Description
A specialised transcriptome is activated early during retinal development. Importantly, numerous splicing altering variants are associated with Inherited retinal diseases (IRD’s), like Leber Congenital Amaurosis (LCA), therefore, an improved understanding of retinal alternative splicing could help reveal underlying disease mechanisms. The inclusion of retina-specific exons is thought to be driven by a group of RNA-binding proteins, including Musashi 1 (MSI1) and MSI2, which are required for mouse photoreceptor development. Furthermore, MSI1 expression enhanced cryptic exon inclusion in human cells. To investigate MSI1 in human photoreceptors, gene edited induced pluripotent stem cell (iPSC) lines for control, MSI1 knock-out (KO), a homozygous LCA splicing mutant, and LCA-MSI1 KO were differentiated to retinal organoids (ROs). The effect of MSI1 ablation on photoreceptor development, outer segment formation, retinal-specific splicing and CEP290 cryptic exon inclusion was investigated. RO photoreceptor development was delayed, and outer segment density was reduced in the MSI1 KO lines. Immunohistochemistry (IHC) analysis confirmed photoreceptor development in MSI1 KO ROs and showed MSI1/2 localisation to the photoreceptor cytoplasm and nucleus. MSI2 upregulation in MSI1 KO ROs was observed by IHC and RNAseq. These data highlight the complexity of alternative splicing in the human retina and how this could affect splicing dysregulation in IRD.
Lay Abstract
Diseases involving progressive blindness, like Leber Congenital Amaurosis (LCA), are often caused by the increasing death and/or dysfunction of specialised nerve cells in the eye that are called photoreceptors. These cells are responsible for sensing light and are a part of the retina, a layer of nerve tissue at the back of the eye. Inherited changes in the DNA can cause photoreceptors to dysfunction and die, resulting in disease. Recent technological advances have enabled the study of photoreceptors in lab-grown models of the human retina, called retinal organoids, or a retina in a dish. My project uses retinal organoids to study how DNA changes can alter a critical step in protein production and explores how photoreceptor function and survival is impacted. Retinal organoids were generated that have been edited to remove an important retinal regulator. My results suggest a potential impact of its removal on photoreceptor maintenance and function. Overall, this project aims to understand processes that have a role in the development of blindness.
| Lay Title | Modelling of disease-associated photoreceptor dysfunction using lab-grown retinal organoids |
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| Role | PhD Student |