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Gene Editing in Chronic Granulomatous Disease
Colin L Sweeney1, Randall K Merling1, Suk See De Ravin1
1Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods in Molecular Biology (Clifton, N.J.)
|June 8, 2019
Summary
Gene editing offers a promising approach for treating chronic granulomatous disease (CGD). This study details methods for correcting the genetic defects in CGD patient stem cells to restore neutrophil function.
Area of Science:
- Immunology
- Molecular Biology
- Stem Cell Therapy
Background:
- Chronic granulomatous disease (CGD) is an inherited immune deficiency.
- CGD results from defects in the phagocytic oxidase (phox) enzyme complex, impairing neutrophil microbicidal activity.
- Restoring NADPH oxidase activity is crucial for treating CGD.
Purpose of the Study:
- To provide detailed protocols for gene editing in stem cells for CGD.
- To describe the differentiation of gene-edited stem cells into functional neutrophils.
- To outline assays for evaluating neutrophil identity and function post-editing.
Main Methods:
- Targeted gene editing using zinc finger nucleases (ZFNs), TALENs, or CRISPR/Cas9.
- Homology-directed repair in human-induced pluripotent stem cells and hematopoietic stem cells.
- Differentiation protocols for generating mature neutrophils from edited stem cells.
- Flow cytometry for surface marker analysis.
- Intracellular staining for phox proteins.
- Reactive oxygen species (ROS) generation assays.
Main Results:
- Successful gene editing of CGD patient stem cells.
- Restoration of phox gene expression and NADPH oxidase activity in differentiated neutrophils.
- Demonstration of functional neutrophil recovery.
Conclusions:
- Targeted gene editing is a viable strategy for CGD therapy.
- Detailed protocols enable the functional restoration of neutrophils in CGD patients.
- This approach holds potential for developing curative treatments for CGD.
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