Poster abstracts

Poster number 19 submitted by Catherine Dominguez

Modifiers of SMN splicing in spinal muscular atrophy

Catherine Dominguez (Department of Pediatrics, The Ohio State University), Thomas Bebee (Department of Pediatrics, The Ohio State University), Jordan Gladman (Department of Pediatrics, The Ohio State University), Dawn Chandler (Department of Pediatrics, The Ohio State University)

Abstract:
Spinal Muscular Atrophy (SMA) is a neurodegenerative disease that is caused by low levels of Survival Motor Neuron (SMN) protein, encoded by two genes, SMN1 and SMN2. SMA is the result of an incomplete rescue by SMN2 expression in individuals lacking SMN1. These two genes differ by a single nucleotide in exon 7. This leads to increased skipping in SMN2 transcripts during pre-mRNA splicing. Therefore, while SMN1 produces almost exclusively full-length protein, SMN2 produces few full-length, functional proteins. Our first aim is to use antisense oligonucleotides (ASOs) to modify SMN splicing to develop a novel mouse model of SMA with an intermediate disease phenotype. Respiratory dysfunction and hypoxia are causes of acceleration in SMA disease progression and the only treatment of the disease is through the use of ventilation support. Therefore, our second aim is to assess the effects of hypoxia as a modifier of SMN splicing.
We've developed a mild SMA mouse model with the mouse gene mutated to resemble human SMN2. This mouse model will be used to produce an intermediate SMA mouse model through increasing Smn exon 7 skipping with ASOs targeted to an important splicing enhancer. To determine hypoxic alteration of splicing, we compared splicing changes under hypoxic and normoxic conditions. In addition, we designed SMN minigenes for mutation analysis to determine important splice sites in hypoxia-induced skipping.
In 293T cells and SMA patient fibroblasts, ASO treatment caused an increase in SMN skipping levels at 24, 48, and 96hrs, and lowered protein. In hypoxic cells hnRNPA1 accumulated after 24hrs and mutation of its binding sites was performed. Upon mutation of these hnRNPA1 binding sites, hypoxic induction was eliminated.
We conclude that the ASO is able to decrease protein levels and will next move into our mouse model. In our hypoxia-treated cells, we determined the primary factor responsible for hypoxia-induced skipping.

Keywords: Spinal Muscular Atrophy, ASO, splicing