Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs

Laugsch, Magdalena; Rostovskaya, Maria; Velychko, Sergiy; Richter, Cornelia; Zimmer, Ariane; Klink, Barbara; Haase, Michael; Neumann, Katrin; Thieme, Sebastian; Roesler, Joachim; Brenner, Sebastian; Anastassiadis, Konstantinos

doi:10.1038/mt.2015.154

Cited by 20 publications

(18 citation statements)

References 57 publications

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“…A previous study by Laugsch et al 17 demonstrated restoration of gp91 phox expression and ROS activity in iPSCs from a patient with a mutation in exon 7 of CYBB, by utilizing a large bacterial artificial chromosome containing the endogenous sequence of exons 6 through 13 together with the intervening introns, for homologous recombination-mediated replacement of the CYBB mutation without nuclease enhancement. In the current study, TALEN-mediated targeted insertion of a minigene consisting of the normal or codon-optimized cDNA of all 13 exons of CYBB without introns into the start site of exon 1 of the endogenous CYBB gene resulted in no detectable gp91 phox expression, indicating that intronic or other downstream non-coding elements are necessary for expression from the CYBB promoter.…”

Section: Discussionmentioning

confidence: 99%

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

et al. 2017

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X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91 phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91 phox expression and ROS production in iPSCderived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN-or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91 phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91 phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.

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Section: Discussionmentioning

confidence: 99%

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

et al. 2017

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“…The WT hiPSCs lines T12 (WT#3) and S24 (WT#2) were provided by the Pediatric Endocrinology and Diabetology laboratory (Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Germany). These hiPSCs lines were obtained from fibroblasts from healthy donors (Kind et al, 2010), reprogrammed by lentiviral transduction vector and validated as described in Laugsch et al (2016). The WT hiPSCs lines NP0040-8 (WT#1) provided by Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany was reprogrammed by episomal reprogramming vectors and validated according to EBiSC standards (NP0040-8 is available at EBiSC under accession name UKKi011-A; https://cells.ebisc.org/ UKKi011-A/).…”

Section: Wt Hipscsmentioning

confidence: 99%

Modeling the Pathological Long-Range Regulatory Effects of Human Structural Variation with Patient-Specific hiPSCs

et al. 2019

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“…Here, we perform a second step toward the translation ability of this strategy, by performing a proof of principle study on a mouse model of X‐linked CGD, a human immunodeficiency which has been the target of many attempts to gene therapy . Viral vector‐based trials for gene correction in humans have been reported, but the occurrence of undesired events and the concomitant introduction of programmable nucleases prompted the investigation of “gene editing” approaches to more precisely correct the nucleotide defect in iPSCs or directly in hematopoietic stem cells.…”

Section: Discussionmentioning

confidence: 99%

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells

et al. 2019

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In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X‐linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell‐mediated chromosome transfer. X‐transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X‐linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

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Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs

Cited by 20 publications

References 57 publications

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction

Modeling the Pathological Long-Range Regulatory Effects of Human Structural Variation with Patient-Specific hiPSCs

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells

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