K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

Area of Science:

• Genetics
• Molecular Biology
• Dermatology

Background:

• Autosomal dominant genetic diseases pose challenges for gene therapy due to the need for precise allele targeting.
• Epidermolysis bullosa simplex-Dowling-Meara (EBS-DM) is caused by a specific mutation in the keratin 14 (K14) gene, leading to severe skin blistering.

Purpose of the Study:

• To develop and validate spliceosome-mediated RNA trans-splicing for targeted correction of the K14 gene mutation in EBS-DM.
• To identify highly functional RNA trans-splicing molecules (RTMs) with optimized binding domains (BDs).

Main Methods:

• Utilized spliceosome-mediated RNA trans-splicing to replace exons 1-7 of the K14 gene with a wild-type sequence via an RTM.
• Developed a fluorescence-based screening system to identify RTMs with effective binding domains from a library.
• Tested the best RTMs in an EBS-DM patient-derived cell line for endogenous trans-splicing efficiency and accuracy.

Main Results:

• Identified highly functional RTMs through a fluorescence-based screening assay.
• Demonstrated efficient and accurate K14 gene trans-splicing at both RNA and protein levels in EBS-DM patient cells.
• Observed phenotypic reversion of skin cells in vitro using scratch assays, indicating successful correction.

Conclusions:

• RNA trans-splicing is a promising strategy for treating autosomal dominant genetic diseases like EBS-DM by simultaneously knocking down and repairing the mutated allele.
• The developed RTM screening method facilitates the identification of effective therapeutic molecules for gene repair.