Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction.
Mol Ther
; 25(2): 321-330, 2017 02 01.
Article
in En
| MEDLINE
| ID: mdl-28153086
ABSTRACT
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 gp91phox 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 gp91phox expression and ROS production in iPSC-derived 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 gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phox 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Membrane Glycoproteins
/
Introns
/
Gene Expression Regulation
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NADPH Oxidases
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Targeted Gene Repair
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Induced Pluripotent Stem Cells
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Granulomatous Disease, Chronic
Limits:
Humans
Language:
En
Journal:
Mol Ther
Journal subject:
BIOLOGIA MOLECULAR
/
TERAPEUTICA
Year:
2017
Type:
Article