Promotion of exon 7 inclusion to treat spinal muscular atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of death in children. The disease is caused by the loss of the survival of motoneuron1 gene (SMN). All patients express an almost identical gene, SMN2. SMN2 is spliced differently from SMN1 and mostly excludes exon 7. This leads to an unstable SMN protein and prohibits SMN2 to substitute for the loss ofSMN1. A possible therapeutic approach for SMA is to promote inclusion of SMN2 exon 7. This exon is regulated by the tra2-beta1 splicing factor that binds to the central exonic splicing enhancer. We showed that protein phosphatase 1 (PP1) regulates alternative splicing by directly binding to the RNA recognition motif of tra2-beta1. Inhibition of PP1 by chemicals such as cantharidin or its natural protein inhibitors, such as NIPP-1, promotes exon inclusion. We therefore postulate that PP1 activity regulates the inclusion of SMN2 exon 7 by acting on tra2-beta1, as well as tra2-beta1 interacting proteins in the splicing complex. A decrease of PP1 activity promotes exon 7 inclusion, leading to the formation of SMN protein, whereas an increase of PP1 has the opposite effect. The overall goal of the proposal is to change PP1 activity in a way that specifically cause exon 7 inclusion, which will be aChieved by pursuing three specific aims: 1. Analyze the interaction of protein phosphatase 1 isoforms with tra2-beta1 and SMN2 pre-mRNA We will determine if PP1 dephosphorylates splicing factors such as tra2-beta1 within the splicing complex and/or if PP1 competes directly for binding to pre-mRNA with the tra2-beta1 splicing factor. We will determine if distinct PP1 isoforms exhibit any differential interaction with tra2-beta1. 2. Test the hypothesis that valproic acid causes a sequestration of PP1, which leads to exon 7 inclusion The HDAC2 inhibitor valproic acid has been shown to promote exon 7 inclusion, but its mechanism of action remains to be determined. It is known that the PP1 inhibitor NIPP1 interacts with histone deacetylase 2 (HDAC2) and that valproic acid causes proteasomal degradation of HDAC2. We will determine if valproic acid causes an increased sequestration of PP1 through NIPP1. 3. Develop selective, targeted inhibitors of PP1 that promote SMN2 exon 7 inclusion We will covalently tether a morpholino-oligonucleotide targeting exon 7 and a modified natural product that specifically inhibits PP1 to selectively promote the inclusion of exon 7 The expected results are a better understanding of signals that regulate exon 7 inclusion; an understanding how valproic acid acts on splice site selection and the generation of compounds that can selectively alter splice site selection.