Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design

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Abstract

Adeno-associated virus (AAV) capsids can deliver transformative gene therapies, but our understanding of AAV biology remains incomplete. We generated the complete first-order AAV2 capsid fitness landscape, characterizing all single-codon substitutions, insertions, and deletions across multiple functions relevant for in vivo delivery. We discovered a frameshifted gene in the VP1 region that expresses a membrane-associated accessory protein that limits AAV production through competitive exclusion. Mutant biodistribution revealed the importance of both surface-exposed and buried residues, with a few phenotypic profiles characterizing most variants. Finally, we algorithmically designed and experimentally verified a diverse in vivo targeted capsid library with viability far exceeding random mutagenesis approaches. These results demonstrate the power of systematic mutagenesis for deciphering complex genomes and the potential of empirical machine-guided protein engineering.

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Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65 -mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial

Stephen Russell, Jean Bennett, Jennifer Wellman … (2017)

Background Phase 1 studies have shown potential benefit of gene replacement in RPE65 -mediated inherited retinal dystrophy. This phase 3 study assessed the efficacy and safety of voretigene neparvovec in participants whose inherited retinal dystrophy would otherwise progress to complete blindness. Methods In this open-label, randomised, controlled phase 3 trial done at two sites in the USA, individuals aged 3 years or older with, in each eye, best corrected visual acuity of 20/60 or worse, or visual field less than 20 degrees in any meridian, or both, with confirmed genetic diagnosis of biallelic RPE65 mutations, sufficient viable retina, and ability to perform standardised multi-luminance mobility testing (MLMT) within the luminance range evaluated, were eligible. Participants were randomly assigned (2:1) to intervention or control using a permuted block design, stratified by age (<10 years and ≥10 years) and baseline mobility testing passing level (pass at ≥125 lux vs <125 lux). Graders assessing primary outcome were masked to treatment group. Intervention was bilateral, subretinal injection of 1·5×10 11 vector genomes of voretigene neparvovec in 0·3 mL total volume. The primary efficacy endpoint was 1-year change in MLMT performance, measuring functional vision at specified light levels. The intention-to-treat (ITT) and modified ITT populations were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00999609, and enrolment is complete. Findings Between Nov 15, 2012, and Nov 21, 2013, 31 individuals were enrolled and randomly assigned to intervention (n=21) or control (n=10). One participant from each group withdrew after consent, before intervention, leaving an mITT population of 20 intervention and nine control participants. At 1 year, mean bilateral MLMT change score was 1·8 (SD 1·1) light levels in the intervention group versus 0·2 (1·0) in the control group (difference of 1·6, 95% CI 0·72–2·41, p=0·0013). 13 (65%) of 20 intervention participants, but no control participants, passed MLMT at the lowest luminance level tested (1 lux), demonstrating maximum possible improvement. No product-related serious adverse events or deleterious immune responses occurred. Two intervention participants, one with a pre-existing complex seizure disorder and another who experienced oral surgery complications, had serious adverse events unrelated to study participation. Most ocular events were mild in severity. Interpretation Voretigene neparvovec gene replacement improved functional vision in RPE65 -mediated inherited retinal dystrophy previously medically untreatable. Funding Spark Therapeutics.

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Clades of Adeno-associated viruses are widely disseminated in human tissues.

Guangping Gao, Luk H Vandenberghe, Mauricio R Alvira … (2004)

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors.

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Identification of a heparin-binding motif on adeno-associated virus type 2 capsids.

Marjanka K. Schmidt, W Van der Pol, O Muller … (2003)

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.