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

Lead Research Organisation: University College London
Department Name: Institute of Ophthalmology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Patterson EJ (2018) Residual Cone Structure in Patients With X-Linked Cone Opsin Mutations. in Investigative ophthalmology & visual science

 
Description Assessment of genotype-phenotype relationship for blue cone monochromacy
Amount £171,000 (GBP)
Organisation BCM Families Foundation 
Sector Charity/Non Profit
Country United States
Start 05/2019 
End 06/2021
 
Title Differentiation of patient derived iPSC to 3D retinal organoids 
Description We have recruited genotyped patients with specific interchange haplotypes of the cone opsin gene array, for differentiation of iPSCs to 3D retinal organoids. Fibroblasts were cultured from skin biopsies, and reprogrammed to iPSC. Differentiation to retinal organoids is underway. A limited number of lines will be differentiated in the first instance. We are also attempting to change the haplotype in single gene arrays using CRISPR/Cas9 in iPSCs. This aspect of the research is ongoing. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact These methods are established in our lab, and other labs, but this is the first time they have been applied to study mechanism of disease for this specific patient group 
 
Description Blue Cone Monochromacy Families Foundation, USA 
Organisation BCM Families Foundation
Country United States 
Sector Charity/Non Profit 
PI Contribution I am PI on a successful grant awarded to UCL by the founder of the Blue Cone Monochromacy Families Foundation, USA, with co-PIs Prof Michel Michaelides and Prof Joe Carroll This award (£171,101 for 2 years) is titled; "Assessment of genotype-phenotype relationship for blue cone monochromacy" This award has enabled a talented postdoctoral fellow who we collaborate with, Dr Emily Patterson, to move from Prof Joe Carroll's lab in the USA to UCL Institute of Ophthalmology, where she will develop her career in this field. Dr Patterson will be performing deep phenotyping on our patient group with different cone opsin mutations, including; i) using SD-OCT to obtain to obtain 3-dimensional images of the retina in patients, to measure thickness, reflectivity and integrity of retinal layers and assess differences across mutations with longitudinal assessment of disease progression/stability. ii) AOSLO to obtain diffraction-limited images of the retina in the living eye to visualise individual photoreceptor cells and to quantify various mosaic metrics for functioning rods, functioning cones, and non-functioning cones. iii) Establish reliable methods and baseline measurements of visual function using psychophysical and colour vision tests such as AVOT to accurately determine the degree of retained of retained visual function tests to assess residual color vision along the protan, deutan and tritan axes. The award supports salary for Dr Emily Patterson and patient imaging costs. The success of this award was based on our track record of collaboration and research excellence on retinal diseases due to mutations in the cone opsin genes, and the MRC project grant award, so that we have critical mass in the highly specialised study of clinical aspects of these conditions using state of the art imaging and visual assessment platforms.
Collaborator Contribution This award has enabled a talented postdoctoral fellow who we collaborate with, Dr Emily Patterson, to move from Prof Joe Carroll's lab in the USA to UCL Institute of Ophthalmology, where she will develop her career in this field.
Impact This award has enabled a talented postdoctoral fellow who we collaborate with, Dr Emily Patterson, to move from Prof Joe Carroll's lab in the USA to UCL Institute of Ophthalmology, where she will develop her career in this field.
Start Year 2019
 
Description UK Inherited Retinal Disease Consortium 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution The prevalence of retinal disease caused by mutations in the cone opsin genes is poorly understood, largely due to the fact that the majority of mutations are intractable by exome and genome sequencing and specific molecular genetic investigation is required. I am the lead PI in London (UCL Institute of Ophthalmology and Moorfields Eye Hospital) for the UK Inherited Retinal Disease Consortium, a funded collaboration (Retina UK, £340,400) of 4 centres that also includes Manchester (led by Prof Graeme Black), Oxford (led by Prof Susan Downes) and Leeds (led by Prof Chris Inglehearn) with the aim of determining the cause of disease in patients who are genetically unsolved. We are actively involved in performing the specific molecular tests and sequencing required to identify and confirm cone opsin mutations as the cause of disease in patients and families in this extended UK cohort.
Collaborator Contribution Recruitment of unsolved cases and families for investigation of cone opsin mutations
Impact Multidisciplinary - Molecular genetics and clinical phenotyping
Start Year 2020
 
Description UK Inherited Retinal Disease Consortium 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution The prevalence of retinal disease caused by mutations in the cone opsin genes is poorly understood, largely due to the fact that the majority of mutations are intractable by exome and genome sequencing and specific molecular genetic investigation is required. I am the lead PI in London (UCL Institute of Ophthalmology and Moorfields Eye Hospital) for the UK Inherited Retinal Disease Consortium, a funded collaboration (Retina UK, £340,400) of 4 centres that also includes Manchester (led by Prof Graeme Black), Oxford (led by Prof Susan Downes) and Leeds (led by Prof Chris Inglehearn) with the aim of determining the cause of disease in patients who are genetically unsolved. We are actively involved in performing the specific molecular tests and sequencing required to identify and confirm cone opsin mutations as the cause of disease in patients and families in this extended UK cohort.
Collaborator Contribution Recruitment of unsolved cases and families for investigation of cone opsin mutations
Impact Multidisciplinary - Molecular genetics and clinical phenotyping
Start Year 2020
 
Description UK Inherited Retinal Disease Consortium 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution The prevalence of retinal disease caused by mutations in the cone opsin genes is poorly understood, largely due to the fact that the majority of mutations are intractable by exome and genome sequencing and specific molecular genetic investigation is required. I am the lead PI in London (UCL Institute of Ophthalmology and Moorfields Eye Hospital) for the UK Inherited Retinal Disease Consortium, a funded collaboration (Retina UK, £340,400) of 4 centres that also includes Manchester (led by Prof Graeme Black), Oxford (led by Prof Susan Downes) and Leeds (led by Prof Chris Inglehearn) with the aim of determining the cause of disease in patients who are genetically unsolved. We are actively involved in performing the specific molecular tests and sequencing required to identify and confirm cone opsin mutations as the cause of disease in patients and families in this extended UK cohort.
Collaborator Contribution Recruitment of unsolved cases and families for investigation of cone opsin mutations
Impact Multidisciplinary - Molecular genetics and clinical phenotyping
Start Year 2020
 
Description collaboration with USA 
Organisation Medical College of Wisconsin
Country United States 
Sector Academic/University 
PI Contribution In collaboration with Prof Joe Carroll (Medical College of Wisconsin, Milwaukee, Wisconsin) and Profs Maureen Neitz and Jay Neitz (University of Washington, Seattle, Washington) we have selected a UK and US (U of Washington) genotyped patient cohort for cross-comparison (UCL/MEH and Wisconsin) of deep phenotyping using confocal adaptive-optics scanning light ophthalmoscopy and optical coherence tomography UK patients we have genetically solved were recruited to this study, and deep phenotyping was performed by Michel Michaelides at Moorfields
Collaborator Contribution Profs Maureen and Jay Neitz recruited genotyped patients to this study, and deep phenotyping was performed by Prof Joe Carroll
Impact Joint publication of research findings, PMID:30128495 Multidisciplinary - Genetics and state-of-the art imaging
Start Year 2018
 
Description collaboration with USA 
Organisation University of Washington
Department Department of Biomedical Informatics and Medical Education
Country United States 
Sector Academic/University 
PI Contribution In collaboration with Prof Joe Carroll (Medical College of Wisconsin, Milwaukee, Wisconsin) and Profs Maureen Neitz and Jay Neitz (University of Washington, Seattle, Washington) we have selected a UK and US (U of Washington) genotyped patient cohort for cross-comparison (UCL/MEH and Wisconsin) of deep phenotyping using confocal adaptive-optics scanning light ophthalmoscopy and optical coherence tomography UK patients we have genetically solved were recruited to this study, and deep phenotyping was performed by Michel Michaelides at Moorfields
Collaborator Contribution Profs Maureen and Jay Neitz recruited genotyped patients to this study, and deep phenotyping was performed by Prof Joe Carroll
Impact Joint publication of research findings, PMID:30128495 Multidisciplinary - Genetics and state-of-the art imaging
Start Year 2018