Background: Whether blood eosinophil counts and exhaled nitric oxide (FeNO) are associated with important outcomes in mild asthma is unclear. Methods: This question was explored in a pre-specified analysis of a 52week, open-label, randomized, parallel-group trial in patients with mild asthma receiving only reliever inhalers, comparing salbutamol 200µg asneeded, maintenance budesonide 200µg twice-daily with salbutamol as needed, and budesonide/formoterol 200/6µg as-needed. Outcomes were compared between patients with blood eosinophils of <0.15, 0.15-<0.3 and ≥0.3x109/L; FeNO of <20, 20-50 and >50ppb; and a composite score based on both. Results: The proportion of patients randomised to as-needed salbutamol having a severe exacerbation increased progressively with increasing blood eosinophil sub-group (4.1%, 6.5% and 19.5%; p=0.014). There were no significant interactions between either biomarker and the effect of as-needed budesonide/formoterol compared with as-needed salbutamol for either exacerbations or severe exacerbations. However, there were significant interactions between blood eosinophil sub-groups and the effect of maintenance budesonide compared with as needed salbutamol for exacerbations (p<0.001) and severe exacerbations (p<0.001). Maintenance budesonide was more effective than as-needed salbutamol in patients with eosinophils ≥0.3x109/L for exacerbations (odds ratio 0.13; 95% CI 0.05-0.33) and severe exacerbations (0.11; 0.03-0.45). This was not the case for eosinophils <0.15x109/L (odds ratio for exacerbations 1.15; 0.51-1.28 and severe exacerbations 5.72; 0.97-33.6). There was no consistent interaction between treatment response and FeNO or the composite score. Conclusions: In patients with mild asthma the effects of as-needed budesonide/formoterol on exacerbations are independent of biomarker profile, whereas the benefits of maintenance inhaled budesonide are greater in patients with high blood eosinophil counts.

Mutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5′ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5′ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5′ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5′ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5′ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5′ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5′ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20–40%) from several mutants of the exon 2 5′ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5′ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs.

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATR context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AON) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATR-allele. Most importantly, upon lentivirus-mediated delivery, the U1 partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATR mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5′ splice site (5′ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.