Is Exon Skipping a Viable Therapeutic Approach for Vascular Ehlers-Danlos Syndrome with Mutations in COL3A1 Exon 10 or 15?

Abstract

Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, 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 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Keywords: COL3A1; Vascular Ehler-Danlos Syndrome; antisense oligonucleotides; collagen III.

Figure 1

Characterisation of skin fibroblasts derived from two vEDS patients carrying the mutations c.766delA and IVS14-2A>G. (a,b) RT-PCR amplification and Sanger sequencing of COL3A1 transcripts (exons 9–20) and GAPDH transcript (exon 1–3) from patient fibroblasts. Normal; unaffected healthy control. Neg ctrl; no template control. (c) Western blot analysis of collagen III expression in cell lysate (intracellular) and concentrated supernatant (extracellular). Data from triplicate samples are shown. The bar graph represents densitometric analyses (n = 3, error bars = SD). (d) Immunofluorescence analysis of intracellular and extracellular collagen III in ECM. Red: collagen III. Blue: nuclei.

Figure 1

Characterisation of skin fibroblasts derived from two vEDS patients carrying the mutations c.766delA and IVS14-2A>G. (a,b) RT-PCR amplification and Sanger sequencing of COL3A1 transcripts (exons 9–20) and GAPDH transcript (exon 1–3) from patient fibroblasts. Normal; unaffected healthy control. Neg ctrl; no template control. (c) Western blot analysis of collagen III expression in cell lysate (intracellular) and concentrated supernatant (extracellular). Data from triplicate samples are shown. The bar graph represents densitometric analyses (n = 3, error bars = SD). (d) Immunofluorescence analysis of intracellular and extracellular collagen III in ECM. Red: collagen III. Blue: nuclei.

Figure 2

Assessment of COL3A1 exon 10 skipping and collagen III expression in patient fibroblasts carrying the COL3A1 c.766delA mutation. (a) The exon 10 map indicates the ASO target sites and the exon-splicing enhancer and silencer motifs predicted by Splice Aid. Uppercase letters indicate exonic nucleotides and lowercase letters intronic nucleotides. The image is adapted from the original Splice Aid output (http://www.introni.it/splicing.html, accessed on 19 March 2024). (b) RT-PCR analysis of COL3A1 mRNA across exons 1–14. (c) Western blot analysis of intracellular and secreted collagen III protein. Collagen III is detected at top band; ~140 kDa, however, the presence of lower band is likely a non-specific band due to prolonged image exposure. (d) Immunofluorescence staining of collagen III deposition, (confocal images) in patient fibroblast cultures, transfected with the exon-skipping PMO cocktail [COL3A1_H10A(+15+39) and COL3A1_H10D(+12−13)] at 50 µM and 10 µM for four days. The bar graphs show average values from three independent transfections (n = 3, error bar = SD). Red: collagen III. Blue: nuclei.

Figure 3

Analysis of COL3A1 exon 15 skipping, collagen III expression and deposition in IVS14-2A>G patient fibroblasts: (a) The exon 15 map indicates the ASO target sites within exon 15 and the exon-splicing enhancer and silencer motifs predicted by Splice Aid output (http://www.introni.it/splicing.html, accessed on 19 March 2024). Uppercase letters indicate exonic nucleotides and lowercase letters intronic nucleotides. The image is adapted from the original Splice Aid output (splice aid). (b) RT-PCR analysis of COL3A1 mRNA across exons 9–20. (c) Western blot analysis of intracellular and secreted collagen III protein from patient fibroblast cultures transfected with COL3A1_H14A(+20+44) PMO at 50 µM and 2 µM, at day 4. Collagen III is detected at top band; ~140 kDa, however, the presence of lower band is likely a non-specific band due to prolonged image exposure. The statistical analysis was performed on data from three independent transfections (n = 3). (d) Immunofluorescence staining of collagen III deposition (confocal images). Red: collagen III. Blue: nuclei.

Figure 3

Analysis of COL3A1 exon 15 skipping, collagen III expression and deposition in IVS14-2A>G patient fibroblasts: (a) The exon 15 map indicates the ASO target sites within exon 15 and the exon-splicing enhancer and silencer motifs predicted by Splice Aid output (http://www.introni.it/splicing.html, accessed on 19 March 2024). Uppercase letters indicate exonic nucleotides and lowercase letters intronic nucleotides. The image is adapted from the original Splice Aid output (splice aid). (b) RT-PCR analysis of COL3A1 mRNA across exons 9–20. (c) Western blot analysis of intracellular and secreted collagen III protein from patient fibroblast cultures transfected with COL3A1_H14A(+20+44) PMO at 50 µM and 2 µM, at day 4. Collagen III is detected at top band; ~140 kDa, however, the presence of lower band is likely a non-specific band due to prolonged image exposure. The statistical analysis was performed on data from three independent transfections (n = 3). (d) Immunofluorescence staining of collagen III deposition (confocal images). Red: collagen III. Blue: nuclei.