AAV ancestral reconstruction library enables selection of broadly infectious viral variants

Abstract

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

“…At present, no experimental or computational approaches are available for engineering future antigenic variants of AAV before they emerge in nature. Earlier engineering strategies to address the problem of NAbs relied on the limited antigenic variability among different natural AAV isolates and yielded chimeric or ancestral AAV capsids (20)(21)(22). Complementarily, our approach explores the entire sequence space for each amino acid residue within antigenic footprints located on any AAV capsid surface.…”

Systemic delivery of adeno-associated viral vectors

“…Several strategies have been used to develop novel capsids for improved systemic delivery. These include (1) isolation and reconstruction from existing or ancestral species [110,111], and (2) modification by rational design and directed evolution [112,113]. …”

In vivo directed evolution of AAV in the primate retina

“…The ITRs in AAV are highly recombinogenic, leading to the possibility of recombination and subsequent replication in the presence of a helper virus when using this strategy, so an intron was inserted in the rep supplied in trans to prevent packaging of recombined genomes (Figure 1). Replication incompetent libraries were constructed, packaged, and included in the primate screen including AAV2-7mer, Ancestral-7mer( 12 ) and LoopSwap( 13 ) libraries.…”