Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response

Area of Science:

• Hematology
• Molecular Biology
• Gene Therapy

Background:

• Hematopoietic stem and progenitor cells (HSPCs) are crucial for treating genetic diseases via gene editing.
• The impact of programmable nucleases on HSPC function and engraftment is not fully understood.
• DNA double-stranded breaks (DSBs) are a key outcome of gene editing, potentially affecting cell viability.

Purpose of the Study:

• To characterize the DNA damage response (DDR) in HSPCs after gene editing.
• To evaluate the effects of DSB induction and repair template delivery on HSPC function.
• To identify strategies for overcoming functional impairments in edited HSPCs.

Main Methods:

• Induction of single or multiple DSBs using zinc-finger and CRISPR/Cas9 nucleases in HSPCs.
• Monitoring of DDR foci, cell-cycle progression, and transcriptional profiles at single-cell resolution.
• Assessment of HSPC proliferation, yield, and engraftment capacity after gene editing.

Main Results:

• p53-mediated DDR pathway activation was the primary response to DSBs in all HSPC subtypes.
• High DSB load or AAV-mediated repair template delivery led to cumulative p53 activation, reducing HSPC proliferation and engraftment.
• Functional impairment due to DDR was reversible with low DSB burden and could be mitigated by transient p53 inhibition.

Conclusions:

• Gene editing in HSPCs elicits a significant p53-dependent DNA damage response.
• DSB load and repair template delivery influence the extent of functional impairment in edited HSPCs.
• Transient p53 inhibition offers a potential strategy to enhance the feasibility and success of HSPC gene editing.