Evaluating targeting of mouse cone photoreceptors for CRISPR-based therapies for inherited retinal diseases

Thesis

Inherited retinal diseases (IRDs) are a heterogenous group of disorders affecting cells of the outer retina. They represent a large socioeconomic burden and are the leading cause of inherited blindness in the world. Cone photoreceptor dysfunction is implicated in many IRDs and several mutations specific to cone cells have been identified. Great strides have been made via gene supplementation for the treatment of recessively inherited IRDs, however, this strategy is insufficient to treat disorders which cause a gain of function or dominant negative effect, including some of the mutations identified in cone related genes. This thesis broadly explores targeting of cone photoreceptors of mice for development of gene editing applications. 

In this thesis I first investigate dosing limitations and broad transduction patterns of AAV5 in combination with a ubiquitous (CAG) vs. photoreceptor-specific (hGRK1) promoter within a transgenic mouse line (CGFP) which expresses enhanced green fluorescent protein (EGFP) in a subset of cone cells. Expression of a red fluorescent protein, DsRed, was driven by either CAG or the hGRK1 promoter. CAG-DsRed and hGRK1-DsRed plasmids were packaged into AAV5 and subretinally injected into eyes of CGFP mice. Both vectors demonstrate in-vivo transduction of photoreceptors of the CGFP mouse, with evidence of toxicity with the ubiquitous CAG promoter. 

Next, activity of CRISPR-Cas9 was assessed in-vivo in the CGFP mouse. To test Cas9 expression, hGRK1-SaCas9 with an EGFP targeting sgRNA, F10, was packaged into AAV5 and subretinally injected into eyes of 6-week old CGFP mice. A dose study was conducted, with viral doses of 5x1011 gc/mL, 5x1012 gc/mL, and 1x1013 gc/mL. Some evidence of EGFP disruption was seen at the mid dose, however, attempts to detect DNA editing in pooled retinal cells were unsuccessful. 

Finally, several sgRNA candidates targeting the opsin locus control region (OPSIN-LCR) of the CGFP mouse were screened in-vitro for use in CRIPSR interference applications. Multiple sgRNA candidates were identified which produced gene repression in a dual-luciferase assay. A novel fibroblast line derived from the CGFP mouse was also used and a preliminary protocol for electroporation was established, demonstrating activity of three ubiquitous promoters in the cell line.

Authors: Rhodes, E

• DOI: 10.5287/ora-j1vq0xe45
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: CRISPR; inherited retinal diseases; gene therapy; retina
• Subjects: Gene therapy; CRISPR (Genetics); Retina; Retina--Abnormalities--Genetic aspects