In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

Abstract

SUMMARY Adeno-associated viral vectors (AAV) have emerged as a gene delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAV, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In silico derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of 9 functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8 and 9 as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

“…In line with previous findings, 18,19,21 DSF experiments showed that AAV5 was stable up to temperatures approaching 90 C (Tm = 87.4 C) and AAV9 denatured at 76.2 C. AAVrh32.33, a serotype that has not been previously studied, denatured at 64.6 C ( Figure 1B). These measurements were validated by IFS ( Figure 1C) and SDS-PAGE ( Figure 1D) experiments.…”

“…18 Major differences between the melting temperatures of naturally occurring AAV serotypes were observed (AAV2: melting temperature [Tm] = 69.6 C ± 0.5 C; AAV5: Tm = 89.7 C ± 0.7 C), 18 whereas reconstructed ancestral AAV particles exhibited enhanced thermostability in comparison to most of their contemporary, naturally occurring homologs. 19 In this study, we explore the possibility of using the biophysical measure of thermostability for AAV serotype identification at the protein level. We show, through the analysis of 67 AAV samples by DSF, that capsid thermostability can be used to discriminate AAV1, AAV2, AAV5, AAV6.2, AAV8, and AAV9 preparations, regardless of the packaged transgene and vector concentration.…”

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

“…Given the spectrum of AAP phenotypes observed across the major clades, we similarly tested 9 putative evolutionary intermediates (AncAAVs) to the major AAV serotypes to gain insight into which elements of VP structure either impose the observed requirement for AAP or impart an ability for some VPs to perform these functions independently (Zinn et al, 2015). As with the nat ural serotypes, a broad range of requirements for AAP was observed for the AAPstop60 AncAAVs (Figure 3A).…”

“…This work was built on the assumption that ancestral AAVs were assembly-competent and that our computational and statistical modeling effort was able to approximate the molecular state of these putative evolutionary intermediates. Indeed, we predicted the primary structure of 9 putative bifurcation points within the lineages of AAV serotypes 1–3 and 6–9, each of which, to varying degrees, demonstrated assembly, viral genome packaging, and in vitro infectivity (Zinn et al, 2015). These reagents now provide us with a toolset to interrogate viral and vector phenotypes across much shorter genetic distances than previously possible.…”

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.…”