Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

Area of Science:

• Neuroscience
• Immunology
• Stem Cell Biology

Background:

• Hematopoietic stem cell transplantation (HSCT) can replace brain microglia but carries high mortality risks.
• Existing methods for microglia replacement are limited by toxicity and efficacy.

Purpose of the Study:

• To engineer a novel, inhibitor-resistant colony-stimulating factor 1 receptor (CSF1R) for enhanced microglia replacement.
• To develop a safer and more effective method for replacing endogenous microglia.

Main Methods:

• Engineered a glycine to alanine substitution at position 795 (G795A) in human CSF1R to confer resistance to CSF1R inhibitors.
• Utilized CRISPR technology to engineer human induced pluripotent stem cell-derived microglia (iMG) with the G795A variant.
• Performed xenotransplantation studies in mice to assess engraftment, persistence, and replacement capacity of engineered microglia.

Main Results:

• The G795A mutation confers resistance to CSF1R inhibitors (PLX3397, PLX5622) without altering receptor function.
• G795A-expressing macrophages efficiently engraft and persist in the mouse brain during inhibitor treatment.
• Engineered G795A-iMG successfully replaced endogenous microglia in a xenotransplantation model, demonstrating functional and gene expression similarity to wildtype iMG.

Conclusions:

• The engineered G795A CSF1R variant enables robust and specific microglia replacement.
• This approach offers a potentially safer and more effective cell-based therapy for neurological conditions requiring microglia modulation.