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

Michael Ablinger,Alfred Klausegger,Stefan Hainzl,Julia Illmer,Ulrich Koller,Thomas Lettner,Hannah Potocki,Verena Wally,Jo Lambert,Johann W Bauer,Manuela Reisenberger,Nicole Friedl,Eline Desmet,Monika Wimmer,Roland Zauner,Mireille Van Gele,Els Van Maelsaeke,Theresa Palmetzhofer,Bernadette Liemberger

doi:10.3390/ijms22073326

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.

Highlights

The observation that the minor residual expression of collagen 17 (C17) is associated with mild phenotypes is a promising indicator for the feasibility of antisense oligonucleotides (AON) therapies, even if locally applied in a liposome-based formulation [16,17]. We show that this method effectively skips the affected exon and excludes it from pre-mRNA processing, thereby restoring the wild-type open reading frame (ORF), which leads to the expression of a slightly shorter but functional C17 protein
This deletion results in a frameshift, and subsequently, the aberrantly spliced mRNA runs into a premature termination codon (PTC) that triggers nonsense-mediated decay (NMD) (Supplementary Materials Figure S1A,B)
Considering the given genetic background, we assumed that the induction of exon 7 skipping (36 nts) using AONs would restore the ORF, resulting in the re-expression of a functional, slightly shortened, C17 protein

Summary

Introduction

Epidermolysis bullosa (EB) represents a group of genetic disorders with severe skin fragility. It is phenotypically characterized by blisters and erosions of the skin and mucous membranes, resulting from mechanical trauma that leads to tissue separation at the dermo–. Depending on the localization of the tissue disruption within the DEJ, four major subtypes can be phenotypically distinguished: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler EB (KEB) [1]. The degree or magnitude of the phenotypic manifestations (severe vs intermediate forms) depends on the genes involved, the type of mutation and, the residual protein expression, the mode of inheritance (dominant vs recessive) and the affected site of the body

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