Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Trans-generational epigenetic regulation of Celf2a, a splicing factor involved in Duchenne Muscular Dystrophy

Among a range of increasingly innovative therapies under clinical trial for Duchenne Muscular Dystrophy (DMD), exon skipping is absolutely one of the more promising options. The idea is to use antisense oligonucleotides to splice out selected exons from the pre-mRNA, at or next to the mutation site, so as to generate a translatable transcript from the mutant dystrophin gene. To day, exon skipping and recover of a correct reading frame has been demonstrated to be effective for many mutations, potentially allowing the conversion of severe Duchenne genotypes into milder Becker phenotypes (Bladen et al, 2015) (Verhaart & Aartsma-Rus, 2019). Due to the large size of introns, splicing enhancers play an important role in dystrophin mRNA maturation and indeed, their binding sequences have been used as targets for effective antisense strategies to induce exon skipping (Incitti et al, 2010) (Niks & Aartsma-Rus, 2017). This PhD project was the logical continuation of the investigation on a previously reported case of a DMD individual (GS44), carrying the deletion of exon 44, who showed a milder phenotype resembling a Becker one due to spontaneous skipping of exon 45 coupled to 7% of dystrophin rescue. Such endogenous exon skipping was due to the lack of Celf2a (Martone et al, 2016), an isoform of the Celf2 splicing factor (Ladd et al, 2001). Here, it is demonstrated that the absence of Celf2a expression in the GS44 patient is due to a trans-generational epigenetic silencing that is inherited from the mother and requires the activity of a repressive long non-coding RNA (lncRNA), DUXAP8. Epigenetic regulation hypothesis is supported by the recovery of Celf2a expression upon the patient’s fibroblasts reprogramming into induced Pluripotent Stem Cells (iPSCs) and the subsequent differentiation into myocytes, resulting in the ablation of exon 45 skipping. As already described, the reprogramming process affect chromatin accessibility dynamics (Li et al, 2017; Apostolou & Hochedlinger, 2013) inducing the modulation of several TFs (Peñalosa-Ruiz et al, 2019). Moreover, supposed the involvement of an epigenetic regulation on Celf2a expression, known the role of several ncRNAs as epigenetic modulators (Böhmdorfer & Wierzbicki, 2015), we focused our analysis on lncRNAs differentially expressed in GS44, filtering out the ones affected by the dystrophic condition. Using this filter, it was possible to identify a ncRNA, with an opposite behaviour, DUXAP8, involved in GS44 Celf2a lack: indeed, depletion of DUXAP8 in GS44 myoblasts rescued Celf2a expression, while its overexpression in control myoblasts repressed it. It was also demonstrated that the inactivation of Celf2a could have a more general therapeutic application since CRISPR/Cas9 inactivation of this factor in an unrelated 44 genetic background resulted in exon 45 skipping and partial rescue of dystrophin synthesis. Then, given the potentiality of Celf2a inhibition as treatment for a large class of DMD patients, a structural dynamic analysis was performed, identifying a putative strategy to target specifically Celf2a isoform.