A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism

Abstract

Significance A challenge with the available synthetic viruses used for the treatment of genetic disorders is that they originate from wild-type viruses. These viruses benefit from infecting as many cells as possible in the body, while therapies should most often target a particular cell type, for example, dopamine neurons in the brain. In this paper, we present a technique for developing targeted synthetic viruses suitable for clinical therapy. We show that such viruses can be designed to target human dopamine neurons in vivo and to transport along the connective pathways in the brain, allowing for unprecedented accuracy of the therapy. Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.