In vivo–directed evolution of adeno-associated virus in the primate retina
Leah C. Byrne, Timothy P. Day, Meike Visel, Jennifer A. Strazzeri, Cécile Fortuny, Deniz Dalkara, William H. Merigan, David V. Schaffer, John G. Flannery
Leah C. Byrne, Timothy P. Day, Meike Visel, Jennifer A. Strazzeri, Cécile Fortuny, Deniz Dalkara, William H. Merigan, David V. Schaffer, John G. Flannery
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Research Article Ophthalmology

In vivo–directed evolution of adeno-associated virus in the primate retina

Abstract

Efficient adeno-associated virus–mediated (AAV-mediated) gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution — guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library — to create AAV viral capsids with the capability to deliver genes to the outer retina in primates. A replication-incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates — involving intravitreal library administration, recovery of genomes from outer retina, and extensive next-generation sequencing of each round — resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These viral vectors expand the toolbox of vectors available for primate retina, and they may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

Authors

Leah C. Byrne, Timothy P. Day, Meike Visel, Jennifer A. Strazzeri, Cécile Fortuny, Deniz Dalkara, William H. Merigan, David V. Schaffer, John G. Flannery

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Figure 1

Replication-incompetent AAV libraries.

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Replication-incompetent AAV libraries. (A) Schematic depicting both reco...
(A) Schematic depicting both recombination and nonrecombination events with the pRepIntronHelper. If a recombination event were to occur, the intron sequence (nebulin intron 8, 774 bp) would push the transgene over the packaging capacity of AAV, leading to incomplete packaging. If recombination does not occur, the mutated rep sequence will be packaged, mitigating the possibility of replication. (B) Titering of the rep in trans system with and without pRepIntronHelper, compared with the transgene with native rep, pSub2Cap2. The rep in trans system leads to similar titers as normal pSub2Cap2 packaging. An adenovirus rescue study determined that the rep in trans system leads to a greater than 10-fold reduction in replication. The abilities of an AAV9 error-prone library and the 7-mer-Ancestral library to replicate with the rep in trans system are shown, compared with an AAV2 with the wild-type genome and a replication-incompetent AAV2 with a CMV-GFP transgene. AAV, adeno-associated virus.