An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA

Spinal muscular atrophy (SMA) is a degenerative motor neuron disorder resul ting from homozygous loss of the SMN1 gene. SMN2, a nearly identical copy g ene, is preserved in SMA patients. A single nucleotide difference between S MN1 and SMN2 causes exon 7 skipping in the majority of SMN2 mRNA. Gene ther apy through modulation of SMN2 gene transcription in SMA patients may be po ssible. We constructed a series of SMN mini-genes comprised of SMN exon 6 t o exon 8 sequences fused to green fluorescence protein (GFP) or luciferase reporters, to monitor SMN exon 7 splicing. These reporters recapitulated th e splicing patterns of the endogenous SMN gene in stable cell lines. The SM N1-luciferase reporter was approximately 3.5-fold more active than SMN2-luc iferase and SMN1-GFP intensities were visually distinguishable from SMN2-GF P. We have screened chemical inducers and inhibitors of kinase pathways usi ng stable SMN-reporter lines and found that the phosphatase inhibitor sodiu m vanadate specifically stimulated exon 7 inclusion within SMN2 mRNAs. This is the first compound identified that can stimulate exon 7 inclusion into transcripts derived from the endogenous SMN2 gene. These results demonstrat e that this system can be utilized to identify small molecules that regulat e the splicing of SMN exon 7.