Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types

Recombinant adeno-associated viruses (rAAVs) are efficient, non-invasive gene delivery vectors via intravenous delivery, however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our previous Cre recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to quickly and accurately identify variants of interest in a given selection landscape through multiple positive and negative selection criteria by incorporating next-generation sequencing, synthetic library generation, and a novel analysis pipeline. In vivo selections for brain endothelial cell-, astrocyte-, and neuron-transducing capsids have identified variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity, or cross the blood-brain barrier across diverse murine strains. Collectively, M-CREATE methodology accelerates the discovery of novel capsids for use in neuroscience and gene therapy applications.