Elucidating splicing factor function and retinal splicing programmes: developing new therapeutic strategies for splicing factor retinitis pigmentosa

Lead Research Organisation: Newcastle University
Department Name: Institute of Human Genetics


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.

Technical Summary

Alternative splicing is a pre-mRNA processing step that removes or includes specific exons/introns enabling generation of multiple isoforms from a single gene. Mutations in pre-mRNA splicing factors (PRPFs) cause 15-20% of autosomal dominant retinitis pigmentosa (adRP). It is unknown how mutations in ubiquitously expressed splicing factors cause retinal disease. In collaborative work, we have used patient-specific induced pluripotent stem cells (iPSC) differentiated to retinal cell-types to show that PRPF31 haploinsufficiency results in reduced spliceosome activity, defective splicing and impaired ciliogenesis in retinal pigment epithelium (RPE) and photoreceptors. Building upon these multidisciplinary skills and expertise, we aim to focus on a key essential structural component of the activated spliceosome, PRPF8, in order to identify genes and functional pathways that are affected by splicing factor dysfunction and their contribution to adRP. Mechanistic insights into splicing regulation of key candidate genes will inform the development of proof-of-concept splice-correcting gene therapies for RP.

The specific aims of this proposal are to:
1) use RNA-seq, quantitative proteomics and cellular assays to determine the effect of PRPF8 mutations on retinal cell structure and function, and identify mis-spliced genes and dysregulated splicing programmes;
2) use biochemical & in vivo splicing assays to assess the effect of PRPF8 mutations on splicing kinetics, and spliceosome assembly, stability, activity and function;
3) use RNA-Seq/iCLIP-Seq to identify the physiological & disease relevant RNA-binding sites of PRPF8 in the nucleus and cytoplasm;
4) use splice-switching morpholino oligonucleotides to validate the candidate mis-spliced transcripts in retinal cells;
5) gain insights into functional & splicing defects in PRPF8-related RP to develop new targeted therapeutic interventions, leading to translation into Phase I clinical trials for PRPF8-RP.

Planned Impact

The completed research will have outputs that will have the following impacts, benefiting both basic and translational research:

1) Reagents
Patient-specific iPSCs and isogenic controls will be deposited in the European Bank of Induced Pluripotent Stem Cells (EbiSC). Various constructs generated during the lifetime of this project will form an essential resource and will be deposited at Addgene.

2) Data sharing
The research programme will generate large amounts of RNA-Seq and proteomic data. These will be stored in standard formats, with standards-compliant metadata, to ensure that data are easily shared with other researchers. All raw sequence data and initially mapped data will be deposited in established public repositories including NCBI SRA (Short Read Archive), NCBI Gene Expression Omnibus and ProteomeXchange. High content imaging data will be in the form of indexed .tiff files and associated meta-data that will be up-loaded to open access microscopy-based phenogenomic resources ("Image Data Repository" https://idr-demo.openmicroscopy.org/, "Mineotaur" http://www.mineotaur.org/).

3) Publications
Data sets will be valuable to other researchers, so we will publish a "marker paper" and other peer-reviewed publication to enable new users to assess our data and existing users to cite their usage of our resource. We will disseminate our research findings and data sets to the scientific community through presentations at national and international conferences. All published work will be open-access and will be deposited in PubMed Central Universities' ePrints facilities, Research Gate, etc. in accordance with RC-UK, HEFCE and EU policies. Publications will acknowledge the support received from RC-UK.

4) Clinical and translational research advances
Retinitis pigmentosa (RP) comprises a heterogeneous group of rare inherited conditions (1 in 2500) and remain difficult to diagnose and treat. In the UK, about 23,000 patients are affected. There are very few preventative treatments or new therapeutic interventions that may modify disease progression or the long-term outlook of patients. The identification of new disease pathways in RP may enable the future rational design of therapeutics to modify or treat retinal degeneration or disease progression, or improve the long-term outlook of patients with RP. Since these conditions result from reduced levels of normal protein or dominant negative effects of mutant protein, they can in principle be corrected by gene-replacement or allele-specific knock-down, therapeutic approaches that are currently undergoing Phase III clinical trials. Patients who carry splicing factor mutations may therefore be given a clearer prognosis and enable them to be prioritized as patients that can most benefit from future targeted therapies. Next generation sequencing projects have shown that somatic mutations in splicing factors (PRPF8, U2AF1, SRSF2, SF3B1 etc.) are common in myeloid neoplasms and associated with specific phenotypes. While the focus of this application is deeper understanding and design of therapeutic interventions for PRPF8-RP, identification of PRPF targets and their functional validation will have far-reaching significance in other common clinical conditions such as blood cancers.

5) Intellectual Property (IP)
The proposed funding will produce foreground IP that will lead directly to 'composition of matter' patent filings. These will protect siRNA sequences that demonstrate the greatest efficacy at restoring the normal phenotype, and will be broad enough to protect any sequences that have 70% sequence identity to the lead targets. Prof. Lako has engaged with the technology transfer officer at Newcastle University and prior art searches have been conducted. These searches indicate that patent filings in this area primarily focus on up-regulation of the wild type allele. Therefore, our proposed strategy in modulating the expression of the mutant allele is innovative.


10 25 50