Duchenne Muscular Dystrophy (DMD) is one of the most severe neuromuscular diseases, affecting 1:3500 males born alive. It is a monogenic disorder caused by mutations in the largest human gene, the one encoding for the dystrophin protein. The lack of the protein affects the activity of skeletal muscles as well as that of heart and diaphragm leading the patients to the wheel chair by the age of 13 and to death by the third decade of age. The proposed project should possibly lead to the identification of therapeutic compounds for the DMD cases in which the open reading frame of dystrophin would be restored by exon 45 skipping; such cases represent almost 10% of the entire DMD population. The project is based on a recently identified DMD patient that, even if carrying a typical Duchenne mutation (exon 44 deletion), displays a Becker-like phenotype thanks to natural exon 45 skipping. The case reported is a 21 years-old boy that can walk and breath autonomously, but is unable to stand up from the ground. We have described (Martone et al. 2016, Nat. Commun. 7:10488) that in this patient, the absence of a specific splicing factor isoform (Celf2a) is able to cause the restoration of a dystrophin in-frame transcript, corresponding to 7% of total dystrophin amount, through endogenous skipping of exon 45. We demonstrated that Celf2a is needed for exon 45 inclusion in the dystrophin transcript. These data suggested that drug dependent inhibition of this factor, that could be applicable to almost 7.000 DMD-affected people in Europe and to 100.000 at a worldwide scale, could become a powerful new therapeutic approach.
The aims of this project will be the finding of a small molecule able to inhibit Celf2a activity and the understanding of the molecular regulation of Celf2a expression.
Due to its complex structure, the dystrophin pre-mRNA splicing makes quite intense use of splicing enhancers, whose function is to recognize short exon sequences, embedded in very complex introns, and to allow their recognition by the splicing machinery. It was previously suggested that different dystrophin exons respond to diverse splicing enhancer proteins (Mirò et al. 2015, NAR 43:2378); however, no idea of whether the alteration of such factors could be toxic to the cell and be compatible with life was available. In the described case report (Martone et al. 2016, Nat. Commun. 7:10488), the lack of the Celf2a splicing factor was able to produce exon 45 skipping. Moreover, the mother of the described patient is also missing Celf2a splicing isoform without any evident negative effect, suggesting that the inhibition of Celf2a could be able to occur without problems for normal life. So far, only for a specific subclass of mutations, those including premature stop codons, a specific drug, Ataluren, is under clinical test even though comprehensive clinical data confirming the positive benefit are not yet available. In this case, the transition to clinics has been favoured not only by the lack of any available treatment but also by the low cost of the product and by the easy administration procedures. The other advanced strategies under investigation, such as gene replacement and exon-skipping, are quite laborious and expensive. This is why alternative treatments, possibly based on oral administration of compounds of simple chemical nature able to provide skipping of specific exons, could be an interesting strategy to set up.
The other innovative aspect of this project resides on the use of patient-derived iPSCs to investigate how changes in chromatin structure can reset gene expression during reprogramming. It is known that reprogramming of human somatic cells to induced Pluripotent Stem Cells requires profound alteration in the epigenetic landscape (Buganim et al. 2013, Nat Rev Genet 14: 427). It seems that complete resetting and erasure of epigenetic memory of the initial somatic cell is required to obtain high quality population of iPS cells, but this could be not enough. In this case report, an epigenetic signature that maintain Celf2a gene transcriptionally inactive, is lost during reprogramming; this evidence suggests that not only the epigenetic memory defined as remaining of residual epigenetic signatures from the original somatic cells can be present in the produced iPS cells (Vaskova et al. 2013, Acta Naturae 5: 15-21) but also that some specific signatures, maintained through generation (the absence of Celf2a in GSD44 patient is inherited from the mother), can be lost during the reprogramming process. The understanding of Celf2a gene regulation could be interesting to shed light on this not well-understood mechanism, consisting on the unexpected alterations in iPSC gene expression patterns, that has an important impact on the idea to use iPSCs as ideal candidates for disease modelling approaches.