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

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.

Therapeutic Prospects of Exon Skipping for Epidermolysis Bullosa

“…Ablinger et al designed ASOs against COL17A1 exon 7 based on a patient with intermediate JEB carrying a variant in this exon. ASO treatment restored type XVII collagen expression in patient-derived primary cultured keratinocytes through exon 7 skipping [19]. The authors were also able to identify type XVII collagen expression at the cutaneous basal membrane in HSEs generated from JEB keratinocytes pretreated with COL17A1 exon 7 ASOs.…”

“…Extensive research into splice-modulating antisense oligonucleotide therapies has led to the approval of several ASO drugs by the FDA [13] and/or EMA, such as Nusinersen for spinal muscular atrophy (OMIM 253300) and Eteplirsen [14] and Viltolarsen [15] for Duchenne muscular dystrophy (OMIM 310200). Preclinical research into exon skipping for COL7A1 and COL17A1 has demonstrated its potential as therapy for EB as well [16][17][18][19].…”

“…Many different chemical modifications of the nucleotide s ribose have been developed. To date, studies on exon skipping for EB have all used ASOs containing 2 -O-methyl modified bases (2 OMe) on a phosphorothioate (PS) backbone (2 OMe-PS), 2 -O-methoxyethyl (2 -MOE)-modified bases with phosphorothioate backbone (2 -MOE-PS), and phosphorodiamidate morpholino oligomer (PMO) [16,17,19,22] (Figure 1). These ASO chemistries have been tested in vivo [21,23], and each can confer different benefits to pharmacokinetics and pharmacodynamics [21].…”

Investigational Treatments for Epidermolysis Bullosa

“…A double-blind, randomized, intra-subject, placebo-controlled clinical trial of an AON targeting this exon in DEB wounds (NCT03605069) is currently being assessed [43]. Although direct delivery of AON to skin cells in wounds is feasible, improving delivery into intact skin is also being addressed using cationic liposomes [44].…”

Molecular Research in Medical Genetics

“…In the last few years, the use of gene-editing technologies has demonstrated promising results. In particular, the use of antisense oligonucleotides was useful to correct some point mutations in Duchenne Muscular Dystrophy [ 17 , 18 ] and Epidermolysis Bullosa [ 19 ].…”