TY - JOUR
T1 - Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa
AU - Hainzl, Stefan
AU - Trattner, Lisa
AU - Liemberger, Bernadette
AU - Bischof, Johannes
AU - Kocher, Thomas
AU - Ablinger, Michael
AU - Nyström, Alexander
AU - Obermayer, Astrid
AU - Klausegger, Alfred
AU - Guttmann-Gruber, Christina
AU - Wally, Verena
AU - Bauer, Johann W
AU - Hofbauer, Josefina Piñón
AU - Koller, Ulrich
N1 - Hainzl, Trattner, Liemberger, Bischof, Kocher, Ablinger, Klausegger, Guttmann, Gruber, Wally, Hofbauer, Koller: EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria; Bauer: Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria
PY - 2024/1/7
Y1 - 2024/1/7
N2 - 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 COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-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 COL7A1 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 COL7A1 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 COL7A1 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 COL7A1 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).
AB - 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 COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-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 COL7A1 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 COL7A1 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 COL7A1 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 COL7A1 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).
KW - Antisense oligonucleotides
KW - Col7a1
KW - Recessive dystrophic epidermolysis bullosa
KW - Splicing modulation
U2 - 10.3390/ijms25020761
DO - 10.3390/ijms25020761
M3 - Original Article
C2 - 38255836
SN - 1422-0067
VL - 25
JO - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
JF - INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
IS - 2
M1 - 761
ER -