MODELLING RIBOFLAVIN TRANSPORTER DEFICIENCY (RTD) USING IPSC-DERIVED MODELS TO TEST GENE THERAPY EFFICACY

Riboflavin transporter deficiency syndrome (RTD) is a rare childhood-onset neurodegenerative disorder caused by mutations in SLC52A2 and SLC52A3 genes, encoding the riboflavin (RF) transporters RFVT2 and RFVT3. In the present study we focused on RTD Type 2, which is due to variants in SLC52A2 gene. There is no cure for RTD patients and, although studies have reported clinical improvements with administration of RF, an effective treatment is still unavailable. Herein, we developed three disease models through 2D motoneurons, spinal cord and retinal organoid differentiation of RTD patients-specific induced pluripotent (iPSC) line carrying several mutations in SLC52A2 gene. First, we tested gene replacement therapy on RTD type 2 patient-derived motoneurons using an adeno-associated viral vector 2/9 (AAV9) carrying the human codon optimized SLC52A2 cDNA. We optimized the in vitro transduction of motoneurons using Sialidase treatment. Our results proved that treated RTD motoneurons showed a significant increase in neurites’ length when compared to pathological untreated samples demonstrating that AAV9-SLC52A2 gene therapy can rescue RTD motoneurons. This leads the path towards more complex in vitro as well as in vivo studies offering a potential treatment for RTD patients. Therefore, RTD iPSC-derived spinal cord organoids were generated. This model displayed similar features of the 2D model of motoneurons, including motoneurons cytoskeletal defects and motoneurons degeneration confirming the hallmarks of the disease. These results showed the feasibility of the 3D model to test AAV9 gene therapy. Additionally, since very little is known regarding the RTD retina and the mechanism leading to patients’ blindness, retinal organoids have been generated. This study started to clarify some of the pathogenesis in RTD retina. Both photoreceptors and retinal ganglion cells seems to be involved in the mechanism leading to the blindness of RTD patients. These results allowed us to consider retinal organoids as a feasible model to further indagate the RTD pathomechanism in retina. This thesis highlights the successful applications of 2D and 3D iPSC-derived models in RTD disease modelling, confirming the suitability of these in vitro models for testing novel therapeutics.