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

Abstract

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.

Human Immune Responses to Adeno-Associated Virus (AAV) Vectors

“…Recently, two different approaches were reported to overcome the limitations of "conventional" methods of vector evolution. Both methods take advantage of the latest advancements in DNA synthesis and sequencing, and are aimed at the reduction of the complexity of the initial library to be screened either by artificial intelligence (6) or by grafting peptides derived from a subset of proteins involved in specific cellular functions (36). Regardless of the method used, the isolation of new rAAV vector capsids responds to a precise need of increased transduction efficacy with optimized biodistribution and reduced immunogenicity, at least in terms of cross-reactivity with preexisting antibodies.…”

AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In‐house AAV Vector Production Based on Chloroform Extraction

“…AAV1,2,3,5,6,7,8, and 9 exhibit high affinity for central nervous system tissue, whereas AAV4 efficiently transduces the eye, and AAV1, 6, and 9 can also infect heart and skeletal muscle (Vance et al, 2015). Even though AAV can efficiently transduce a wide array of tissues and cell types, huge efforts are being put in to generating new and improved viral capsids (Davidsson et al, 2019;Deverman et al, 2016;Ojala et al, 2018). Another way to improve transduction efficiency regardless of serotype used was the development of self-complementary AAVs (scAAV;McCarty, Monahan, & Samulski, 2001).…”

Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role forSptbn2in neurulation and abdominal wall closure

“…Transduction of virus encoding sgRNA into Cas9-expressing mice would allow targeted knockouts or gene editing, either to introduce specific disease-associated mutations, or to correct mutant alleles and test therapeutic gene editing. The rise of AAV evolution (28,29), and thus engineering viral tropism, may also allow targeted transduction of specific organs during embryonic development.…”