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. 2021 Mar 24;22(7):3326.
doi: 10.3390/ijms22073326.

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

Michael Ablinger  1 , Thomas Lettner  1 , Nicole Friedl  1 , Hannah Potocki  1 , Theresa Palmetzhofer  1 , Ulrich Koller  1 , Julia Illmer  1 , Bernadette Liemberger  1 , Stefan Hainzl  1 , Alfred Klausegger  1 , Manuela Reisenberger  1   2 , Jo Lambert  3 , Mireille Van Gele  3 , Eline Desmet  3 , Els Van Maelsaeke  3 , Monika Wimmer  1 , Roland Zauner  1 , Johann W Bauer  1   2 , Verena Wally  1
Affiliations

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

Michael Ablinger et al. Int J Mol Sci. .

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.

Keywords: antisense oligonucleotides; exon skipping; junctional epidermolysis bullosa; liposomes; molecular therapy; splice mutation; topical therapy; type XVII collagen.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Antisense oligonucleotides (AON)-induced exon skipping. (A) Three AONs were designed to bind complementary to the cryptic exon 7 splice acceptor site (red upper case letters). The mutation at the wild-type splice junction isindicated as red lower case letter. (B) Exon skipping patterns observed in AON-treated vs. untreated (−AON) junctional epidermolysis bullosa patient-derived keratinocyte line (JEB-KC) compared to healthy control KC (hKC). Untreated JEB-KC show a distinct band that corresponds to the deletion of 16 nts of exon 7 (∆16 nt). All AON-treated samples show splicing products corresponding to the skipping of exon 7 (∆7) or exon 6 and exon 7 in combination (∆6+7). The latter was also found in untreated JEB-KC. All bands were analyzed by Sanger sequencing. (C) Next-generation sequencing (NGS) results of COL17A1 mRNA splice variants. All numbers are given as percentages, and grey lines indicate that transcripts have an open reading frame (ORF) divergent from the wild type.
Figure 2
Figure 2
AON-mediated type XVII collagen expression. (A) Semiquantitative (Sq)RT-PCR showed the restoration of COL17A1 expression in JEB-KC upon AON treatment. GAPDH was used as the reference. Fold change over untreated JEB-KC (-AON) was calculated using the 2−∆∆Ct method [23]. Unpaired, two-sided Student’s t-test was performed (n = 6), and p ≤ 0.05 was considered significant (** p < 0.01). (B) Western blot analysis of whole-cell lysates. Wild-type hKC showed two distinct type XVII collagen bands at 180 and 120 kDa that are absent in JEB-KC and reappear upon AON treatment. Annexin I (ANXAI) was used as the loading control. (C) The 120-kDa band reappeared in a conditioned culture medium of AON-treated JEB-KC. (D) In immunofluorescence microscopy, the images show that the expression of type XVII collagen is restored upon AON treatment. Scale bars: 50 µm. (E) Western blot analysis showed the expression of both the 120-kDa and the 180-kDa collagen 17 (C17) variants from the C17 lacking exons 6 and 7 (Δ6Δ7) expression cassette. (F) In addition, JEB∆6∆7 stained positive for type XVII collagen and showed C17 integration into the cell membrane (magnified section, white arrows). Scale bars: 50 µm, magnification: 10 µm.
Figure 3
Figure 3
Generation of 3D skin equivalents from AON-treated JEB-KC. Skin equivalents were generated from hKC and JEB-KC. Upon treatment with AON3, the deposition of C17 became detectable within the basal layer of the epidermis. Scale bar: 50 µm.
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