Abstract
Understanding the genetic basis of hereditary kidney diseases is crucial for diagnosis, management, and treatment of patients. Furthermore, this can provide valuable insights into the fundamental biological processes involved in kidney development and its functions. Therefore, this thesis aims to generate, validate, and characterize advanced in vitro models for genetic
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kidney disorders to provide novel insights in the disease pathophysiology and to eventually offer novel therapies for these diseases. The approach includes developing advanced, microphysiological culture systems and kidney organoid models, along with the application of gene editing technologies (CRISPR/Cas system) for gene mutation as well as restoration. Following this introduction, in chapter 2, a novel 3D microfluidic system is presented to model nephropathic cystinosis in vitro. We employed a HFM system as 3D scaffold, and we used a CRISPR-engineered (CTNS-/-) and a patient-derived (CTNSPatient) cystinotic conditionally immortalized PTEC line (ciPTECs) to generate cystinotic tubules. We aimed to offer a novel advanced model to study cystinosis at a proximal tubule level that fully recapitulates the disease phenotype, viz. renal Fanconi syndrome. In chapter 3, CRISPR technology is used to generate kidney organoids depleted for the KCNJ16 gene, a novel candidate gene recently described to be related to a tubular disease phenotype in the kidney. We focused on the characterization of described and novel phenotypes upon KCNJ16 depletion, as well as on the potential phenotype restoration using a pharmacological approach. Similarly, chapter 4 presents NPHP1-depleted kidney organoids using CRISPR technology with the aim of phenotypically characterizing and further investigating the missing link between NPHP1 loss and the characteristics and symptoms of nephronophthisis-1. In chapter 5, a novel gene editing method to restore defects in the CTNS gene in the two cystinotic ciPTEC lines mentioned earlier is described. Our novel approach includes the use of Homology-Independent Targeted Integration (HITI), a novel enhanced CRISPR version for highly efficient and precise DNA insertions, coupled with a novel non-viral peptide-mediated delivery method (LAH5), as well as several repair constructs designed to be able to repair any mutation within the first 10 exons of the CTNS gene. We aimed to introduce the CRISPR repair system into both cystinotic ciPTEC lines genome efficiently and precisely, which would lead to the downstream lysosomal cystine restoration. Additionally, we aimed to explore whether the phenotypical restoration upon gene repair extends beyond lysosomal cystine accumulation by evaluating the mitochondrial bioenergetics before and after gene repair of selected clones. In chapter 6, the results presented in this thesis are summarized and discussed in the context of the latest innovations in the field of advanced in vitro modelling for kidney diseases, providing a comprehensive overview of the findings of this thesis as well as current developments in the field. Lastly, the future directions to further develop CRISPR and kidney organoids as a platform for disease modelling and their value in future medicine are presented from a reflective perspective.
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