Optogenetic gene therapy for vision restoration

Thesis

Inherited retinal dystrophies are a heterogeneous group of retinal diseases that often lead to blindness. The diverse range of causative gene mutations poses great challenges to developing therapies to improve the vision of affected people. One technique being investigated is optogenetics for vision restoration, a technique that aims to deliver light-sensitising molecules to the affected retina. This research project provides data on a range of variables that need to be optimised in order to successfully restore vision using an optogenetic gene therapy approach.

Fifteen wild type, chimeric and fusion constructs based on human opsins were compared by in vitro and in vivo tests to select the optimal light-sensitising construct. Of these, wild type rhodopsin, melanopsin and melanopsin–Gα subunit fusion constructs show the most promise for future studies.

For delivery of the light-sensitising construct, the adeno-associated virus (AAV) serotype AAV2/2(7m8) was shown to perform best compared to two other AAV serotypes when tested in degenerate mouse eyes and macaque and human retinal explants. The importance of the delivery route to the eye, the use of a targeted versus non-targeted approach and the AAV vector concentration were all tested in in vivo experiments.

In addition to testing such variables, the gene expression changes in target retinal bipolar cells in late-stage retinal degeneration were examined by microarray analysis of bipolar cell enriched samples isolated by fluorescence-activated cell sorting. Genes important for G protein signalling were generally expressed at a similar level in the degenerate state, while genes associated with oxidative stress and metabolism were differentially expressed.

Collectively, these data will inform the choice of construct, delivery method and target cell in future research into optogenetic strategies for vision restoration.

Authors: Hickey, D

• Alternative title: Light-sensitive proteins, viral vectors and bipolar cell adaptation to a diseased state
• DOI: 10.5287/ora-beqr4g17z
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Bipolar cell; Optogenetics; Opsin; Retinal degeneration; Microarray; Gene therapy; Retina
• Subjects: Transcriptomics; Retina; Optogenetics; Gene therapy