Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

Abstract

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell–derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Tissue-specific macrophages: how they develop and choreograph tissue biology

“…Introducing a diverse microbiome in experimental mice, as done in the 'wildling' mice, has been shown to represent a much closer model to humans than using specificpathogen free mice and might be also important to study in the context of macrophage-targeted therapies 172,173 . Recent mouse studies using macrophage replacement models by using genetic or pharmacological inhibition of the colony-stimulating factor 1 receptor have shown promise for translation to patients, for example, in the context of replacing dysfunctional microglia during neurodegenerative diseases 231 . Finally, although in humanized mice the human immune system development can be recapitulated, the niche-specific imprinting on human macrophages will be derived from mouse tissues.…”

A Trisomy 21-linked Hematopoietic Gene Variant in Microglia Confers Resilience in Human iPSC Models of Alzheimer’s Disease

“…The engulfment of WT microglia by A455D microglia following exposure to p-Tau is additionally substantiated by scRNA-seq ligand-receptor interaction analysis of the interplay between WT and A455D microglia. As demonstrated in recent studies 72,73,83,84 , efficient microglia replacement by peripheral cell sources or through direct microglia transplantation requires the treatment with destructive agents, such as CSF1R inhibitors and/or toxic preconditioning of the bone marrow niche. Our results suggest that in the context of AD, eliminating endogenous senescent/dystrophic microglia may not be necessary.…”

Microglial Ffar4 deficiency promotes cognitive impairment in the context of metabolic syndrome

“…6I). To elucidate the role of microglia in behavioral abnormalities of cKO/HFD mice, we took advantage of pharmacological microglia deletion using PLX3397, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, to assess the consequence of microglia elimination on cognitive function and anxiety behavior (27)(28)(29). Results showed that PLX3397 treatment markedly reduced the num ber of Iba-1-positive microglia in the hippocampus (fig.…”