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

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.

Induced alternative splicing: opportunity to study PCSK9 protein isoforms at physiologically relevant concentrations

“…The proposed study on protein isoforms using splice modulation is most suitable for genes where alternative transcript isoforms are naturally present. Previously, we reported AO-mediated exon skipping studies for several genes [27][28][29][30]. We consistently observed e cient exon skipping for alternatively spliced exons and variable exon skipping e ciencies for other exons.…”

Induced alternative splicing an opportunity to study PCSK9 protein isoforms at physiologically relevant concentrations

“…The proposed study on protein isoforms using splice modulation is most suitable for genes where alternative transcript isoforms are naturally present. Previously, we reported AO-mediated exon skipping studies for several genes [27][28][29][30] . We consistently observed efficient exon skipping for alternatively spliced exons and variable exon skipping efficiencies for other exons.…”

Growth charts for Marfan syndrome in the Netherlands and analysis of genotype–phenotype relationships

“…The classification of HI and DN is a prediction in most cases, thus FBN1 variant outcome may be surprising. As an example of unexpected findings, exon skipping of exon 52 in 50% of the fibrillin‐1 mRNA resulted in complete loss of fibrillin‐1 fiber formation in the extracellular matrix, while fibers reappeared when >80% of exon skipping was achieved, revealing that a mismatch in size of the fibrillin‐1 protein may hamper fibrillin‐1 fiber formation (Cale et al, 2021 ). Thus, among the FBN1 DN variant type, tall individuals may indeed have reduced fibrillin‐1 fiber formation (HI phenotype), while producing the fibrillin‐1 protein.…”