Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells

It is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations

RNA Editing Approaches for the correction of nonsense mutation in a cell model for Cystic Fibrosis

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. In particular, CFTR nonsense (STOP) mutations generate a premature termination codon (PTC) in the mRNA, leading to the production of a shortened and non-functional protein1. Currently, there is no pharmacological therapy that specifically targets nonsense mutations in CF. In this regard, we are exploring the possibility to correct the PTC using different RNA-based editing tools. These systems exploit the Adenosine Deaminases Acting on RNA (ADAR) to convert the adenosine within the PTC into inosine and allow the full-length protein synthesis2. Among these, a compact REPAIRv2 system uses a modified and truncated dCAS13x.1 fused with ADAR2DD that is recruited to the adenosine of PTC by means of a specifically designed guide RNA1. A different system named RESTORE uses specific antisense RNA oligonucleotides (ASOs), complementary to the CFTR mRNA region with the PTC, except for a cytidine-adenosine mismatch that promotes ADAR recruitment3. In addition, we also evaluated phenotypical anomalies of CFTR mutated cells showing morphological differences in comparison to wild type cells4. Our results pave the way to new therapeutical strategies potentially able to correct the nonsense mutations in cystic fibrosis

Chromosome Instability, friend or foe: genetic and epigenetic causes in cancer

Chromosome Instability (CI) compromises the fidelity of the transmission of the genetic material, and thus, is a grave risk for health. Many genetic diseases including cancer are characterised by CI. However, the leading causes identified so far can be different in each condition. Understanding the specific underlying mechanisms and how cells learn to deal with CI is of great relevance to design ad hoc and personalised therapeutic strategies. With this aim, we have been focused on three research lines: 1) investigating the involvement of DNA methylation in centromere stability and function, 2) unveiling a new player in chromosomal common fragile sites (CFSs) stability, 3) exploiting CI to induce senescence and allow cancer cells’ clearance. By using molecular and cell biology approaches, as well as microscopy techniques, we observed that global DNA methylation loss, a frequent event in cancer cells, undermines the correct loading of centromere proteins resulting in mitotic defects and CI. We also identified a key element, an helicase protein involved in the resolution of RNA:DNA hybrids at CFSs during mitosis, that appears to be critical for cancer cells’ survival. Moreover, we showed that curcumin treatment leads to senescence only tumor cells by increasing their endogenous CI. We also demonstrated that the combination of curcumin treatment and senolytic molecules is able to deplete cancer cells [1]. Our results pave the way for new therapeutic strategies against cancer progression that take advantage of the CI distinctive for each tumor context. We also set the course for DNA methylation essential to maintain centromere functionality and thus chromosomal stability

Specific Irreversible Cell-Cycle Arrest and Depletion of Cancer Cells Obtained by Combining Curcumin and the Flavonoids Quercetin and Fisetin

Background: Induced senescence could be exploited to selectively counteract the proliferation of cancer cells and target them for senolysis. We examined the cellular senescence induced by curcumin and whether it could be targeted by fisetin and quercetin, flavonoids with senolytic activity. Methods: Cell-cycle profiles, chromosome number and structure, and heterochromatin markers were evaluated via flow cytometry, metaphase spreads, and immunofluorescence, respectively. The activation of p21(waf1/cip1) was assessed via RT-qPCR and immunoblotting. Senescent cells were detected via SA-beta-Galactosidase staining. Results: We report that curcumin treatment specifically triggers senescence in cancer cells by inducing mitotic slippage and DNA damage. We show that curcumin-induced senescence is p21(waf1/cip1)-dependent and characterized by heterochromatin loss. Finally, we found that flavonoids clear curcumin-induced senescent cancer cells. Conclusions: Our findings expand the characterization of curcumin-induced cellular senescence in cancer cells and lay the foundation for the combination of curcumin and flavonoids as a possible anti-cancer therapy