Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

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 repl...

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Bibliographic Details
Published in:	The Journal of experimental medicine Vol. 220; no. 3
Main Authors:	Chadarevian, Jean Paul, Lombroso, Sonia I, Peet, Graham C, Hasselmann, Jonathan, Tu, Christina, Marzan, Dave E, Capocchi, Joia, Purnell, Freddy S, Nemec, Kelsey M, Lahian, Alina, Escobar, Adrian, England, Whitney, Chaluvadi, Sai, O'Brien, Carleigh A, Yaqoob, Fazeela, Aisenberg, William H, Porras-Paniagua, Matias, Bennett, Mariko L, Davtyan, Hayk, Spitale, Robert C, Blurton-Jones, Mathew, Bennett, F Chris
Format:	Journal Article
Language:	English
Published:	United States Rockefeller University Press 06-03-2023
Subjects:	Aminopyridines - pharmacology Animals Brain - metabolism Brief Definitive Report Cell- and Tissue-Based Therapy - methods Humans Mice Microglia - metabolism Neuroinflammation Neuroscience Protein Engineering - methods Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - genetics Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - metabolism
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Disclosures: M.L. Bennett and F.C. Bennett are co-inventors on a pending patent filed by The Board of Trustees of The Leland Stanford Junior University (application 16/566,675) related to methods of microglia replacement. M. Blurton-Jones, J.P. Chadarevian, H. Davtyan, J. Hasselmann, W. England, and R.C. Spitale are co-inventors on a pending patent filed by the University of California Regents (application 63/169,578) related to genetic modification of cells to confer resistance to CSF1R antagonists. M. Blurton-Jones is a co-inventor of patent WO/2018/160496, related to the differentiation of human pluripotent stem cells into microglia. F.C. Bennett is a consultant for and shareholder in Glia Biotherapeutics Inc. M. Blurton-Jones and R.C. Spitale are co-founders of NovoGlia Inc. No other disclosures were reported. J.P. Chadarevian and S.I. Lombroso contributed equally to this paper and have the right to list their names first in their CV. M. Blurton-Jones and F.C. Bennett contributed equally to this paper.
ISSN:	0022-1007 1540-9538
DOI:	10.1084/jem.20220857