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 3

Directed evolution of AAV in primate retina.

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Directed evolution of AAV in primate retina. (A) Deep sequencing of libr...
(A) Deep sequencing of libraries revealed convergence of variants over rounds of selection. C, central; P, peripheral. (B) In each of the libraries evaluated, a small proportion of variants were overrepresented in the plasmid library. (C) Scatterplots illustrate the behavior of individual variants over all rounds of selection for the ~588 LoopSwap library for all rounds of selection and at the final round of selection for AAV2-7-mer and 7-mer-Ancestral libraries. Additional scatter plots are shown in Supplemental Figure 2. Black dots in the LoopSwap plots indicate variant NHP#26, validated in Figure 6. The black dot in the AAV2-7-mer plot indicates variant NHP#9, validated in Figure 5. A pseudocount of 1 was added to each variant prior to plotting. x axis is the percent of the library made up by each variant in the original library and y axis is the percent of total library at the indicated round of selection. As variants increase in representation, they rise on the y axis. Variants overrepresented in the original library are colored blue. Variants that had the greatest fold increase in representation in the final round of selection are shown in magenta. Variants that were overrepresented in the original library and increased significantly in representation over rounds of selection are colored orange. From the last round of selection, sequencing was performed on samples from central (R5C, Supplemental Figure 2) and peripheral (R5P) samples separately. AAV, adeno-associated virus.