Nonhuman primate-derived enhancers for studying neural circuit functions

The National Institute of Mental Health awarded the University of Pittsburgh a Cooperative Agreement totaling $4,028,357 on September 1, 2026, under the Mental Health Research Grants program (CFDA 93.242) to develop and validate nonhuman primate-derived enhancers for cell type-specific neural circuit studies.

The University of Pittsburgh will conduct high-throughput in vivo screening of 3,600 enhancers over the five-year award period (720 per year), testing each in two macaques and one marmoset. Researchers will use spatial transcriptomics to identify top-performing enhancer sequences and package the best 175 enhancers into adeno-associated virus (AAV) capsids paired with optogenetic and chemogenetic actuators. The work builds on prior discovery and validation of enhancer-driven AAVs for targeting neurons in nonhuman primate cortex and striatum. Machine learning methods incorporating comparative genomic tools will predict cell type-specific enhancers from multi-omic data (whole transcriptome and open chromatin regions) collected from approximately two million brain cell nuclei across three species—rhesus, capuchin, and marmoset—spanning 21 brain regions.

The award funds collaborative validation work at the University of Pittsburgh and Emory University, where three labs will clone enhancers paired with excitatory opsins and chemogenetic actuators into AAV backbones, package the constructs, and inject them at different titers into rhesus and marmosets. Work is performed in Pittsburgh, Pennsylvania. The period of performance runs through August 31, 2031.

Abstract

Nonhuman primate (NHP) research using cutting-edge molecular reagents, including optogenetic and chemogenetic actuators, is crucial for understanding the mechanisms of cognition and the causes of neurodegenerative and neuropsychiatric diseases. However, cell type specific molecular access to NHP neurons is impeded by the lack of available tools, which are currently limited to a handful of well-validated reagents. Our armamentarium pilot project discovered and validated enhancer-driven adeno-associated viruses (AAVS) for cell type specific targeting of neurons in the NHP cortex and striatum. Our armamentarium 2.0 aims will vastly expand this toolkit to enable unique systems neuroscience studies in NHP. We collected 'multi-omic' - whole transcriptome and open chromatin regions (OCRS) - data from ~2 million brain cell nuclei from three NHP species: rhesus, capuchin, and marmosets, and across 21 brain regions. We developed cutting-edge machine learning (ML) methods incorporating comparative genomic tools and bayesian confidence estimation methods to predict - from thousands of NHP genome-derived cell type specific OCRS - the enhancers that will work best in nhps. We developed and validated a screening approach that combines a massively parallel reporter assay with spatial transcriptomics. We will perform high-throughput in vivo screening of 720 enhancers per year (3,600 total), each in two macaques and one marmoset, and we will use spatial transcriptomics to identify the enhancers that perform best in both NHP species. The top performing NHP enhancer sequences (175 over 5 years) will be packaged into AAV capsids and validated in parallel by three labs working at the university of pittsburgh and emory university. We will clone enhancers paired with excitatory opsins and chemogenetic actuators into AAV backbones, package the constructs, and inject them at different titers into rhesus and marmoset brains. We will perform in vivo physiology and post-mortem immunohistochemistry experiments to validate functionality, cell type specificity, and efficiency. Specificity and efficiency will be quantified using a high-throughput whole NHP brain mapping pipeline that automatically registers multimodal imaging data and uses ML algorithms to detect AAV expression. Data on validated enhancers will be systematically cataloged in a new online open-science macaque and marmoset brain tool resource - the circuitbank atlas (aim 3). We will use the same framework as our recently developed marmoset neuroscientific apparatus (MNSA), an online open-access resource for marmoset neuroscience. The circuitbank atlas will allow users to view detailed, zoomable microscopy images that are seamlessly aligned with MRI templates directly within their web browsers. Users can select from our catalog of validated constructs and access the associated expression data, optimized usage protocols, and links to directly order the constructs. Altogether, our research plan satisfies all criteria of the armamentarium for precision brain cell access. This work will provide comprehensive access to NHP brains and enable circuit-based studies of primate brain functions.