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.