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MODULATING ALTERNATIVE SPLICING OF FIBRONECTIN WITH ANTISENSE OLIGONUCLEOTIDES
Felicia Heidebrecht1, Vaishnavi Gnanananthan1, Benjaman To1, Nadja Utz1, Frank Rigo2, Sue Freier2, Mark Dockrell1
1 SWT Institute for Renal Research, London, UK
2 ISIS Pharmaceuticals, Carlsbad, CA, USA
INTRODUCTION: The EDA+ splice variant of fibronectin (FN EDA+) is an important component of the extracellular matrix found in fibrosis, facilitating fibroblast activation and collagen deposition. Directing the alternative splicing away from EDA inclusion could provide a novel therapy to limit renal fibrosis. The aim of this study was to test the possibility of modulating the TGFβ1-induced fibronectin splicing using RNase-H independent antisense oligonucleotides (ASO) and to investigate the presence of sweet spots on the exon that would prove better targets.
METHODS: In a first study 62 ASOs targeting overlapping sequences covering the whole exon and adjacent intron regions were administered before TGFβ1 challenge and assessed for their effects on both EDA+ and EDA- splice variants in primary human proximal tubule epithelial cells (PTEC).
In a second study selected ASOs binding: the 5’ and 3’ intron-exon junctions; previously described Splicing Enhancer Sites and Splicing Silencer Sites; as well as ASOs showing strongest knock down in the first study were further tested after the administration of TGFβ, as a model of therapeutic intervention after onset of disease.
RESULTS: Many ASOs that induced a switch in the splicing (reducing EDA+ more than 3 fold and inducing the EDA- 2 to 3.5 fold) did so in the presence of TGFβ1 stimulation or the effect in shifting the balance away from EDA+ was stronger with TGFβ1. Thus, this approach is attractive for therapeutic purposes since they alter the ratio EDA+/EDA- when TGFβ is present. The best sites for ASO-mediated splicing modulation were not predicted a priori and differences in the efficacy of the ASO when given pre compared to post TGFβ were observed. This might reflect the role of the cellular context on the three-dimensional structure of the mRNA and the accessibility of a certain target sequence.
CONCLUSION: These observations together with striking differences between various ASOs including ASOs with a high degree of overlap demonstrate the importance of wide screening in the appropriate cellular context for finding the most effective sequence to target. This type of screening using ASOs that cover the whole exon in overlapping steps could highlight unknown regulatory sequences along the exon.