RESTORING MMACHC EXPRESSION: TRIDs AS A PROMISING APPROACH FOR cblC DEFICIENCY TREATMENT

Rare genetic diseases, often defined as ''orphan diseases'' due to their low influence and limited therapeutic options, have always represented a demanding challenge for research and medicine. Recent attention has focused on stop mutations, one of the different classes of mutations responsible for these pathologies. This gene defect, considered among the most harmful, determines the formation of premature termination codons (PTCs), whose presence on the coding mRNA results in the synthesis of a non-functional truncated protein or with alterations that can compromise its functioning biological activity. One of the most promising approaches to counteracting these mutations is based on the use of a new class of molecules, known as Translational Readthrough-Inducing Drugs (TRIDs), which interfere with protein translation, allowing the synthesis of a complete and potentially functional protein. This study aimed to evaluate the readthrough activity of 3 new TRID molecules (NV848, NV914, and NV930) to rescue the expression of the cobalamin chaperone MMACHC protein associated with the metabolism of vitamin B12. We engineered HCT116 cells with two vectors harboring the R132X and Y222X mutations in the MMACHC cDNA and treated them with TRIDs at different time points. After 24, 48, and 72 hours of TRID treatments, Real-Time RT-PCR analysis revealed an increase in the expression levels of mutated MMACHC mRNA, suggesting a possible transcript stabilization. Immunofluorescence analyses further supported these data, confirming the protein's partial rescue. These results suggest that the analyzed TRIDs could represent a promising therapeutic option for the treatment of cobalamin C (cblC) deficiency disease. Furthermore, such evidence paves the way for developing personalized therapeutic strategies based on the readthrough of premature stop codons, offering an innovative perspective for treating numerous rare genetic diseases currently without effective treatments.