Adeno-associated viruses (AAVs) are potent gene delivery vectors that are widely used in laboratory animals and in a growing number of clinical trials. However, AAVs are not naturally cell type specific; this limitation prevents the widespread use of genetically coded tools to study circuit function in genetically intractable species, like non-human primates (NHPs), and hinders the development of cell type specific and circuit targeted therapeutic strategies.
In this 4-year project funded by the NIH’s National Institute of Mental Health, researchers will use cutting edge experimental, genomic, and computational tools to create and validate a suite of next generation AAVs that drive cell type specific expression in the NHP cognitive and reward systems and establish a foundation for circuit-targeted translational applications. McGowan Institute for Regenerative Medicine affiliated faculty member Leah Byrne, PhD (pictured), Assistant Professor, Department of Ophthalmology, University of Pittsburgh, with secondary appointments in the Departments of Neurobiology and Bioengineering, is a co-principal investigator on this project which began on September 1, 2022.
The abstract for this project reads:
Adeno-associated viruses (AAVs) are potent gene delivery vectors for neuroscience studies and gene therapy applications. However, naturally occurring AAVs are not cell type specific: they must be combined with other technologies, such as transgenic animals, to achieve cell type specific gene expression. This requirement limits the use of genetically coded ‘circuit-breaking’ tools to study behavior in nonhuman primates (NHPs) – the experimental animal model with the greatest similarity to humans – and hinders development of cell type specific targeting strategies for achieving direct clinical benefits. To expand cell type specific access in NHPs and lay the foundation for circuit specific gene therapy, we propose to create, test, and validate next generation, cell type specific AAVs. First, we will define cell type specific enhancers – distal regulatory elements that have demonstrated considerable promise as cell type specific AAV drivers. In preliminary data, we collected transcriptomic and chromatin accessibility (i.e., “multi-omic”) single cell data from the striatum, dorsolateral prefrontal cortex (dlPFC), primary motor cortex (M1), insula, and ventral midbrain of 2 rhesus macaque monkeys. We combined the rhesus monkey data set with existing human and mouse data and used convolutional neural networks (CNNs) to rank open chromatin sequences according to their potential as cell type specific enhancers. We packaged AAVs with the top candidate enhancers, injected them into NHP striatum, and observed cell type specific, enhancer driven expression in striosomes – a cell type specific striatal compartment related to reward processing. To broadly advance this agenda and develop AAVs that drive robust, cell type-specific expression, we propose to expand our multi-omic single cell database with additional data from macaque and marmoset. We will leverage this updated, sex-balanced database, which will include data from 8 NHPs to identify cell type specific enhancers that are likely to drive robust expression in primates. In parallel, we will use our validated scAAVengr pipeline to screen AAV capsid mutants for cell-type biased infection patterns in the NHP cognitive and reward systems. We will combine the top cell type specific enhancers with the most biased AAV capsids to generate new, cell type specific AAVs for targeting neurons in the NHP cognitive and reward systems. We will validate AAV specificity using Fluorescent in situ hybridization (FISH). This data will be combined with ultra-high resolution MRI scans to create a rhesus macaque brain atlas, and the validated vectors will be stored and distributed by The University of Pittsburgh BioForge Initiative. NHPs are critical for studying human cognition and disease, and thus there is a pressing need to define the molecular properties of NHP cell types and study their behavioral functions. This proposal will generate a unique NHP multi-omic single cell database, provide cell type specific AAVs for neuron types in cognitive and reward systems, and establish a new multimodal rhesus brain atlas. These contributions will significantly advance circuit manipulation capabilities in the primate brain and promote fundamental research in basic and preclinical science.
Congratulations, Dr. Byrne!
NIH Reporter: Cell type specific AAVs to study reward and cognition
UPMC News Release