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Developing of molecular therapies for collagen 3A1 mutations: Ehlers-Danlos syndrome

Utama, Sasiwimon (2022) Developing of molecular therapies for collagen 3A1 mutations: Ehlers-Danlos syndrome. PhD thesis, Murdoch University.

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Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS), a connective tissue disorder characterized by skin hyperextensibility, joint hypermobility and fatal vascular rupture, is caused by COL3A1 mutations that disrupt collagen III expression and structure. Collagen III is a fibrillar collagen and, along with collagens I, II, V and XI, plays an important role in the extracellular matrix (ECM), particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 demonstrated reduced collagen III deposition in the ECM, presumably due to compromised COL3A1 homo-trimer fibril assembly. The study explored the potential of an antisense oligonucleotide (AO) mediated splice switching strategy to bypass COL3A1 mutations affecting in-frame exons. Antisense oligonucleotides, designed to redirect COL3A1 pre-mRNA processing to excise specific exons were transfected into vEDS patient and healthy control fibroblasts. Exons 10, 15 and 40 could be excised from the mature COL3A1 mRNA, although variable levels of exon skipping were observed in transfected patient fibroblasts. Intracellular collagen III expression was increased after AO transfection and induction of the internally truncated isoforms, leading to a reduction in collagen III deposition in the ECM, compared to that in sham and untreated fibroblasts as assessed by immunofluorescence staining. In conclusion, this study explored skipping of specific in-frame COL3A1 exons, as a potential therapy for selected vEDS cases. While efficient exon skipping, and therefore induced isoform generation was achieved, the resultant collagen III protein isoforms showed extracellular matrix defects. The outcome of altering collagen III repeat structure on trimerization suggests that newly induced collagen III isoforms missing only one exon will not tolerate such changes, and this therapeutic avenue does not appear viable for vEDS.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Centre for Molecular Medicine and Innovative Therapeutics
Supervisor(s): Wilton, Steve, Fletcher, Sue, Aung-Htut, May and Mitrpant, C.
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