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.
Highlights
Inherited retinal degenerations (RDs) are caused by mutations in more than 200 genes [1], the majority of which are expressed in photoreceptors or retinal pigment epithelium (RPE) of the outer retina, leading to vision loss and decreased quality of life [2, 3]
To mitigate the possibility of replication of the associated virus (AAV) libraries in the primate retina and subsequent spread, a rep in trans strategy was developed in which the library rep sequence is mutated while leaving intact regulatory elements, including the P40 promoter, which is necessary for cap protein expression
The inverted terminal repeats (ITRs) in AAV are highly recombinogenic, leading to the possibility of recombination and subsequent replication in the presence of a helper virus when using this strategy, so an intron was inserted in the rep supplied in trans to prevent packaging of recombined genomes (Figure 1)
Summary
Inherited retinal degenerations (RDs) are caused by mutations in more than 200 genes [1], the majority of which are expressed in photoreceptors or retinal pigment epithelium (RPE) of the outer retina, leading to vision loss and decreased quality of life [2, 3]. Directed evolution has enabled the creation of new, efficient viral vectors for outer retinal gene delivery in mice [7,8,9]. We used directed evolution [13], guided by insights into the population dynamics of viral selections that were derived from deep sequencing, to create adeno-associated viruses (AAVs) capable of delivering genes to outer retina after intravitreal injection in cynomolgus macaques, which have eye structures similar to humans. Secondary screening of subsets of the resulting viral variants revealed the most efficient AAVs for photoreceptors and RPE of the primate retina. The outer retinal gene delivery offered by these variants may enable less invasive delivery of therapeutic genes to the retina and potentially provide more expansive transduction at a similar dosage as that currently used in clinical trials
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
