Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa

• Published in: International journal of molecular sciences 2024-01, Vol.25 (2), p.761
• Main Authors: Hainzl, Stefan, Trattner, Lisa, Liemberger, Bernadette, Bischof, Johannes, Kocher, Thomas, Ablinger, Michael, Nyström, Alexander, Obermayer, Astrid, Klausegger, Alfred, Guttmann-Gruber, Christina, Wally, Verena, Bauer, Johann W, Hofbauer, Josefina Piñón, Koller, Ulrich
• Format: Article
• Language: English
• Subjects: antisense oligonucleotides; COL7A1; Collagen; Collagen Type VII - genetics; Development and progression; Epidemiology; Epidermolysis bullosa; Epidermolysis Bullosa Dystrophica - genetics; Epidermolysis Bullosa Dystrophica - therapy; Fibroblasts; Gene expression; Genes; Genetic disorders; Health aspects; Humans; Introns; Messenger RNA; Mutation; Oligonucleotides, Antisense - genetics; Oligonucleotides, Antisense - therapeutic use; Pharmaceutical industry; Protein expression; Proteins; recessive dystrophic epidermolysis bullosa; Retention; RNA Precursors; RNA Splicing; Skin; splicing modulation
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms25020761

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). -encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).