Directed evolution identifies novel AAV capsid variants for targeting the canine outer retina following intravitreal delivery

Abstract

Purpose : The delivery of AAV vectors across physical barriers to target the outer retina is key to the success of retinal gene therapies. Subretinal injections are the current approach used to deliver AAV vectors to photoreceptors and RPE. However, directed evolution has been shown to be a promising method for selecting new vectors with enhanced ability to cross physical barriers. We show here results from a screen performed in the canine retina, a large animal model with eye structure and size similar to the human eye.

Methods : A highly diverse library of millions of distinct capsid variant AAV vectors was injected intravitreally into the eye of a wildtype dog. A subset of the AAV vectors injected was recovered from samples of outer retina by PCR amplification. Isolated variants were then pooled and re-injected in additional wildtype canine eyes, and following 5 total rounds of selection the pool had converged to a relatively small number of variants. High throughput sequencing was then used to characterize the convergence of variants over all rounds of selection. The final candidate variants were then evaluated by packaging a GFP reporter controlled by a ubiquitous CAG promoter. Their retinal distribution and cellular tropism was examined by cSLO (AF mode) imaging, histology and immunocytochemistry.

Results : Nineteen variants were identified as candidate vectors for targeting the canine outer retina. Intravitreal injection of a mixture of equal amounts of these variants carrying GFP resulted in efficient targeting of all retinal layers including photoreceptors and RPE in 6 eyes of 3 wildtype dogs.

Conclusions : Directed evolution performed in a large animal model, with an eye structure similar to that of humans, resulted in new AAV capsid variants with improved abilities to bypass significant structural barriers, and efficiently transduce the outer retina. These vectors show promise for gene therapy delivery via the intravitreal route of administration, thus potentially providing a safer approach for targeting a larger area of outer retina than currently achieved by the more traumatic and focal subretinal injection method.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.