Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52
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 <i>FBN1</i> mutations. Mutations in <i>FBN1</i> result in reduced fibrillin-1 expression, or the productio...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/7/3479 |
| _version_ | 1797539790076248064 |
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| author | Jessica M. Cale Kane Greer Sue Fletcher Steve D. Wilton |
| author_facet | Jessica M. Cale Kane Greer Sue Fletcher Steve D. Wilton |
| author_sort | Jessica M. Cale |
| collection | DOAJ |
| description | 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 <i>FBN1</i> mutations. Mutations in <i>FBN1</i> 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 <i>FBN1</i> 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 <i>FBN1</i> 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 <i>FBN1</i> 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. |
| first_indexed | 2024-03-10T12:50:51Z |
| format | Article |
| id | doaj.art-f8f1a53ab9c64a7aaedad45ab070b39e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T12:50:51Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f8f1a53ab9c64a7aaedad45ab070b39e2023-11-21T13:06:06ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-03-01227347910.3390/ijms22073479Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52Jessica M. Cale0Kane Greer1Sue Fletcher2Steve D. Wilton3Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch, WA 6150, AustraliaCentre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch, WA 6150, AustraliaMarfan 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 <i>FBN1</i> mutations. Mutations in <i>FBN1</i> 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 <i>FBN1</i> 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 <i>FBN1</i> 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 <i>FBN1</i> 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.https://www.mdpi.com/1422-0067/22/7/3479Marfan syndromefibrillin-1antisense oligonucleotidesexon skippingsplice-switching |
| spellingShingle | Jessica M. Cale Kane Greer Sue Fletcher Steve D. Wilton Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 International Journal of Molecular Sciences Marfan syndrome fibrillin-1 antisense oligonucleotides exon skipping splice-switching |
| title | Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 |
| title_full | Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 |
| title_fullStr | Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 |
| title_full_unstemmed | Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 |
| title_short | Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52 |
| title_sort | proof of concept antisense oligonucleotide mediated skipping of fibrillin 1 exon 52 |
| topic | Marfan syndrome fibrillin-1 antisense oligonucleotides exon skipping splice-switching |
| url | https://www.mdpi.com/1422-0067/22/7/3479 |
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