Small molecule-based treatments for rare diseases. In vitro and in vivo studies.

In the last years, the CFTR corrector C17 has emerged as a promising pharmacological agent for treating the α-sarcoglycanopathy (LGMDR3), a rare form of limb-girdle muscular dystrophy. This disease is mainly caused by missense mutations in sarcoglycan (SG) genes (overall about 60%), and it is characterized by progressive weakness and degeneration of skeletal muscle due to the disruption of the SG-complex. Gene replacement therapy trials for LGMDR3 have shown potential but also limitations, such as the amount of viral vector required, the one-shot application, and long-term outcomes. In parallel, pharmacological approaches utilizing small molecules like CFTR corrector C17 have demonstrated to be effective in the rescue of the SG-complex in a model of LGMDR3. In this mouse, in which hind-limbs expressed a mutated form of human α-SG, it was possible to observe a significant improvement of muscle strength, as the consequence of the amelioration of the dystrophic phenotype. Besides LGMDR3, this study explored the therapeutic potential of the small molecule C17 in addressing protein misfolding and trafficking defects in another rare genetic disorder, the Allan-Herndon-Dudley Syndrome (AHDS), caused by mutations in the SLC16A2 gene encoding the monocarboxylate transporter 8 (MCT8), responsible for thyroid hormone transport. MCT8 mutations disrupt thyroid hormone signalling, leading to severe developmental impairments. C17, belonging to the class of bithiazole derivatives effective in rescuing CFTR mutants, is emerging as a promising drug candidate. While the pharmacological studies showed efficacy, pharmacokinetic (PK) studies revealed that after an acute i.p. injection, C17 distributed primarily to highly irrorated organs. Then, it is redistributed towards target tissues, like skeletal muscle and heart. On these bases, a novel C17 chronic treatment in the LGMDR3 mouse model was applied, resulting in improved muscle function and sarcolemma fragility, as proven by the reduced blood level of CK, marker of muscle damage. The safety profile was further supported by the similarity of C17 to other CFTR correctors in terms of metabolism and excretion. Concerning the pharmacological profile, we observed that C17 was promising in treating also the AHDS. In cells stably expressing different MCT8 mutants, C17 promoted the re-localization of the defective proteins to the surface and the recovery of the transport activity. This was proven by the increased uptake of radiolabelled T3 upon cell treatment. Notably, pharmacokinetic studies showed that C17 crosses the BBB, a key aspect in the treatment of AHDS since, in addition to peripheral tissue problems, TH transport defects affect mainly the CNS leading to impairment of cognitive and motor functions. We also investigated the LGMDR3 related cellular dysfunctions during the attempt of developing a three-dimensional muscle model. We studied the proliferation, engraftment, and differentiation of LGMDR3 myogenic cells by using utilised decellularized extracellular matrix (dECM) scaffolds. While healthy myogenic cells successfully repopulated dECM, LGMDR3 cells exhibited impaired adhesion and migration properties that could be related to SGCA gene mutations. The C17 treatment was applied when myogenic cells were still proliferating, since α-SG protein is present at the cell membrane in a very low amount, aiming to correct the α-SG protein and improve its function. Such an early treatment, in 2D culture conditions, slightly improved migration properties, interactions between LGMDR3 cells and ECM proteins in term of improved resistance to detachment, and the organization and formation of myotubes. This suggest that such a treatment could be of benefits in enhancing cellular behaviour also during engraftment of decellularized scaffold (ongoing experiments). This study deepened our understanding of the molecular mechanisms behind LGMDR3 and showed significant promise for personalized medicine.