First, we defined the ABCA4 transcriptional unit and of gene regulatory networks controlling ABCA4. Moreover, we generated a cis-regulatory map in human adult retina and in model organisms combining chromatin interaction mapping such as HiChIP and UMI-4C, and other epigenomic signatures such as ATAC-seq and ChIP-seq, and bulk and single-cell transcriptomics. Furthermore, we functionally assessed cis-regulatory elements (CREs) of ABCA4 using in vitro studies in human retinal cells and in vivo enhancer assays in aquatic animal models. Overall, this research has contributed to the annotation of the non-coding genome in retina that may be a target for mutations in many other IRD.
Second, we aimed to identify and characterize missing heritability in the ABCA4 gene in STGD1. Despite genomic advances, 20-35% of STGD1 cases remained genetically unexplained, rendering ABCA4 an excellent model gene to identify and functionally assess missing heritability. Novel therapies are being developed for STGD1 cases that have an unequivocal genetic diagnosis. Here, we identified structural and sequence variants of the ABCA4 locus in monoallelic STGD1 cases. We performed in vitro analyses of ABCA4 splice defects and we identified ABCA4 RNA defects in retinal stem cells of monoallelic STGD1 cases. Lastly, we performed functional studies of ABCA4 cis-regulatory variants such as 5’UTR variants.
Third, we developed innovative therapeutic strategies that could overcome limitations of currently used lentiviral-based delivery of ABCA4 and develop new concepts to treat ABCA4-related retinal disease. These included antisense oligonucleotide (AON)-based splice modulation, dual AAV-based delivery of ABCA4 cDNA in a pig model, CRISPR/Cas9-based genome editing of deep intronic variants, mitochondrial gene therapy to modulate energy metabolism in the degenerating retina, and pharmacological modulation of ABCA4 protein folding to restore protein expression and function. The preclinical efficacy of these approaches was assessed in state-of-the-art cellular models, such as patient-derived iPSCs and iPSC-derived photoreceptor precursor cells and retinal organoids, as well as animal model systems, including mouse and pig.
The training network was designed to train 14 ESRs to become excellent interdisciplinary research scientists with in-depth knowledge on innovation in academic, industrial and non-profit organisations. Network-wide training was provided in the form of seven Complementary Skills (CS) courses and four Research Training (RT) workshops. Due to the COVID-19 pandemic, some events were moved online or postponed to enable in person meetings.
The StarT network aimed to disseminate its research results to the scientific community via publication in peer reviewed scientific journals and presentations at national and international meetings.
StarT organised an ‘Excellence in Science Communication’ course as part of the network training schedule and additional training on how to communicate research to the broad public. A StarT website was created and maintained, with publications and outreach activities. ESRs performed personal outreach activities: they wrote layman’s contributions about their research for the general public, and prepared infographics and videos on their research topics.
