Harnessing human iPSC-microglia for CNS-wide delivery of disease-modifying proteins in Alzheimer's disease mice
Many studies have sought to circumvent the protective qualities of the blood brain barrier to deliver therapeutic proteins to the brain for the treatment of neurodegenerative diseases. Yet, brain specific uptake, peripheral toxicity, off-target effects, and repeated dosing continue to present considerable challenges. As proof of principle, we sought to determine whether human iPSC-microglia (iMG) could be genetically engineered ex vivo to enable pathology-responsive delivery of amyloid-targeting proteins to the brain. Human iPSCs were CRISPR-edited to express the beta-amyloid degrading enzyme neprilysin (NEP) or secreted neprilysin (sNEP) under control of the endogenous CD9 promoter, a microglial gene that is specifically upregulated in plaque-associated microglia. Following adult-transplantation into xenotolerant, amyloid-accumulating mice (5x-MITRG), beta-amyloid peptides, synaptic markers, and off-target substrate levels were examined. To further determine whether increased engraftment of therapeutic microglia could provide additional disease-modifying efficacy, sNEP iMGs were further modified to confer resistance to CSF1R antagonists, enabling brain-wide engraftment of human microglia. Amyloid pathology and synaptic, inflammatory, and neurodegeneration biomarkers were then assessed. We demonstrate that microglia can be engineered to enable pathology-responsive delivery of therapeutic proteins to the brain. NEP and sNEP xenografted microglia reduced levels of amyloid peptides and oligomers, prevented pathology-associated reductions in synaptic markers, and lowered astrogliosis within the hippocampus and overlying cortex of 5x-MITRG mice without off-target degradation. Surprisingly, sNEP delivery by microglia adjacent to the injection site alone was sufficient to achieve almost all disease-modifying outcomes as effectively as CNS-wide sNEP-microglia engraftment. However, significant reductions in amyloid pathology, dystrophic neurites, and perineuronal nets within the plaque-dense subiculum of 5x-MITRG mice was only achieved following CNS-wide microglial replacement. Taken together, these results demonstrate that human microglia can be engineered as a promising new immune cell therapeutic platform to provide widespread and pathology-responsive delivery of biological therapeutics for the treatment of neurodegenerative disease. Overall design: To identify human microglia populations that are altered in response to in vivo cuprizone treatment, immuno-deficient hCSF1 mice xenotransplanted with human iPSC-derived hematopoietic progenitor cells were treated with cuprizone for four weeks. scRNA-seq was preformed on mice that received normal chow (n=3) or cuprizone chow (n=3).