PROJECT SUMMARY/ABSTRACT
Neuropsychiatric disorders represent a leading cause of disability, affecting nearly 19% of the US population.
Only 9% of neuropsychiatric drugs entering clinical trials reach the market, which is one of the lowest success
rates across all therapeutic areas. Fundamental differences between the neurobiology of rodents and humans
have been proposed to account for translational failures in development of effective therapeutic strategies to
mitigate neurological or neurodegenerative diseases or disorders. Rodent behavioral assays are also variably
effective in predicting clinically effective neuropsychiatric drugs. Nonhuman primates (NHPs) are recognized as
a valuable, clinically relevant alternative to span the gap between rodents and humans in the development of
therapies designed to advance brain health. Among NHPs, the common marmoset [Callithrix jacchus (cj)] affords
a highly tractable option because of its small size, short lifespan, production of multiple offspring/year and
accurate recapitulation of human neuroanatomy. However, the ultimate utility of the marmoset model remains in
its infancy due to the paucity of efficient tools to facilitate studies requiring genetic modification, especially those
needed to recapitulate complex aspects of brain health. To address this urgent need, we propose an innovative,
more efficient approach to achieve gene editing and transgenesis in marmosets based on the novel use of highly
manipulable induced pluripotent stem cells (iPSCs) that can be differentiated to form male germ cells that can
ultimately be used to produce transgenic offspring carrying precisely edited alleles of genes relevant to brain
health and disease. Specifically, we will combine 1) close proximity to one of two NIH-designated Marmoset
Breeding Colonies, maintained at the Southwest National Primate Research Center, 2) experience with NHP
pluripotent stem cells, iPSC derivation, and CRISPR/Cas9 editing, 3) a novel strategy to produce transplantable
male germ cells from edited cjiPSCs, 4) documented expertise transplanting NHP germ cells into testes to
produce sperm, 5) published experience in the use of cutting-edge single-cell genomics and multiparametric
integrative epigenomics to assess normality of any cell type, and 6) leading expertise in brain health and disease
in general and the neurogenetics of epilepsy in particular. In Aim 1, we will use CRISPR editing to generate
mutant ARX alleles and reporter transgenes in cjiPSCs. In Aim 2, we will optimize derivation and transplantation
of male cjiPSC-derived germ cells into recipient testes and grafts to foster development of transgenic sperm. In
Aim 3, we will assess the impact of ARX mutations on marmoset cortical neuron development and migration.
Together, these aims are designed to advance the utility of the marmoset model for brain research based on
CRISPR/Cas9 editing of cjiPSCs, male germline-mediated transgenesis, development of cjiPSC-derived brain
organoids, and specific knowledge of the neurological impact of ARX mutations.