A faulty editing machine inside cells is shredding the wrong genetic instructions in the retina, slowly destroying the sight of people with a common inherited form of blindness. This matters because retinitis pigmentosa caused by mutations in splicing factors—proteins that trim RNA messages—is a puzzle: the splicing machinery is defective in every cell, yet only the retina degenerates. The researchers have found that the problem may be a self-reinforcing loop where the splicing machinery damages its own components, and this loop appears unique to retinal cells. They now want to test this mechanism for a second splicing factor, PRPF8, using retinal cells grown from patient stem cells—a far better model than skin cells or mice, which do not mimic the human disease. If the work succeeds, it could reveal exactly which genes are mis-spliced first and which splicing defects drive degeneration. That knowledge would underpin future gene therapies that knock down the mutant protein only in the retina. Such treatments would address a large unmet need: adult-onset RP, where patients retain useful vision into their forties but have no approved therapy. The research is fundamental science, but it directly targets a specific disease mechanism, making clinical translation a realistic long-term goal.
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Retinitis pigmentosa (RP) is a common form of hereditary, progressive sight loss: it has a prevalence of 1 in 2500 and more than 1 million people affected worldwide. A major form of RP is caused by defects ("mutations") in genes that encode protein components ("splicing factors") of the "spliceosome". The spliceosome is a complex of proteins that ensure the new RNA transcripts formed ("transcribed") from genes are then correctly spliced to form the final messenger or mRNA. The cell then uses the final mRNA to encode the production of proteins. Splicing removes non-coding RNA ("introns") from the essential coding regions ("exons") that encode proteins. An analogy is the editing of unwanted or nonsensical passages out of a set of instructions, so that only intelligible words and sentences remain in the final text. The spliceosome is the cellular apparatus that performs the editing and ensures the fidelity and specificity of splicing. RP caused by mutations in splicing factors is a perplexing condition because splicing is ubiquitous in cells, but the condition only causes the degeneration of retinal cells. Previous work has suggested that mis-splicing of genes that encode retinal proteins may be important. However, our recently published work suggests that defective splicing of components of the splicing apparatus itself is the fundamental molecular defect. This positive feedback loop appears to only occur in retinal cells, suggesting that the targeting of this process might be particularly effective as a possible treatment. This work developed experimental methods and applied them to understand the function of PRPF31 in RP. In the present proposal, we now wish to broaden our investigations to include PRPF8, since this is a key structural component at the heart of the spliceosome and is essential for correct splicing. Mutations in PRPF8 are also a major cause of splicing factor RP. In order to understand this mechanism of disease in greater depth and to assess potential treatments, we will use special cell systems that closely model human retinal tissue. We will use patient-specific human stem cells differentiated into retinal cells, allowing us to study cellular structures and functions in retinal tissue derived from patients with splicing factor RP. These investigations would be impossible if we were to rely on the very limited clinical resources of patient tissue, inappropriate cell models such as skin fibroblasts, or the available mouse mutants that do not recapitulate the human disease. We will use biochemical methods to understand the effect of PRPF8 mutations on the structure and function of the spliceosome. We will combine these studies with "next generation" or clonal sequencing to determine the nucleotide sequences of RNA from patient-derived retinal tissue. This will determine which tissue is first affected during retinal degeneration, what types of splicing defects occur and which genes are affected. These studies will then inform the design of pre-clinical studies into potential treatments of PRPF8-related RP, for example by specific ablation ("knock-down") of the mutant form of the protein in retinal cells. The outcome of this proposed research will establish the disease mechanisms for RP caused by mutations in PRPFs, specifically PRPF8 and PRPF31, enabling the development of future therapeutic strategies to treat splicing factor RP. Current clinical trials for ocular gene therapies have focused on severe, early-onset disorders such as retinal dystrophies. However, there remains a large and unmet clinical need for the treatment of adult-onset RP, a large proportion of which are due to defects in PRFPs. These conditions present a particular challenge because patients can have useful residual vision into their fifth decade. A clear understanding of disease mechanism and greater requirement to demonstrate safety is therefore required before proceeding to clinical trials.
Colin Johnson (Co-Investigator)Henning Urlaub (Co-Investigator)Majlinda Lako (Principal Investigator)Reinhard Lührmann (Co-Investigator)Robin Ali (Co-Investigator)Sina Mozaffari-Jovin (Co-Investigator)Sushma Nagaraja Grellscheid (Co-Investigator)
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