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.

PROJECT NARRATIVE A major hurdle in developing effective neurological treatments has been translation of scientific discoveries from rodent models to humans. To close this critical translational gap, we will use pluripotent stem cell-, CRISPR/Cas9-based gene editing-, male germ line-, 3D organoid-, and marmoset tissue-derived-tools to explore ARX-associated mutations linked to brain cortex malformation, epilepsy, and intellectual disability. The overarching goals of this project are to develop a marmoset model system to represent the neurobiology of the human brain and to develop a novel pluripotent stem cell/male germline-mediated transgenesis approach to advance the utility of the marmoset model to benefit the brain health research community.