Generation of isogenic lines using CRISPR/Cas9 technology from induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cells (iPSCs), introduced by Yamanaka in 2006, have revolutionized the field of regenerative medicine, providing unprecedented opportunities to model and study human diseases in vitro. From the perspective of neurological disorders, iPSC models have facilitated an essential link between neurobiology, neural function and disease-associated cellular changes, addressing research questions with translational potential. A promising application of iPSC technology is the generation of isogenic lines, i.e. genetically identical cell lines differing only by a specific genetic modification. A critical aspect of generating isogenic lines is the ability to identify the genetic cause that defines the cellular phenotype and thus causes the disease. This is not possible when comparing iPSC lines derived from healthy individuals with those derived from patients with the disease under investigation, due to the inheritance patterns of related SNPs. The integration of iPSCs and genome editing technologies will undoubtedly provide much insight into disease mechanisms and therapeutic targets. This, together with the application of iPSC-derived cell types for cell-based therapies and CRISPR-based reagents for in vivo therapies, offers exciting possibilities for personalised genetic medicines in the not too distant future. 1 In the first part of my PhD studies, I focused on the application of precision gene editing to iPSC modelling of PEBAT, a neurological disorder due to variants in the TBCD gene. Specifically, we investigated the best experimental strategy to combine CRISPR/Cas9 and piggyBac transposon technologies to generate an isogenic knock-in iPSC line without imprinting. We successfully generated an isogenic human model from a patient carrying a bi-allelic pathogenic variant c.3365C>T, p.Pro1122Leu. We have shown that the corrected isogenic iPSC line obtained is able to maintain its pluripotent character, has a normal karyotype without the occurrence of chromosomal aberrations and is able to differentiate into cells of the three embryonic layers. The isogenic iPSC line obtained showed a rescue of the TBCD mutated phenotype: i.e., rescued TBCD protein levels, correct mitotic spindle morphology and reduced apoptotic rate. This innovative in vitro model confirmed the pathogenic role of the TBCD variant identified in the patient and allowed us to study the patho-mechanisms underlying PEBAT (4). Thereafter, in the second year I investigated the mechanisms underlying Noonan Syndrome – 14, a recessive developmental disorder within the RASopathy clinical spectrum. In particular, we developed an in vitro model based on patient-derived iPSCs derived from two patients with two different variants in the SPRED2 gene (c.299T˃C and c.1142_1143delTT). After reprogramming patient fibroblasts by introducing episomal vectors through nucleoporation, we generated and characterised multiple patient-derived iPSC clones. First, we decided to assess the levels of cell death in the iPSCs derived from the patients and from controls using TUNEL cell death assays. These analyses revealed a significant increase in DNA fragmentation in the iPSCs from NS-14 patients, indicating an increase in the number of dead, TUNEL-positive cells. Notwithstanding, MTT assay showed essentially unaltered cell metabolism in both patient-derived cells compared to the control line, suggesting that the alive cells are metabolically comparable to the control cells.