witten/Herdecke (Cologne), Cologne, Germany.
Gene correction at specific target loci provides a powerful strategy to overcome diseases caused by genetic disorders like hemophilia B. This disease is caused by mutations in the coagulation factor IX (FIX) gene leading to a dysfunction of the resulting protein and to bleeding disorders. In this study we aim at using a dog model for hemophilia B containing a single point mutation in the canine FIX (cFIX) which we aimed at correcting via homologous recombination (HR). We designed target specific designer nucleases including transcriptionactivator-like-effector nucleases (TALENs) and CRISPR(clustered regularly interspaced short Palindromic repeats)/Cas9 for introduction of a double strand break directly at the mutation. Both nuclease variants bind to the identical target sequence for a direct comparison and are expressed under control of constitutive promoters (CMV promoter for TALEN constructs and CBh promoter for CRISPR/ Cas9 system). Confirmation of efficiency showed around ~15% mutation rate in hetero-duplex based assays (T7E1) for three TALEN candidates. Efficiency was also confirmed via qPCR based detection method developed in our group. One CRISPR candidate showed even higher efficiency in T7E1 assays compared to TALEN candidates. For gene correction we constructed a HR cassette containing a 2 kb fragment of wild type (WT) cFIX-sequence for replacement of the mutation with this functional sequence. For efficient delivery in vitro in canine cells and in vivo in liver, we produced gene deleted highcapacity adenoviral vectors (HCAdV) containing these TALEN or CRISPR/Cas9 expression cassettes. For efficient delivery of the HR cassettes we produced adenoviral and adeno-associated viral vector systems containing the cFIX WT sequence. We are currently testing ratios and combinations of the different delivery systems in vitro. For this purpose we set up a specifically established pyrosequencing protocol for detection of the efficiency of HR in canine cells. For additional confirmation we also set up a restriction enzyme digest based detection assay based on the restriction enzyme BmrI which specifically detects positive HR events by only binding to the wild type sequence. For HR experiments we are using different sets of cell lines including canine cells (MDCK, 183ccl) and a specifically constructed cell line were we stably integrated the mutated cFIX locus. First experiments showed an increased number of HR events in presence of the TALEN candidates in the BmrI assay. The best vector combination will be used in vivo in canine liver and functionality of the corrected cFIX gene in vivo will be confirmed by phenotypic correction, cFIX expression levels measured via ELISA and molecular analysis will be performed by pyrosequencing. In summary we produced and are setting up a vector system for gene correction in a large animal model for hemophilia B.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Marie Pastor,1 Sandra Johnen,2 Mickael Quiviger,1 Zsuzsanna Izsvak,3 Daniel Scherman,1 Gabriele Thumann,4 Corinne Marie.1 1 UTCBS, INSERM U1022, CNRS UMR8258, Paris, France; 2 Department of Ophthalmology, University Hospital RWTH Aachen, Aachen, Germany; 3Max Delbruck Center for Molecular Medicine, Berlin, Germany; 4Departement des Neurosciences Cliniques, Hopitaux Universitaires de Geneve, Geneva, Switzerland. New generations of plasmid vectors used for non-viral gene therapy have a reduced size, thus allowing an increase in transfection efficiency and expression levels. We designed a novel vector, called pFAR4, which is Free of Antibiotic Resistance markers. In bacteria, the production of pFAR4 derivatives relies on a plasmid-borne function that suppresses a nonsense mutation introduced into an essential Escherichia coli gene. Thanks to its reduced size, pFAR4 appears to be an efficient gene vector as it displays a superior transgene expression level either after transfection of cultured B16 melanoma cells or electrotransfer into skin or tumors. Furthermore, hydrodynamic delivery into liver revealed that pFAR4 allowed a prolonged expression level, in comparison with kanamycin resistant plasmids that promote transgene silencing. We took advantage of the pFAR4 superiority in liver to assess a novel therapeutic approach for the Mucopolysaccharidosis type IIIA (MPS-IIIA) or Sanfilippo A syndrome. MPS-IIIA is a lysosomal storage genetic disease which results from the deficiency of the N-sulfoglucosamine sulfohydrolase (SGSH) protein, a sulfamidase required for the degradation of heparan sulfate glycosaminoglycans (GAGs). The accumulation of these macromolecules leads to somatic organ pathologies, severe neurodegeneration and patients’ death. In MPS-IIIA mice, hydrodynamic delivery of a pFAR4 derivative expressing the deficient enzyme from a liver-specific promoter allowed high and prolonged serum levels of sulfamidase protein that was efficiently taken-up by neighboring organs after its endocytosis by mannose 6-phosphate receptors-expressing cells. This led to the correction of GAG accumulation in all peripheral organs as well as in the brain, albeit at early stages of the disease. Improvement of our approach is provided by fusing the SGSH protein with peptides mediating transcytocis across the blood-brain barrier. Having established that pFAR4 is a potent expression vector in a non-integrative approach, our next step consisted in combining the pFAR4 technology and the Sleeping Beauty (SB) transposon system, which is composed of a transposase that excises the transgenecontaining transposon from a donor plasmid and mediates its integration into the host cells’ genome. The reduced size of pFAR4 allowed an efficient delivery of SB components into human cells. Our current goal is to take the pFAR4 and Sleeping Beauty technologies to the clinic, in the context of an ex vivo gene therapy approach involving the transfection of autologous primary pigment epithelial cells for the treatment of neovascular age-related macular degeneration disease, in two phase I/II clinical trials.
S228
573. Towards Gene Correction in a Large Animal Model: Gene Correction of Canine Coagulation Factor IX Deficiency Via Homologous Recombination Mediated By Designer Nucleases
Thorsten Bergmann,1 Eric Schulz,2 Verena Schildgen,1 Maren Gebbing,1 Stephan David,1 Oliver Schildgen,2 Anja Ehrhardt.1 1 Institute of Virology and Microbiology, Center for Biological Education and Research, Department of Human Medicine, University Witten/Herdecke, Witten, Germany; 2Institute of Pathology, Kliniken der Stadt Köln gGmbH, Hospital of the University Witten/Herdecke (Cologne), Cologne, Germany.
Gene correction at specific target loci provides a powerful strategy to overcome diseases caused by genetic disorders like hemophilia B. This disease is caused by mutations in the coagulation factor IX (FIX) gene leading to a dysfunction of the resulting protein and to bleeding disorders. In this study we aim at using a dog model for hemophilia B containing a single point mutation in the canine FIX (cFIX) which we aimed at correcting via homologous recombination (HR). We designed target specific designer nucleases including transcriptionactivator-like-effector nucleases (TALENs) and CRISPR(clustered regularly interspaced short Palindromic repeats)/Cas9 for introduction of a double strand break directly at the mutation. Both nuclease variants bind to the identical target sequence for a direct comparison and are expressed under control of constitutive promoters (CMV promoter for TALEN constructs and CBh promoter for CRISPR/ Cas9 system). Confirmation of efficiency showed around ~15% mutation rate in hetero-duplex based assays (T7E1) for three TALEN candidates. Efficiency was also confirmed via qPCR based detection method developed in our group. One CRISPR candidate showed even higher efficiency in T7E1 assays compared to TALEN candidates. For gene correction we constructed a HR cassette containing a 2 kb fragment of wild type (WT) cFIX-sequence for replacement of the mutation with this functional sequence. For efficient delivery in vitro in canine cells and in vivo in liver, we produced gene deleted highcapacity adenoviral vectors (HCAdV) containing these TALEN or CRISPR/Cas9 expression cassettes. For efficient delivery of the HR cassettes we produced adenoviral and adeno-associated viral vector systems containing the cFIX WT sequence. We are currently testing ratios and combinations of the different delivery systems in vitro. For this purpose we set up a specifically established pyrosequencing protocol for detection of the efficiency of HR in canine cells. For additional confirmation we also set up a restriction enzyme digest based detection assay based on the restriction enzyme BmrI which specifically detects positive HR events by only binding to the wild type sequence. For HR experiments we are using different sets of cell lines including canine cells (MDCK, 183ccl) and a specifically constructed cell line were we stably integrated the mutated cFIX locus. First experiments showed an increased number of HR events in presence of the TALEN candidates in the BmrI assay. The best vector combination will be used in vivo in canine liver and functionality of the corrected cFIX gene in vivo will be confirmed by phenotypic correction, cFIX expression levels measured via ELISA and molecular analysis will be performed by pyrosequencing. In summary we produced and are setting up a vector system for gene correction in a large animal model for hemophilia B.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy