Technologies

The diversity of the nervous system arises from neuronal populations defined by combinations of molecular identity, connectivity, developmental origin, anatomy, and activity. Yet most genetic targeting strategies rely on a single defining feature, limiting experimental precision. INTRSECT (INTronic Recombinase Sites Enabling Combinatorial Targeting) overcomes this limitation by enabling programmable Boolean logic within viral vectors, allowing researchers to access neuronal populations defined by combinations of biological features rather than single markers. This framework established a general approach for programmable molecular targeting and continues to inform our development of next-generation technologies that expand the precision, versatility, and therapeutic potential of viral gene delivery.

VESPA overcomes the ~4.8 kb packaging limit of adeno-associated virus (AAV) by splitting oversized genes across multiple viral vectors and precisely reassembling them inside the cell. The platform uses PhiC31 serine integrase to irreversibly reconstruct full-length DNA, avoiding the reversibility of Cre-based approaches and the residual protein fragments produced by intein-based splitting. Synthetic introns conceal the small DNA scar left by integration, yielding a normal, full-length protein after splicing. Applied to SCN1A, a gene too large for conventional AAV delivery and mutated in Dravet syndrome, VESPA restored interneuron function and improved survival in a mouse model, demonstrating its potential to expand the range of diseases addressable by AAV gene therapy.

This platform packages a Cre-dependent SaCas9 and a U6-driven sgRNA cassette into a single AAV, restricting CRISPR/Cas9 gene editing to genetically defined cell types in vivo without a separate Cas9 mouse line. A rapid in vitro sgRNA screening pipeline predicts in vivo editing efficiency ahead of virus production, so candidate guides can be ranked before committing to an animal experiment, and independent guides can be combined to push knockdown efficiency higher. As a first application, this approach was used to disrupt the circadian gene Clock selectively in dopamine neurons of the ventral tegmental area, producing titer-dependent gene knockdown and revealing causal links between circadian gene function in a single neural cell type and bipolar disorder-relevant behavioral, sleep, and electrophysiological phenotypes.