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

Ravindra Kumar et al, 2020 (unpublished)Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of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 Cre recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation, and a dedicated analysis pipeline. We have identified capsid 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, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene therapy applications.The next-generation sequencing data included in this project are the AAV capsid libraries that were recovered from the Round 1 selection's AAV DNA and virus libraries; Round 2 selection's DNA, virus and mouse brain libraries as per the synthetic pool design and PCR pool design described in the M-CREATE method.