Prime editing links the split integrated stress response to pathogenic eIF2B mutations and white matter degeneration

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Vanishing White Matter Disease (VWMD) is a fatal neurodevelopmental disorder impacting white matter, currently without a cure.
• The canonical integrated stress response (c-ISR) is implicated, but a novel pathway, the split ISR (s-ISR), has been observed only in mouse models.
• Uncertainty remains regarding s-ISR activation in human VWMD cells and its specific role in disease pathogenesis.

Purpose of the Study:

• To investigate the activation and role of the split ISR (s-ISR) pathway in human cells harboring VWMD-associated mutations.
• To establish human cell models for VWMD using prime editing (PE) technology.
• To determine if s-ISR activation is a common feature across different VWMD mutations and its impact on cell differentiation.

Main Methods:

• Engineered multiple pathogenic eIF2B mutations into HEK293T and induced pluripotent stem cells (iPSCs) using prime editing (PE).
• Assessed the activation of the split ISR (s-ISR) pathway in the engineered human cell models.
• Investigated the effect of s-ISR on protein synthesis and differentiation, particularly in astrocytes, and tested ISRIB for suppression.

Main Results:

• Successfully generated human VWMD models using prime editing, demonstrating PE's applicability and safety.
• All engineered VWMD mutations consistently activated the s-ISR pathway in human cells.
• s-ISR activation was found to impair protein synthesis necessary for iPSC differentiation into astrocytes, linking it to VWMD pathology.
• The s-ISR response could be amplified by stress-induced c-ISR and effectively suppressed by ISRIB.

Conclusions:

• The split ISR (s-ISR) pathway is activated by VWMD-causing mutations in human cells, representing a conserved disease mechanism.
• s-ISR activation directly hinders the maturation of crucial glial cells like astrocytes, contributing to white matter abnormalities in VWMD.
• Prime editing offers a viable method for creating accurate human VWMD models for further research and therapeutic development.