COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents.

Area of Science:

• Molecular Biology
• Genetic Engineering
• Dermatology

Background:

• Mutations in the COL7A1 gene cause defective type VII collagen (C7), leading to severe skin fragility in dystrophic epidermolysis bullosa (DEB).
• Over 800 COL7A1 mutations are known, predisposing DEB patients to aggressive squamous cell carcinoma.

Purpose of the Study:

• To develop and evaluate a non-viral, non-invasive RNA therapy for correcting COL7A1 mutations using spliceosome-mediated RNA trans-splicing (SMaRT).
• To assess the efficiency of the 3'-RTMS6m repair molecule in restoring C7 protein expression in vitro and in skin equivalents.

Main Methods:

• Developed a 3'-RTMS6m repair molecule cloned into a minicircle-GFP vector for COL7A1 mutation correction via SMaRT.
• Transfected recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes and fibroblasts with the RNA therapy.
• Delivered the therapy topically to RDEB skin equivalents complexed with a DDC642 liposomal carrier.
• Confirmed splicing efficiency using next-generation sequencing (NGS) and C7 protein restoration via immunofluorescence (IF) and Western blot.

Main Results:

• Achieved approximately 1.5% trans-splicing efficiency in RDEB keratinocytes and 0.6% in fibroblasts at the mRNA level.
• Demonstrated restoration of full-length C7 protein expression in transfected cells.
• Successfully detected restored C7 accumulation in the basement membrane zone (BMZ) of topical-treated RDEB skin equivalents.

Conclusions:

• The non-viral 3'-RTMS6m RNA molecule effectively and transiently corrects COL7A1 mutations in vitro.
• This SMaRT-based approach shows potential as a therapeutic strategy for RDEB by restoring C7 protein in skin.
• Further development could lead to a non-invasive topical treatment for this severe genetic skin disorder.