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A Mouse Model of X-Linked Chronic Granulomatous Disease for the Development of CRISPR/Cas9 Gene Therapy
Seren Sevim-Wunderlich1, Tu Dang1, Jana Rossius1
1Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany.
Abstract:
Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked CYBB gene that abrogate reactive oxygen species (ROS) production in phagocytes and microbial defense. Gene repair using the CRISPR/Cas9 system in hematopoietic stem and progenitor cells (HSPCs) is a promising technology for therapy for CGD. To support the establishment of efficient and safe gene therapies for CGD, we generated a mouse model harboring a patient-derived mutation in the CYBB gene. Our CybbC517del mouse line shows the hallmarks of CGD and provides a source for Cybb-deficient HSPCs that can be used to evaluate gene-therapy approaches in vitro and in vivo. In a setup using Cas9 RNPs and an AAV repair vector in HSPCs, we show that the mutation can be repaired in 19% of treated cells and that treatment restores ROS production by macrophages. In conclusion, our CybbC517del mouse line provides a new platform for refining and evaluating novel gene therapies and studying X-CGD pathophysiology.
Insights
Researchers developed a new mouse model for Chronic Granulomatous Disease (CGD) by introducing a patient mutation into the CYBB gene. This model aids in testing CRISPR gene therapies to restore microbial defense in CGD patients.
Area of Science:
- Immunology
- Genetics
- Hematology
Background:
- Chronic Granulomatous Disease (CGD) is an inherited immunodeficiency.
- Caused by mutations in the X-linked CYBB gene, leading to impaired phagocyte reactive oxygen species (ROS) production and microbial defense.
- CRISPR/Cas9 gene editing in hematopoietic stem and progenitor cells (HSPCs) shows promise for CGD therapy.
Purpose of the Study:
- To generate a novel mouse model for CGD research.
- To evaluate CRISPR/Cas9-mediated gene repair for CGD therapy.
- To provide a platform for refining and assessing gene-based therapeutic strategies for X-linked CGD.
Main Methods:
- Generation of a CybbC517del mouse line with a patient-derived CYBB mutation.
- Utilized CRISPR/Cas9 ribonucleoprotein (RNP) complexes and an adeno-associated virus (AAV) repair vector.
- Assessed gene repair efficiency and restoration of ROS production in HSPCs and macrophages.
Main Results:
- The CybbC517del mouse line exhibits key characteristics of CGD.
- CRISPR/Cas9 gene editing successfully repaired the CYBB mutation in 19% of treated HSPCs.
- Restored ROS production in macrophages following gene repair was demonstrated.
Conclusions:
- The established CybbC517del mouse model is a valuable tool for CGD research.
- This model facilitates the evaluation of in vitro and in vivo gene therapy approaches for CGD.
- The study validates CRISPR/Cas9 as a potential therapeutic strategy for restoring immune function in X-CGD.
