Antisense genes to induce exon inclusion.

Many inherited diseases are associated with changed splicing patterns, and alternative splicing influences several biological processes as well as the replication of certain viral pathogens. For this reason, there is a broad interest in modulating individual splicing events for therapeutic purposes. Based on the small nuclear RNA (snRNA) U7, we have developed expression vectors for short antisense RNAs that accumulate in the cell nucleus where splicing occurs and that can very specifically modulate the splicing of individual exons. More specifically, in the context of the fatal neuromuscular disorder Spinal Muscular Atrophy (SMA), we have shown that U7 snRNA constructs can restore the inclusion of exon 7 in the SMN2 gene and thereby alleviate or even fully cure disease symptoms in a severe mouse model for SMA. Here we describe more generally procedures to produce U7 constructs to induce exon inclusion and to test their efficiency in cell culture experiments at the level of RNA as well as protein. The analytical methods comprise reverse transcription (RT-)PCR to detect the splicing changes, quantitative real-time RT-PCR to measure U7 snRNA expression levels and western blot and immunofluorescence methods to detect a restoration of protein expression. Additionally, we indicate how U7 cassettes can be introduced into gene transfer vectors for in vivo experiments in animal models or to transduce cell systems that are not readily amenable to DNA transfection.