Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Alternative splicing of human cystic fibrosis transmembrane conductance reg ulator (CFTR) exon 9 is regulated by a combination of cis-acting elements d istributed through the exon and both flanking introns (IVS8 and IVS9). Seve ral studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At p resent, no cellular factors have been identified that recognize this elemen t. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transi ent TDP-43 overexpression in Hep3B cells results in an increase in exon 9 s kipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results i n increased inclusion of exon 9, providing a new therapeutic target to corr ect aberrant splicing of exon 9 in CF patients. The: clinical and biologica l relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(Delta F508)/ TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.