Inherited Retinal Diseases (IRD)
An Integrated CRISPR/iPSC-based approach to elucidate uncertain variants in RPE65, a target for gene therapy
This project is being carried out by PhD student Eline Van Vooren and supervised by Prof. Dr. Elfride De Baere and Dr. Miriam Bauwens.
The project is funded by an FWO fundamental research fellowship (2022-2026). |
In 2017 and 2018, Luxturna received FDA and EMA approval as the first gene therapy product to treat patients with biallelic RPE65-mutations, causing severe inherited blindness. Eligibility for gene therapy requires a complete molecular diagnosis, which is often hampered by the identification of variants of uncertain significance (VUS). To this end, we first will set up a biochemical assay to assess the pathogenicity of coding RPE65 VUS, by simulating the visual cycle in HEK293-F cells via overexpression of relevant genes, incl. RPE65. RPE65 is a key enzyme in the visual cycle, uniquely expressed in the retinal pigment epithelium (RPE), which is characterized by tissue-specific splicing and expression patterns. Expanding on the first assay, a second, biologically relevant disease model will be developed using human iPSC-derived RPE and CRISPR-editing. In addition, we will investigate the regulatory network underlying RPE65 expression by interrogating 9 candidate cis-regulatory elements of RPE65 via genome editing in ARPE-19 cells. This integrated approach, using established and novel, biologically relevant cellular models and genome editing will elucidate the regulation and pathomechanisms of coding and non-coding variants in RPE65.
The set-up will improve eligibility of patients with uncertain RPE65 variants for gene therapy (trials) but can ultimately serve as platform to assess all genomic variation in RPE-expressed genes and impact a substantially larger group of patients. |
Role of the ultraconserved IRXA cluster in North Carolina Macular Dystrophy, a retinal enhanceropathy
The function of ultraconserved non-coding elements (UCNEs) in the genome is still poorly understood. A recent example of an ultraconserved genomic region implicated in human disease are duplications of a gene desert downstream of the IRXA cluster, found in patients with North Carolina Macular Dystrophy (NCMD). NCMD is a rare autosomal dominant developmental maculopathy impairing central vision. The shared duplicated region harbours a UCNE that is a candidate cis-regulatory element (cCRE) during macular development. Although we have recently provided evidence that NCMD is a retinal enhanceropathy, no NCMD disease models have been investigated so far.
The general aim of my research is to dissect the mechanism underlying IRXA-associated NCMD. Key objectives are: (1) to functionally validate tissue-specific cCREs of the ultraconserved IRXA cluster using CRISPRi perturbation (2) to establish patient-derived disease models for IRXA-associated NCMD (3) to assess the impact of a dysregulated IRXA cluster on 3D topology using chromatin interaction profiling (4) on gene expression and regulatory networks using bulk and single-cell transcriptomics. By elucidating the molecular pathways and regulatory networks impaired in IRXA-associated NCMD, we will gain insight into the role of this ultraconserved cluster in human macular development and disease. |
This project is being carried out by PhD student Lieselot Vincke and supervised by Prof. Dr. Elfride De Baere and Dr. Miriam Bauwens.
The project is funded by an FWO fundamental research fellowship (2023-2027). |
Mapping of 3D genome topologies in human retina, RPE, PPCs and ROs
Using chromatin interaction mapping (Hi-C) we have previously shown a differential 3D genome architecture of human retina and retinal pigment epithelium (RPE). Despite, non-coding structural variants (SVs) and regulatory single-nucleotide variants (SNVs) are still underrepresented in the mutation spectrum of IRD, often due to an interpretation gap. This project aims to:
(1) characterize IRD associated loci through the recent long-read sequencing-based Pore-C technology, and (2) shed light into the non-coding genome. We will use easy-accessible tissue for IRD patients, RPE and Retinal Organoids to generate the Genome Interaction Map. This data will be used to enhance non-coding variants interpretation and ultimately improve IRD genetic diagnoses. |
This project is being carried out by PhD student Nelson Martins and supervised by Prof. Dr. Elfride De Baere, Dr. Miriam Bauwens, Dr. Eva D'haene, and Dr. Ir. Mattias Van Heetvelde. The project is funded by a MSCA Doctoral Network fellowship (2024-2027).