Introduction
Cystic fibrosis (CF; MIM #219700) is the most common autosomal recessive genetic disease in European descents which affects about 1:2000-4000 new-borns in the US having higher morbidity in some European countries (Burgel et al., 2015; Farrell et al., 2017; Kosorok et al., 1996; Palomaki et al., 2004). The pathology is characterised by a wide spectrum of clinical complications of differing severity varying from classic multi-organ disease to atypical mild monosymptomatic CF forms (Schram, 2012; Zielenski, 2000). In classic CF, lung involvement is the major cause of mortality, dominated by recurrent infections and airway obstruction, finally leading to respiratory failure (Marcorelles et al., 2014). CF is caused by mutation in the Cystic Fibrosis Transmembrane conductance Regulator gene (CFTR, MIM #602421; GenBank NM 000492.3) (Ontalus et al. 1996; Choi et al. 2001), which codes for a transmembrane Cl- and HCO3- channel. CFTR gene is located on human chromosome 7, contains 27 exons and generates a 6.4 kb mRNA (Gregory et al., 1990) and have been grouped into functional classes which have recently evolved into theratypes (De Boeck & Amaral, 2016). CFTR disease-causing mutations can affect diverse cellular events including transcription, RNA splicing and protein function (Aissat et al., 2013). In the last years, several studies focusing on how to recover pharmacologically the function of mutated CFTR proteins have identified new drugs that have been approved for the treatment of CF (Hoy, 2019; Strug et al., 2018). These drugs, working as potentiators or correctors recover the defective proteins acting on different CFTR processing steps with a mutation-specific efficacy. This innovative approach has been successfully for developing mutation-oriented strategies for personalized medicine. However, this strategy was not specifically conceived for those CFTR mutations that affect precursor mRNA (pre-mRNA) splicing. Among reported mutations, those affecting pre-mRNA splicing represent 12.3 % and many induce exon skipping (Dujardin et al., 2011). Identification of exonic sequences on pre-mRNA requires the splice site consensus sequences as well as a series of exonic or intronic splicing regulatory elements with enhancer or silencer activity (Exonic Splicing Enhancer ESE, Exonic Splicing Silencer ESS, Intronic Splicing Enhancer ISE and Intronic Splicing Silencer ISS). A core of five small nuclear RNPs (snRNP, including U1 snRNP) and several splicing factors along with pre-mRNA secondary structures are important players that act in a coordinated manner to promote the correct exon recognition (Abbink and Berkhout 2008, Zychlinski et al. 2009). Considering the complexity of the splicing process, different types of mutations can induce exon skipping. These mutations are mechanistically different as they can affect consensus sequences at the splice sites, the polypyrimidine tract as well as exonic regulatory elements. As a consequence of this heterogeneity, their rescue may not be an easy task and might require specific approaches. Exon Specific U1s are modified U1 snRNAs that have been shown to rescue different types of exon skipping defects in several diseases. ExSpeU1s have the same composition of normal U1 snRNPs but while U1 snRNPs interact with the 5’splice site (5’ss), ExSpeU1s target intronic sequences by means of their engineered 5’ tail (Rogalska et al., 2016). It was previously shown that their binding downstream affected exons corrects aberrant splicing in several cellular (Alanis et al., 2012; Dal Mas, Fortugno, et al., 2015; Nizzardo et al., 2015; Tajnik et al., 2016) and mouse models (Balestra et al. 2014; Dal Mas, Rogalska, et al. 2015; Balestra et al. 2016; Rogalska et al. 2016; Donadon et al. 2018). Strikingly, an ExSpeU1 delivered by Adeno Associated Virus (AAV) resulted to an effective and safe therapy in a Spinal Muscular Atrophy (SMA) mouse model extending the survival from 10 days to ~ 6 months (Donadon et al., 2019).
In this study, to prove the potential therapeutic activity of these molecules on CFTR and to establish a useful platform that can be applied to CF, we focussed on ten relatively frequent splicing mutations/variants that cause skipping of corresponding exons 5, 9, 13, 16 and 18. Splicing mutations 711+3A>C/G and 711+5G>A are located in the 5’ss consensus of exon 5; 1863C>T (Y577Y) and 1898+3A>G in an exonic regulatory element and in the 5’ss consensus of exon 13, respectively; 2789+5G>A and 3120G>A are located at the 5’ss consensus of exon 16 and 18 respectively, whereas TG13T3, TG13T5, TG12T5 are variants at the polypyrimidine tract of exon 9. Using minigene splicing assay we prove that all these mutations can be efficiently corrected by ExSpeU1s in vitro . In addition, detailed analysis of exon 13 suggests that the difficult splicing rescue of this exon might be due to the presence of an Intronic Splicing Silencer (ISS) controlling an unfavourable RNA secondary structure. This result represents the first step towards the development of a personalized approach based on the ExSpeU1 strategy for rescuing CFTR splicing mutations.