Understanding the mechanisms of cone opsin mediated disease to develop new therapies

Cone photoreceptors are the light sensitive cells at the back of the eye that enable us to see fine detail in daylight and perceive colour. We have three types of cone photoreceptors, red, green and blue, that enable us to perceive a spectrum of colours. The genes that encode the light sensing molecules for the red and green photoreceptors, called cone opsin genes, are very similar and close together in the human genome which makes them prone to genetic faults that cause different forms of visual loss or progressive blindness. Currently there are no treatments for these blinding conditions.
Through the discovery of these genetic faults, we have found that 'skipping' of important parts of the genes is a common cause of disease. Now we have discovered the genetic faults, we want to understand how these genes are switched on in cones and how the genetic faults impair cone photoreceptor function and structure. This will be achieved by state-of-the-art-techniques using patient stem cells to form a 'retina in a dish'. We will then use our 'retina in a dish' to test a new custom-designed potential therapeutic approach in this experimental system to see if we can silence this disease mechanism and restore normal cone opsin gene expression and function. Our genetic studies to determine the faults in the cone opsin genes in our patient cohort will provide an accurate diagnosis that we can correlate with the pattern of visual loss for earlier disease detection, and to identify the optimal window for potential therapeutic intervention. This study will pave the way to advance treatment of cone-opsin mediated blinding disease.

Mutations in the long-wavelength (LW) and middle-wavelength (MW) cone opsin genes are associated with a wide range of visual defects including red-green color vision deficiency, blue cone monochromacy and X-linked cone dystrophy. Advances in our understanding of the genetic basis of disease has revealed that different genetic mechanisms underlie the spectrum of cone photoreceptor defects, with different effects on cone photoreceptor viability and function. We have shown that interchange haplotypes are a common cause of disease, resulting in aberrant splicing and exon 3 skipping in vitro. The interchange haplotypes abolish Exonic Splice Enhancer (ESE) motifs and create Exonic Splice Silencer (ESS) motifs. Here, we will use the mini-gene constructs we have created for each disease associated interchange haplotype to test the potential of antisense oligonucleotides (AONs) to suppress exon 3 skipping and restore normal splicing of the cone opsin genes. We will create models of the human retina with LW and MW cone opsin mutations in genomic and cellular context by differentiating patient derived iPSC organoid optic cups. Using these models, we plan to define the number of cone opsin genes expressed in a human retina, investigate the effect of interchange haplotypes on the extent of mis-splicing and mutant opsin protein products, and test the most successful AONs from the mini-gene assay on human cone photoreceptors. We will correlate the context and consequence of mutations with the variable phenotypic spectrum of disease, to assess if cone photoreceptor survival, or the rate of photoreceptor loss and functional deterioration, correlates with cone opsin genotype. This knowledge is important for selecting the target cone opsin genes and context of mutations most likely to respond to a therapeutic intervention for potential restoration of visual function in patients with cone opsin mutations.

Currently there are no effective treatments for BCM and X-linked cone dystrophy caused by mutations in the cone opsin array. A molecular diagnosis for patients is a pre-requisite for considering any gene directed therapeutic approach. Correlation of genotype with deep phenotype data for cone opsin conditions will address the fundamental questions of how cone preservation varies between individuals, how this relates to genotype, and the progressive nature of these disorders, helping to establish the natural history of disease and identifying the optimal time to intervene. The main criterion for identifying suitable subjects for trials of rescuing cone photoreceptors is based on the structural integrity of the cones, so this study will inform on selecting the context of mutations most likely to respond to a gene directed therapeutic intervention for potential restoration of visual function in patients with cone opsin mutations.
Due to the evolution of trichromatic colour vision, animal models for these conditions are lacking, and appropriate models need to be established to explore disease mechanisms and to test potential therapeutic approaches. The development of patient iPSC derived organoid optic cups as models of disease outlined in this proposal, will advance our understanding of cone-opsin mediated disease, and as pre-clinical disease models will pave the way to explore potential treatments with translational potential, such as the AON strategy outlined in this application and potentially other therapeutic approaches, such as AAV mediated gene therapy. Our investigation is likely to have wider impact due to the fact that strategies directed at rescuing cones and developing relevant outcome metrics are relevant to all inherited retinal disorders, where preservation/restoration of cone function is also the primary therapeutic goal. We envisage that the data generated will be of direct relevance to other academic researchers, clinicians and pharmaceutical companies with an interest in developing therapies for other inherited retinal diseases.
The information gained will also be fundamentally important for understanding LCR driven expression of cone opsin genes in the array and the consequence of splicing defects caused by interchange haplotypes in a human retina. Our study will, for the first time, define the disease mechanism and the consequence of splicing defects caused by interchange haplotypes in the developing human retina. Broader and long term impact is also possible from data generated based on RNA-seq analysis of the developing optic cups, such as identifying transcriptional networks that govern photoreceptor subtype differentiation (rod, S-cone and L/M cone), and cone photoreceptor mosaic patterning in the developing human retina. Impact will be realised through the engagement and disseminated plans highlighted in the Communications Plan.