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.

Low doses of urban air particles from Buenos Aires promote oxidative stress and apoptosis in mice lungs.

UNLABELLED: Air pollution consists of a wide range of gaseous and particulate pollutants. Exposure to particulate matter (PM) can cause oxidative stress within the lung, which in turn can negatively impact health. The mechanisms by which PM causes oxidative stress include the release of trace metals or organic components from the particle. Previously, we have characterized urban air particles from downtown Buenos Aires (UAP-BA) and, by using in vivo animal studies, found that they are able to generate lung inflammation. PURPOSE: We studied lung responses to low doses of UAP-BA (15 µg), with special emphasis on oxidative balance. METHODS: We assessed cell viability, total cell number (TCN) and cell differential (CD) on bronchoalveolar lavages (BAL), oxidative metabolism in lung homogenates by tertbutylhydroperoxide-initiated chemiluminescence (CL), thiobarbituric reactive substances (TBARS), total reactive antioxidant potential (TRAP), reduced glutathione (GSH), and apoptosis in lung sections. RESULTS: We found that low UAP-BA exposure increases TCN, modifies CD, and decreases cell viability in the BAL. In lung homogenates, TBARS and CL rose while TRAP and GSH showed no alteration when compared to controls. Occurrence of apoptosis evaluated by TUNEL assay was markedly augmented in UAP-BA exposed animals. CONCLUSIONS: Our data further implicate oxidative stress as a possible inducer of apoptosis in lungs from animals exposed to low concentrations of this urban environmental contaminant.

Approaches to study CFTR pre-mRNA splicing defects.

In cystic fibrosis, genomic variants can result in defective processing of the CFTR precursor mRNA. Due to the complexity of the splicing process, the evaluation of their pathological effect is an important aspect both in the diagnostic field and in the study of basic regulatory mechanism. Efficient and correct splicing of CFTR relies on a complex process that includes recognition within the nascent transcripts of a series of different splicing regulatory elements that frequently overlap with the coding sequences. Identification of these elements is essential to determine the pathological impact of splicing-affecting genomic variants. In this chapter, to evaluate the effect of CFTR DNA variations on the pre-mRNA splicing process, different tools based on hybrid minigenes will be described.