| Summary: | A significant number of mutations that change the splicing process and lead to aberrant mRNA production have been identified in Lysosomal Storage Disorders (LSDs). Mucopolysaccharidosis type IIIC (MPS IIIC) is a very rare LSD caused by mutations in the HGSNAT gene which encodes an enzyme involved in heparan sulphate degradation. Splicing mutations are one of the most frequent (~20%) genetic defects in MPS IIIC. Around 55% correspond to 5' splice-site (ss) mutations thus constituting a good target for splicing therapeutics. Recently, we have demonstrated that a modified U1snRNA vector designed to improve the definition of the HGSNAT exon 2 5’ss can restore splicing impaired by the mutation c.234+1G>A. Currently, our goal is to evaluate in vivo the therapeutic potential of that modified U1 snRNA by testing it in mice expressing the human splicing defect. For this purpose, two full-length constructs were generated by cloning the wt or the mutated HGSNAT splicing-competent cassettes in the pcDNA 3.1 vector. Then, the wt and the mutant constructs were transfected in Hep3B and COS-7 cells. Both minigenes reproduce the healthy control and patient cDNA’s splicing pattern. Therefore, they were used to generate C57BL/6 mice expressing the wt (c.241+1G) or mutant (c.234+1A) alleles in the liver. These mice can be used for testing the modified U1 snRNA efficacy in vivo. Thus, wt or mutant minigenes were administrated in mice by hydrodynamic injection following a protocol described by Balestra et al. After 48h, animals were sacrificed, the liver was collected, and the molecular analysis was performed. Preliminary results showed the expression of the mutant construct in the liver of at least one animal. Thus, further tests will be carried out to optimize experimental conditions, by testing other forms of minigenes administration and using other mice strains.
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