High-throughput characterization of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases have remained largely unexplored. Here, we firstly comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC-derivatives in mice. We then generated a high-resolution single cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature which is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases. Overall design: We combined cell sorting with high-throughput single-cell RNA (scRNA) sequencing to characterize long-term engrafted Microglia-Like Cells (MLCs) and endogenous microglia from the same animals. We isolated endogenous microglia (GFP-negative) and MLCs (GFP-positive) from enzymatically dissociated whole brains by FACS based on the expression of cell surface markers (CD45+, CD11b+, CX3CR1+) from one male and one female mouse 12-13 months after either IV or ICV administration of hematopoietic stem/progenitor cells (HSPCs). We also generated comparator datasets from a 12-week-old, treatment naïve C57BL/6J animal for several cell populations: microglia (CD45+, CD11b+, CX3XCR1+), neurons (CD45-, Thy1+, ACSA2+), astrocytes (CD45-, Thy1-, ACSA2+) and peripheral blood mononuclear cells (PBMCs)