Periodic Reporting for period 4 - TREATCilia (Novel Therapeutic Avenues for dynein-related Ciliopathies) Reporting period: 2021-08-01 to 2022-01-31 Summary of the context and overall objectives of the project Cilia are antenna like projections extending from the cell surface. Two main types can be distinguished: motile cilia essential for movement of fluids and non-motile cilia crucial for cell signalling pathways, enabling proper embryonic development and maintaining organ function. Motile cilia dysfunction can cause left-right body axis defects, heart defects as well as recurrent respirator infections and reduced fertility in humans. malfunction of non-motile cilia often results in complex developmental defects affecting the kidneys, eyes, brain, heart, liver and the skeleton. Over 100 genes have been found to be defective in ciliopathies, however a large number of cases is still genetically unexplained. As many as 1 in 1000 persons in Europe is affected by a cilia related genetic diseases, imposing huge health and socioeconomic challenges. There is currently no causative treatment option available. TREATCilia aimed to uncover underlying molecular pathophysiology in dynein based ciliopathies from gene to mechanism and we hope to identify future pharmacological entry points to ameliorate ciliary dynein-related phenotypes. Emplyoing exome sequencing, we were able to identify several novel human disease genes, such as PIH1D3, MNS1 and DNAH9, causing cilia motility defects and/or laterality defects when mutated as well as several novel genes associated with centra nervous system defects. In addition, we were able to establish a number of phenotype genotype correlation for motile ciliopathies/laterality disorders, inherited renal and skeletal phenotypes and neuro-developmental disorders By creating several of dynein-2- and IFT-mutant cell lines harboring human disease alleles using CRISPR base editing technologies, the project achieved generation of unique in-vitro ciliary condrodysplasia disease models. Multi-omics analyses of these model suggest disturbed Golgi-transport, actin-cytoskeleton modifications and changed extracellular matrix composition in mutants. We further identified a number of affected cell signalling pathways, offering putative future therapeutic targets. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We are using Next Generation Sequencing (NGS) technologies, mainly Exome Sequencing to unravel the underlying genetic cause of human ciliary disorders. This has led to the identification of a number of novel disease causing genes and provided novel insight into ciliary protein organisation and functions. For example, we found that mutations in the outer dynein arm protein DNAH9 result in laterality defects associated with no or only very mild respiratory symptoms. As DNAH9 is only required for movement of parts of the cilium, cilia motility impairment is mild. Therefore, patients with DNAH9 protein dysfunction are difficult to diagnose and genetic diagnosis is required. Further, our results point stress that ciliary motility complexes such as dynein arms are organised in a compartementalised manner. With regards to non-motile ciliopathies, we focus on reno-skeletal disorders such as Jeune Syndrom and Short-Rib-Polydactyly Syndrome. In addition to gene identification and genotype-phenotype studies, we have recreated several hypomorphic patient alleles using CRISPR/Cas technologies and we are investigating downstream effects using Transcriptomics and Proteomics approaches. Chlamoydomonas drugscreening is currently prepared. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Using cutting edge technologies such as CRISPR base editing approaches enables us to gain deeper insight into ciliary protein networks and dissect the molecular defects resulting from human patient mutations. Our longterm goal is to identify novel therapeutic approaches.