Development of a stem cell platform for the treatment of neurodegenerativemicrogliopathies

Project SummaryWe 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 andamp; 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 andgt; 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.Project Narrative
Our project seeks to develop our platform technology to produce microglia from human induced pluripotent stem
cells for the treatment of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia. This fatal
neurodegenerative disease is caused by mutations in the microglia gene CSF1R. In this work we will use a
mouse model of this disease to demonstrate the safety and efficacy of our microglia transplantation therapy, with
our future goals focused on developing a much needed treatment for human patients.