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Updated: Apr 5, 2026
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent
Anne-Kathrin Dreyer1, Dirk Hoffmann1, Nico Lachmann2
1Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Abstract:
X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.
Insights
Gene therapy for X-linked chronic granulomatous disease (X-CGD) shows promise using TALENs to insert a corrective gene into patient stem cells. This approach restores immune cell function and offers a safe, efficient treatment strategy for X-CGD.
Area of Science:
- Immunology
- Genetic Engineering
- Stem Cell Biology
Background:
- X-linked chronic granulomatous disease (X-CGD) is an inherited immune disorder caused by defective reactive oxygen species (ROS) production in phagocytes due to NOX2 gene mutations.
- Previous gene therapy attempts for X-CGD faced challenges with proto-oncogene activation from random retroviral insertions.
- Targeting safe harbor sites like AAVS1 for transgene insertion offers a potentially safer alternative for X-CGD gene therapy.
Purpose of the Study:
- To evaluate the efficacy and safety of using gene editing nucleases (ZFNs and TALENs) to insert a gp91phox transgene into the AAVS1 safe harbor site in X-CGD patient-derived induced pluripotent stem cells (iPSCs).
- To assess the functional restoration of phagocytic cells derived from corrected iPSCs.
Main Methods:
- Generated X-CGD patient-derived iPSCs that exhibited the disease phenotype upon differentiation.
- Utilized AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target transgene insertion.
- Differentiated corrected iPSCs into granulocytes and assessed gp91phox mRNA levels, ROS production, and neutrophil extracellular trap (NET) formation.
Main Results:
- TALENs demonstrated higher efficiency and lower cytotoxicity than ZFNs in generating correctly targeted iPSC colonies.
- No mutations were detected at the top-ten predicted off-target sites in the corrected iPSC clones.
- Differentiation of corrected iPSCs led to restored ROS production and NET formation in derived granulocytes, with high gp91phox mRNA expression.
Conclusions:
- TALEN-mediated integration of a myelo-specific gp91phox transgene into the AAVS1 locus of patient-derived iPSCs is a safe and effective strategy.
- This approach enables the generation of autologous, functionally corrected granulocytes for treating X-CGD.
- Targeted gene insertion into safe harbor sites represents a promising advancement in the gene therapy of inherited immune disorders.
