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. However, enhanced vectors are required to extend these landmark successes to other indications 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 use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Figure 1

Ancestral AAV sequence reconstruction. a) A phylogenetic tree relating a subset of extant AAV variants at node 27. Curly braced numbers indicate clade posterior probabilities . The phylogenetic tree graphic was generated in Dendroscope . b) A multiple sequence alignment of a subset of AAV variants with column-specific confidence annotated along the top with single digits. Confidence ranges from above 0.9 (shaded grey) to 0.3–0.4 (shaded white). c) A distribution of predicted ancestral amino acid sequences for node 27, residues 451–481. The character height of each amino acid is proportional to its posterior probability.

Figure 2

Variable residues mapped to the crystal structure of homologous AAV1, the closest AAV relative with an available structure. A three-dimensional molecular model of the AAV1 capsid was generated in PyMOL . An amino acid alignment of the ancestral AAV sequence with AAV1 was used to map the highlighted residues to the a) individual asymmetric unit and b) full biological assembly.

Figure 3

Dominant amino acids at variable positions after six rounds of selection. A heat map was generated based on the frequency of the most common amino acid at each position in the different libraries. The dominant amino acid and frequency at each position were determined based on sequencing results from individual clones n = 61 (synthesized library), n = 23 (post-packaging), and n=14 (for each ancestral library after selection on respective cell lines).

Figure 4

Change in amino acid frequency at variable positions after six rounds of selection. The percent change in amino acid frequency between the post-packaging library and evolved libraries after six rounds of selection on each cell line was calculated. If the identity of the dominant amino acid did not change, the increase (blue) or decrease (red) in frequency is displayed. If selection resulted in a change in amino acid identity at that position, the new amino acid and frequency is shown (yellow).

Figure 5

Identification of key variable residues by Bayesian Dirichlet-multinomial model comparison tests. A comparison of the two sets of variable amino acids was conducted to identify positions that changed significantly during selection. The posterior probability that the two sets of amino acids come from two different probability distributions was calculated assuming probability parameters that are Dirichlet-distributed with low pseudocounts to reflect sparse observed sequences. Results colored green indicate a >95% chance that the sets came from different distributions, yellow a >50% chance, red a >5% chance, and no color a <5% chance. Synth, synthesized library; PP, post-packaging; R3, round three of selection; R6, round six of selection.

Figure 6

Transduction efficiency of ancestral libraries benchmarked against natural AAV serotypes. After six rounds of selection, viral genomic DNA was recovered from ancestral libraries and packaged as rAAV scCMV-GFP along with wild type AAV 1–6, 8, and 9. Cell lines were infected at a genomic multiplicity of infection (MOI) of 2,000 (293T, IB3, B16- F10, GBM) or 32,000 (C2C12). The fraction of GFP expressing cells was quantified by flow cytometry 72 hours later. Data are presented as mean ± SEM, n = 3. AL, ancestral library.

Figure 7

Candidate ancestral variants display higher thermostability than natural serotypes. The thermostability of the ancestral library selected on C2C12 cells and of the representative ancestral variant C7 was characterized and compared to that of natural serotypes 1, 2, 5, and 6. Virions packaged with scCMV-GFP were incubated at temperatures ranging from 59.6°C to 78°C for 10 minutes before being cooled down to 37°C and used to infect 293T cells. The fraction of GFP expressing cells was quantified by flow cytometry 72 hours later. Data are presented, after being normalized to the fraction of GFP expressing cells after incubation at 37°, as mean ± SEM, n = 3.

Figure 8

Glycan dependency of candidate ancestral AAV variants. a) After six rounds of selection, the transduction efficiency of ancestral libraries carrying scCMV-GFP was quantified by flow cytometry 72 hours after infection at a genomic MOI of 2,000 (Pro5, Lec1, Lec2) and 50,000 (CHO-K1, pgsA). The CHO-K1/pgsA comparison examines heparan sulfate proteoglycan dependence, while Pro5/Lec1 and Pro5/Lec2 probe sialic acid dependence. Data are presented as mean ± SEM, n = 3. b) Glycans present on CHO glycosylation mutants. AL, ancestral library.

Figure 9

Evaluation of gastrocnemius muscle transduction. Luciferase activity measured in relative light units (RLU) per mg protein was determined in gastrocnemius tissue homogenate 48 days after intramuscular administration of 5 × 10¹⁰ viral particles of ancestral clones C4, C7, G4, or AAV1 in adult mice. Controls injected with phosphate-buffered saline displayed no activity (data not shown). *, statistical difference of P < 0.05 by two-tailed Student’s t-test.