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

遗传性视网膜营养不良的基因治疗试验数量正在稳步增加。重要的是要确保这些患者的疾病是真正和完全由基因治疗的靶基因突变引起的。目前根据基因型或有效性选择患者进行基因治疗的做法既不客观也不规范。基因诊断中的变异解释指南需要在特定学科的基础上进一步制定和标准化。需要对个体变异和患者基因型进行客观可靠的致病性评估，作为选择患者进行基于基因型的治疗试验的基本标准。在第一个资助期，我们将个体变异和患者基因型的评分系统作为概念验证与我们进一步开发的用于CNGA3错义变异功能测试的体外aequorin生物测定的功能数据相关联。在后续应用中，我们将首先将该生物测定转移到CNGB3，然后转移到CNGA1和CNGB1。我们将在HEK293细胞中以中等通量格式再次测试CNG通道的功能，其中包括所有先前未表征的假定致病错义变体。通过这种方法，我们可以评估所有非功能特征的变体，并确认或拒绝计算机预测。在SPP结束时，我们期望对我们的患者队列、文献和各种突变数据库中观察到的所有CNGA3、CNGB3、CNGA1和CNGB1变体进行客观的功能确认的致病性评估。这将允许优先选择患者进行未来基因治疗试验，以治疗常染色体隐性隐性色盲（在CNGA3或CNGB3双等位基因突变的情况下）和常染色体隐性遗传性视网膜色素变性（在CNGA1或CNGB1双等位基因突变的情况下）。

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.