Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

Generation of three isogenic control lines from patient-derived iPSCs carrying bi-allelic ABCA4 variants underlying Stargardt disease.

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4. Employing the CRISPR/Cas9 approach, we generated isogenic control lines (RMCGENi005-A-1, RMCGENi018-A-1, RMCGENi017-A-1) for each of three induced pluripotent stem cell lines (RMCGENi005-A, RMCGENi018-A, RMCGENi017-A) derived from Stargardt patients carrying compound heterozygous ABCA4 variants. All of the generated lines showed pluripotent characteristics, no chromosomal aberrations and no indication of off-targets. The availability of these isogenic control lines will allow us to have a fair comparison between health and disease state within our studies on Stargardt disease.

Generation of iPSC lines from three Stargardt patients carrying bi-allelic ABCA4 variants.

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4, a protein that is expressed in the retina. Induced pluripotent stem cell lines (RMCGENi005-A, SCTCi018-A, SCTCi017-A) were generated by lentivirus reprogramming of fibroblasts derived from Stargardt patients carrying different bi-allelic ABCA4 variants. All the generated lines showed pluripotent characteristics and no chromosomal aberrations. The availability of these lines will allow us to generate patient-derived photoreceptor precursor cells and retinal organoids to further study ABCA4 and thereby, Stargardt disease in relevant model systems carrying the patient's genetic background.

TGF-ß is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells.

BACKGROUND: Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1ß and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-ß pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-ß in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs). METHODS: TGF-ß mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with normouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-ß priming experiments were performed with three inhibitors of TGF-ß signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-ß receptor II. RESULTS: TGF-ß mRNA levels were elevated in gout patients compared to healthy controls. TGF-ß-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-ß correlated positively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-ß but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-ß. CONCLUSIONS: TGF-ß is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-ß signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-ß relation.