Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

Author:

Chadarevian Jean Paul123ORCID,Lombroso Sonia I.4567ORCID,Peet Graham C.458ORCID,Hasselmann Jonathan13ORCID,Tu Christina23ORCID,Marzan Dave E.9ORCID,Capocchi Joia2ORCID,Purnell Freddy S.45ORCID,Nemec Kelsey M.4510ORCID,Lahian Alina23ORCID,Escobar Adrian3ORCID,England Whitney11ORCID,Chaluvadi Sai4510ORCID,O’Brien Carleigh A.45ORCID,Yaqoob Fazeela45ORCID,Aisenberg William H.45ORCID,Porras-Paniagua Matias12ORCID,Bennett Mariko L.1013ORCID,Davtyan Hayk23ORCID,Spitale Robert C.11ORCID,Blurton-Jones Mathew123ORCID,Bennett F. Chris4513ORCID

Affiliation:

1. Department of Neurobiology & Behavior, University of California, Irvine 1 , Irvine, CA, USA

2. Institute for Memory Impairments and Neurological Disorders, University of California, Irvine 2 , Irvine, CA, USA

3. Sue and Bill Gross Stem Cell Research Center, University of California, Irvine 3 , Irvine, CA, USA

4. Department of Psychiatry, Perelman School of Medicine 4 , , Philadelphia, PA, USA

5. University of Pennsylvania 4 , , Philadelphia, PA, USA

6. Pharmacology Graduate Group, Biomedical Graduate Studies Program, University of Pennsylvania 5 , Philadelphia, PA, USA

7. Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania 6 , Philadelphia, PA, USA

8. Neuroscience Graduate Program and Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus 7 , Aurora, CO, USA

9. Department of Biology, The College of New Jersey 8 , Ewing, NJ, USA

10. Department of Neuroscience, Perelman School of Medicine 9 , Philadelphia, PA, USA

11. Department of Pharmaceutical Sciences, University of California, Irvine 10 , Irvine, CA, USA

12. Department of Bioengineering, University of Pennsylvania 11 , Philadelphia, PA, USA

13. Division of Neurology, Children's Hospital of Philadelphia 12 , Philadelphia, PA, USA

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.

Funder

National Cancer Institute

University of California, Irvine

National Institutes of Health

National Science Foundation

National Institute of Neurological Disorders and Stroke

Paul Allen Frontiers Group

The Klingenstein-Simons fellowship in neuroscience

Susan Scott Foundation

Publisher

Rockefeller University Press

Subject

Immunology,Immunology and Allergy

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