Abstract
Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disorder of children with an incidence of 1 in 10,000 live births and a carrier frequency of 1 in 40-50 adults. SMA is attributable to a deficiency in the survival of motor neuron protein (SMN) caused in most patients by mutation of the SMN1 gene. Deficiency of SMN protein results in degeneration of anterior horn cells leading to hypotonia, symmetrical muscle weakness, fasciculation of the tongue muscles and tremors of the fingers and hands.
There are two genomic copies of the SMN gene (SMN1 and SMN2) and expression of the full length SMN2 gene product has been shown to partly compensate for the lack of SMN1 product. However, a single base difference (C/T) at the sixth nucleotide in exon 7 of the SMN2 gene promotes excision of that exon from the mature transcript leading to production of only a minimal amount of full-length protein.
The promotion of exon 7 inclusion in the SMN2 transcript by masking splice silencing motifs with antisense oligonucleotides (AO) is a potential intervention to increase the level of full-length SMN protein. Using a panel of modified 2'-O-methyl AO’s (phosphorothioate backbone) targeted across exon 7 of the SMN2 pre-mRNA we aimed to identify possible splice silencing motifs that would lead to exon 7 inclusion during SMN2 expression in SMA fibroblasts.
While our results failed to demonstrate a strong exonic silencing motif which could be masked to promote exon 7 inclusion parallel experiments in normal control cells showed robust exon 7 skipping could be induced by AO’s targeted near the exon 7 donor splice site. Therefore, this study has provided additional insight into exon splicing as it relates to SMN exon 7 providing a potential model with which to study the functionality of alternatively spliced SMN proteins.