Evolution and Investigation of Engineered AAV Capsids Exhibiting Enhanced Transduction of the Central Nervous System with or without Murine Strain Specificity

Abstract

Recombinant adeno-associated viral (rAAV) capsids are increasingly used as gene delivery vectors in science and in therapeutics. However, there is room for further improvement on the efficiency and specificity to transduce the central nervous system (CNS) via non-invasive systemic delivery. We have shown in the past that marked improvement is possible using a Cre recombination-based AAV targeted evolution (CREATE) platform to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). While CREATE was successful in identifying efficient CNS vectors in two rounds of evolution, the method was limited by its ability to identify only a few top candidates from the selection. To truly utilize the potential of a large selection design (such as a library of ~1.28 billion theoretical size using 7-mer-NNK mutagenesis strategy) across multiple different selection targets (such as endothelial cells, neurons and astrocytes), we developed Multiplexed-CREATE (M-CREATE). This method involves: (1) application of a positive selection pressure for on-target delivery and post-hoc negative selection against off-target delivery using next generation sequencing, (2) an unbiased selection design, and (3) a novel data analysis platform. M-CREATE identified distinct families of variants based on shared amino acid motifs. These include a family of PHP.B-like variants that appear to be dominant across in vivo selections for CNS transduction in Cre-transgenic adult mice. While some variants from this family showed similar tropism to PHP.B as expected, two variants (PHP.Vs) were distinct by exhibiting biased transduction to the vascular cells forming the blood-brain barrier (BBB). In addition, the new analysis platform allowed us to further mine the selection that yielded PHP.eB from its PHP.B parent (Chan et al, 2017) and therefore uncover PHP.N, that differs by 3 amino acids from PHP.B and shows greater specificity in transducing neurons over other CNS cell-types. This further demonstrates that small sequence variations within a family can yield different tropisms. Recent reports on the non-permissibility of PHP.B or PHP.eB in BALB/cJ (Hordeaux et al, 2018; Matsuzaki et al, 2019), and the subsequent identification of LY6A as the receptor underlying the improved BBB entry (Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) has motivated us to investigate the non-dominant families with distinct amino acid signatures in our 7-mer NNK library. We identified several non-dominant families (PHP.Cs), exhibiting better transduction of the CNS compared to parent AAV9 while also being efficient in crossing the BBB across mouse strains, including BALB/cJ. This suggests that variants with different amino acid motifs from the PHP.C-like families may have different mechanisms of BBB transmission, thereby making them promising candidates for further study toward translation of AAV vectors across strains and species.