Semester

Fall

Date of Graduation

2012

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Neurology

Committee Chair

Visvanathan Ramamurthy

Committee Co-Chair

Peter Mathers

Committee Member

Rae Matsumoto

Committee Member

James M. O Donnell

Committee Member

Maxim Sokolov.

Abstract

Since groundbreaking clinical trials treating Leber congenital amaurosis (LCA) patients with retinal pigmented epithelium-specific 65 kDa protein (Rpe65) defects, which were the most successful gene therapy studies to date, much effort has been put into finding other candidate genes associated with inherited retinal degeneration to treat. Animal models with Rpe65 defects demonstrate an unusual circumstance that greatly favors treatment, where dramatic vision loss is disproportionately greater than the ensuing slow photoreceptor degeneration. This pattern is also believed to occur in Rpe65-LCA patients, and stems from the primary defect in retinal pigmented epithelial (RPE) cells that subsequently perturbs photoreceptors. However, a majority of inherited retinal degenerations are associated with photoreceptor gene defects, and do not show this advantageous disparity between visual dysfunction and photoreceptor loss. To combat this, efforts have concentrated on improving photoreceptor transduction efficiency with viral vectors, and delivering gene replacement to slower progressing retinal dystrophies.;Aryl hydrocarbon receptor interacting protein-like1 (Aipl1) is mainly associated with the severe retinal degeneration, LCA. However, reports of Aipl1 defects in patients showing slower progressing retinal disease, retinitis pigmentosa (RP) and cone-rod dystrophy (CORD), points to a subset of Aipl1 patients as potential candidates for future AAV-mediated gene therapy trials. Multiple mouse models with Aipl1 defects have been developed and contribute to the ease of conducting pre-clinical rescue studies to examine the rescue potential of Aipl1..;To investigate the rescue potential of Aipl1 defects, we conducted AAV-mediated gene replacement studies in Aipl1-null mice, a model for Aipl1-LCA, the most rapidly progressing of any retinal degeneration mouse model. Our study examines the recent advancements in AAV viral vectors, using a combined self-complementary Y733F capsid mutant AAV8 (sc-Y733F-AAV8), to evaluate whether these advancements would provide functional benefits to a model of retinal degeneration. We found that the sc-Y733F-AAV8 viral vector significantly improved vision rescue in Aipl1-null mice as compared to conventional single-stranded AAV8, even when treatment was administered at postnatal day 10, after initiation of photoreceptor degeneration. From these studies, we also conclude that the Aipl1-null mouse provides a good model to test the functional benefits of ongoing and future advancements in AAV viral vectors.;In addition to LCA, Aipl1 has been reported in patients with CORD and RP. We were particularly interested in a mutation, P351Delta12 hAipl1 because of its association with CORD and our ongoing interest in the role of Aipl1 in cone photoreceptors. We generated a transgenic mouse model expressing P351Delta12 hAIPL1, which showed a cone-rod dystrophy phenotype similar to a case of Aipl1-CORD in a patient with this mutation. The mice demonstrated early and rapidly declining cone-mediated vision with slow progressing rod defects. Additionally, in our studies of double transgenic mice expressing both mutant P351Delta12 hAIPL1 and WT hAIPL1, we conclude that the P351Delta12 hAipl1 mutation does exert dominant effects that explain the autosomal dominant inheritance pattern observed in the Aipl1 patients with this mutation.;Interestingly, despite the dominant phenotype, we were able to rescue cone dysfunction and degeneration following AAV-mediated gene delivery of WT hAIPL1. We believe this is due to the drastic overexpression of WT hAIPL1 that is achieved with AAV gene delivery. Further work with this novel model of Aipl1-CORD is needed to elucidate the dominant mechanism of the P351Delta12 hAIPL1 protein, which appear to behave differently in rods and cones, as rod dysfunction was not rescued with AAV-mediated overexpression of WT hAIPL1. With these successes in pre-clinical Aipl1 rescue studies, which have also been independently demonstrated by two other groups, we conclude that Aipl1 defects show great potential for movement into AAV-mediated gene therapy clinical trials.

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