Development of a stem cell platform for the treatment of neurodegenerativemicrogliopathies

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Project Summary We have developed a platform technology that uses induced human pluripotent stem cells (iPSCs) to generate genetically corrected microglia at scale and with high purity for transplantation. In this study, we will demonstrate the safety and therapeutic efficacy of our microglia transplantation technology using a mouse genetic model of adult-onset leukoencephalopathy with axonal spheroids & pigmented glia (ALSP), a fatal disease with no effective treatments. The three overlapping aims to be investigated in this Phase I project are: Aim 1: Optimize microglial transplantation delivery parameters in a mouse genetic model of microglial insufficiency. We have preliminary data showing that our iPSC-derived microglia can repopulate the brains of microglia-deficient mice. We will test three different microglia injection locations, and after determining the optimal injection location using whole brain clearing and quantification, we will then evaluate different numbers of cells at this optimized location. Aim 2: Determine the safety of microglial transplantation in the mouse model of microglial insufficiency. To assess the safety of our microglial repopulation, we will determine the percentage of cells that express non- microglial markers, examine brain tissue using immunohistochemistry for markers of undifferentiated iPSCs and proliferative cell markers, and determine the presence of any non-native human cells in relevant peripheral tissues. Aim 3: Demonstrate the efficacy of microglial transplantation on reduction of pathological ALSP hallmarks in the mouse model brain. Brain calcification, axonal spheroids, and astrogliosis are hallmarks of ALSP in human cases, as well as the mouse model. We have preliminary data demonstrating that microglial transplantation reduces these hallmark pathologies. Our delivery parameters from Aim 1 will be used for microglial transplantation. We will assess pathology reduction in the Aim 1 optimized delivery paradigm mice using confocal imaging and quantification of: osteopontin and Alazarin Red staining to identify calcified tissue, neurofilament light chain staining to identify axonal spheroids, and GFAP staining to identify astrogliosis. We expect a > 30% reduction in all of these pathologies with microglial transplantation. Our Phase I results will provide proof-of-concept data to support the development of iPSC-derived microglial cell therapy for ALSP and related neurodegenerative microgliopathies.