Alport syndrome (ATS) is an inherited genetic disorder characterized by glomerular basement membrane (GBM) abnormalities up to end-stage renal disease. Usually, in the most severe forms nephropathy is associated with involvement of eyes and ears because of COL4 chains expression being restricted to kidney, ear and eye. Podocytes, key component of the glomerular structure, are the only cells in the kidney able to produce the COLIVα3-α4-α5 heterotrimer and thus, they are extensively defined as key-players in the pathogenesis of the renal disease. We have demonstrated how it is possible to isolate podocyte-lineage cells from urines of ATS patients and healthy carriers, providing an easily available cell system closer to podocytes’ physiological conditions. Our cellular model represents a novel tool and it turned out to be useful not only to characterize the effect of spliceogenic intronic variants but also to identify the presence of cryptic mosaicism confined to the involved tissue and undetectable on peripheral blood samples. This finding dramatically increases the possibility to implement, theoretically with no limits, the molecular genetic test that we can offer to patients. On the basis of the RNA-Seq data analysis, we have investigated the involvement of IL-32 in ATS. Being convinced about the fundamental role of IL-32 in ATS, and confirming the real involvement of the IL-32/IL-6/IL-8 pathway in podocytes, we performed the most sensitive ELISA assay to detect IL-32 release into urines of ATS patients. Although no such generous amounts of IL-32 were found in the urines, the values found are reported as sufficient to define a peculiar activation of the IL-32 pathway and more in general inflammatory pathway definitely related to the disease. At the end, we have explored the innovative gene therapy approach, that we hope will be definitive in the treatment of the disease, directly on the affected cells isolated from patients, exploring the possibility of reverting collagen IV causative mutations in ATS injured podocytes. With this work and with the achieved results we have demonstrated that gene therapy through gene editing approach is not anymore an unexplored frontier, but it can become an increasingly convincing reality. Although preliminary, the achieved results demonstrate how it is possible to obtain a partial recovery of the causative COL4A3 and COL4A5 mutations, unbalancing the heterozygous state towards the wild type condition. All the results taken together will be fundamental to open new frontiers of management and treatment of the disorder, also in preclinical model, like in dog model through the use of easy-deliverable AAVs system.
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|Titolo:||CRISPR/Cas9 gene therapy on urine-derived-podocyte-lineage cells and novel biomarker identification: new perspectives in Alport Syndrome (ATS)|
|Citazione:||Daga, S. (2019). CRISPR/Cas9 gene therapy on urine-derived-podocyte-lineage cells and novel biomarker identification: new perspectives in Alport Syndrome (ATS).|
|Appare nelle tipologie:||8.1 Tesi Dottorato|