Selecting the right patients for gene-based therapies: Development and implementation of a pathogenicity scoring system combined with functional in vitro validation of gene variants and genotypes in patients with inherited retinal dystrophy as a criterion

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The number of gene therapy trials for inherited retinal dystrophies is steadily increasing. It is important to ensure that the disease in these patients is truly and exclusively caused by the mutations in the target gene of gene therapy. Current practice for selecting patients for gene therapy in terms of genotype or its validity is neither objective nor standardised. Guidelines for variant interpretation in genetic diagnostics need to be further developed and standardised on a subject-specific basis. An objective and reliable pathogenicity assessment for individual variants and patient genotypes needs to be undertaken as an essential criterion for selecting patients into genotype-based therapy trials. In the first funding period, we correlated the scoring system for individual variants and patient genotypes as a proof-of-concept with functional data from our further developed in vitro aequorin-based bioassay for functional testing of missense variants in CNGA3. In the sequel application, we will first transfer this bioassay to CNGB3, and then to CNGA1 and CNGB1. We will again test the functionality of CNG channels with all previously uncharacterised putative disease-causing missense variants in a medium-throughput format in HEK293 cells. With this approach, we can evaluate all non-functionally characterised variants and confirm or reject the in silico prediction. At the end of the SPP, we expect to have an objective functionally confirmed pathogenicity assessment for all CNGA3, CNGB3, CNGA1 and CNGB1 variants observed in our patient cohort, literature and various mutation databases. This will allow patients to be prioritized and selected for future gene therapy trials for autosomal recessive achromatopsia – in the case of biallelic mutations in CNGA3 or CNGB3, and autosomal recessive Retinitis pigmentosa – in the case of biallelic mutations in CNGA1 or CNGB1.