Development of marmoset models of neurodegenerative disease using embryonic stem cell-based gene-editing approaches

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PROJECT SUMMARY The long-term goal of this project is to model human neurodegenerative diseases in marmosets via gene- editing in embryonic stem cells (ESCs). The mouse system is a powerful tool for medical research due to the ability to manipulate the mouse genome. However, considerable anatomical, physiological, cognitive, and behavioral differences between mice and humans limit the degree to which insights from mouse models shed light on human diseases. This is reflected in the high number of failed clinical trails for drugs that were effective in treating mouse models of human disease. Several lines of evidence suggest that the marmoset represents an improved animal system for studying a range of human diseases, including stroke and age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Marmosets are the shortest-lived of the anthropoid primates (average lifespan of 5–7 years compared with 25 years for the rhesus macaque) and exhibit age-related changes that are similar to those seen in humans, including β-amyloid deposition in the cerebral cortex, loss of cholinergic innervation, and reduced neurogenesis, as observed in AD. In addition, marmosets are highly social and communicative and have demonstrated the capacity to learn sophisticated cognitive behaviors. Therefore, marmosets represent an ideal genetic platform for generating models of neurodegenerative diseases that more accurately reflect the human condition and enable the testing of potential autologous (the-same-species) stem cell-based regenerative therapies. Initial efforts will focus on generating a marmoset model of AD. The recent emergence of gene-editing and stem-cell technologies in primates pave the way toward generating marmoset disease models, but improvements in both areas are necessary to make this approach viable. Here, both conventional homologous recombination and CRISPR/Cas9 genome-editing technologies will be employed to modify marmoset ESCs. As genetic evidence demonstrates that mutations in the amyloid precursor protein (APP) gene result in increased β-amyloid production, the formation of plaques, and cognitive impairment, the marmoset APP will be edited to carry human point mutations. Genetic tools for studying neuronal cell type-specific circuits underlying cognitive impairment and neuropathology in AD will also be generated by inserting a Cre recombinase cassette into 3' end non-translated regions of the parvalbumin and choline acetyltransferase genes. These Cre driver lines will enable the visualization and functional manipulation of these cell types. Successful completion of the proposed Aims will generate a greatly improved animal model of AD, enable testing of stem cell-based regenerative methods for treating AD, and pave the way toward applying these genetic tools for analyzing neuronal circuitry of healthy brains. Establishing gene-editing in marmoset ESCs will also enable the development of additional primate models of human diseases, providing critical experimental resources for research supported by multiple NIH Institutes (e.g., NINDS, NIA, NIMH, NEI, NIAAA, NIDA, NICHG, NIGMS).