Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52.

Steve D Wilton,Sue Fletcher,Jessica M Cale,Kane Greer

doi:10.3390/ijms22073479

Abstract

Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.

Highlights

Marfan syndrome (MFS, MIM 154700) is one of the most common dominantly inherited connective tissue diseases, affecting an estimated 2–3 in 10,000 individuals [1,2], in a family of disorders called the type-1 fibrillinopathies [3]
We propose that removal of a mutation-associated exon from all fibrillin-1 gene (FBN1) transcripts during the splicing process could result in the production of fibrillin-1 proteins that are able to form functional microfibrils, restoring extracellular matrix (ECM) stability
Marfan syndrome is well established as an inherited connective tissue dissyndrome well established as an inherited connective tissue disororderAlthough caused byMarfan mutations in theisfibrillin-1 gene, the exact mechanism of pathogenesis der caused by mutations in the fibrillin-1 gene, the exact mechanism of pathogenesis has not has not been fully resolved

Summary

Introduction

Marfan syndrome (MFS, MIM 154700) is one of the most common dominantly inherited connective tissue diseases, affecting an estimated 2–3 in 10,000 individuals [1,2], in a family of disorders called the type-1 fibrillinopathies [3]. Marfan syndrome is characterised by extreme height with disproportionate limb and digit length in comparison to the torso, coupled with a myriad of other skeletal, ocular, skin and cardiovascular abnormalities [4]. It is the progressive growth of the aorta often eventuating into aortic dissection and rupture that is the most common cause of death [5]. Fibrillin-1 multimers form the backbone of microfibrils [6] that are essential in the majority of connective tissues and to which many microfibril associated proteins bind [11]. It is in the microfibril form that fibrillin-1 exerts its structural and regulatory roles, providing a backbone for microfibrils [12], maintaining the stability of elastic

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