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  • Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

International journal of molecular sciences (2021-04-04)
Michael Ablinger, Thomas Lettner, Nicole Friedl, Hannah Potocki, Theresa Palmetzhofer, Ulrich Koller, Julia Illmer, Bernadette Liemberger, Stefan Hainzl, Alfred Klausegger, Manuela Reisenberger, Jo Lambert, Mireille Van Gele, Eline Desmet, Els Van Maelsaeke, Monika Wimmer, Roland Zauner, Johann W Bauer, Verena Wally
ABSTRACT

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, pH 7, ≥98%
Roche
cOmplete, Mini Protease Inhibitor Cocktail, Tablets provided in a glass vial
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1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, ≥99.0% (10 mg phospholipid per ml CHCl3, TLC)
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Thrombin from human plasma, lyophilized powder, Suitable for routine use in the thrombin time test
Sigma-Aldrich
Fibrinogen from human plasma, 35-65% protein (≥90% of protein is clottable).
Sigma-Aldrich
DAPI, for nucleic acid staining