An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition

AN EXON-SPECIFIC U1 SMALL NUCLEAR RNA (snRNA) STRATEGY TO CORRECT SPLICING MUTATIONS ASSOCIATED TO HEMOPHILIA B

Background: Limitations of replacement therapy for coagulation deficiencies encourage research toward alternative strategies. The spliceosomal U1snRNA, having key role in pre-mRNA processing, represents an attractive molecule because of its ability to rescue splicing impaired by mutations, a frequent cause of coagulation factor defects (~15%). Though an engineered U1snRNA we have previously rescued factor VII function impaired by the IVS7+5G/A mutation in cellular models. Here, we exploited this approach to correct factor IX (FIX) mutations either at the donor (IVS5-2A/T, IVS5-2A/C, IVS5-2A/G) or acceptor (IVS5-8G,IVS5-9G) splice sites, and causing severe hemophilia B. Materials and Methods: Transfection of BHK cells with splicing-competent FIX cDNA constructs and modified U1snRNA. Evaluation of FIX mRNA (RT-PCR) and protein (ELISA. aPTT, WB) levels. Results: All mutations induced exon 5 skipping from the FIX mRNA, thus resulting in secretion of unactive FIX molecules lacking the EGF2 domain. A single U1snRNA designed to bind by complementarity to the normal FIX IVS5 donor splice site (5’ss) was able to restore exon 5 inclusion and secretion of functional FIX in the presence of all mutations. To improve specificity for F9 gene we subsequently tested a panel of U1snRNAs (Exon Specific U1snRNA) targeting the non-conserved intronic sequences instead of the 5’ss. Intriguingly, we found a gradient of rescue efficacy, which decreased with the distance from the 5’ss. The best ExSpeU1, targeting intronic positions +9 through +18, rescued exon 5 inclusion (from undetectable to 70-80%) and resulted in increased secretion (~2-3 fold) of FIX molecules with procoagulant activity (from undetectable to ~100%). Conclusions: These results demonstrate for the first time that a unique ExSpeU1 is able to restore gene expression impaired by different splicing mutants. The extent of rescue of the procoagulant function, if achieved in patients, would be beyond the therapeutic threshold

SEOM-SERAM-SEMNIM guidelines on the use of functional and molecular imaging techniques in advanced non-small-cell lung cancer

Imaging in oncology is an essential tool for patient management but its potential is being profoundly underutilized. Each of the techniques used in the diagnostic process also conveys functional information that can be relevant in treatment decision-making. New imaging algorithms and techniques enhance our knowledge about the phenotype of the tumor and its potential response to different therapies. Functional imaging can be defined as the one that provides information beyond the purely morphological data, and include all the techniques that make it possible to measure specific physiological functions of the tumor, whereas molecular imaging would include techniques that allow us to measure metabolic changes. Functional and molecular techniques included in this document are based on multi-detector computed tomography (CT), 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET), magnetic resonance imaging (MRI), and hybrid equipments, integrating PET with CT (PET/CT) or MRI (PET-MRI). Lung cancer is one of the most frequent and deadly tumors although survival is increasing thanks to advances in diagnostic methods and new treatments. This increased survival poises challenges in terms of proper follow-up and definitions of response and progression, as exemplified by immune therapy-related pseudoprogression. In this consensus document, the use of functional and molecular imaging techniques will be addressed to exploit their current potential and explore future applications in the diagnosis, evaluation of response and detection of recurrence of advanced NSCLC

The somatic FAH C.1061C>A change counteracts the frequent FAH c.1062+5G>A mutation and permits U1snRNA-based splicing correction

In tyrosinaemia type 1(HT1), a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue has been reported in many patients. This aspect is generally explained by a spontaneous reversion of the mutation into a normal genotype. In one HT1 patient carrying the frequent FAH c.1062+5G>A mutation, a second somatic change (c.1061C>A) has been reported in the same allele, and found in immunopositive nodules. Here, we demonstrated that the c.1062+5G>A prevents usage of the exon 12 5' splice site (ss), even when forced by an engineered U1snRNA specifically designed on the FAH 5'ss to strengthen its recognition. Noticeably the new somatic c.1061C>A change, in linkage with the c.1062+5G>A mutation, partially rescues the defective 5'ss and is associated to trace level (~5%) of correct transcripts. Interestingly, this combined genetic condition strongly favored the rescue by the engineered U1snRNA, with correct transcripts reaching up to 60%. Altogether, these findings elucidate the molecular basis of HT1 caused by the frequent FAH c.1062+5G>A mutation, and demonstrate the compensatory effect of the c.1061C>A change in promoting exon definition, thus unraveling a rare mechanism leading to FAH immune-reactive mosaicis