Abstract
Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.
Highlights
The RNA-based technology spliceosome-mediated RNA trans-splicing (SMaRT) has evolved into a potential tool to repair mutations at the mRNA level and has been successfully applied to various in vitro disease models including epidermolysis bullosa (EB), cystic fibrosis and hemophilia [1,2,3,4,5].One of the most obvious advantages of SMaRT over gene replacement strategies is the size reduction of the transgene to deliver [6]
COL7A1-MG together with individually selected RNA trans-splicing molecules (RTMs) into human embryonic kidney cells
Its target binding position within the 5' portion of intron 102 prevents interference with antisense oligonucleotides (AS RNAs), which bind to splicing elements and possible splicing regulators on the target molecule
Summary
The RNA-based technology spliceosome-mediated RNA trans-splicing (SMaRT) has evolved into a potential tool to repair mutations at the mRNA level and has been successfully applied to various in vitro disease models including epidermolysis bullosa (EB), cystic fibrosis and hemophilia [1,2,3,4,5].One of the most obvious advantages of SMaRT over gene replacement strategies is the size reduction of the transgene to deliver [6]. The RNA-based technology spliceosome-mediated RNA trans-splicing (SMaRT) has evolved into a potential tool to repair mutations at the mRNA level and has been successfully applied to various in vitro disease models including epidermolysis bullosa (EB), cystic fibrosis and hemophilia [1,2,3,4,5]. The COL7A1 gene is a condign target for SMaRT due to its huge size of more than 9 kb when transcribed into mRNA. RNA trans-splicing molecule (RTM), which primarily base-pairs with a defined sequence on the target pre-mRNA of interest, thereby inducing the recombination of the two pre-mRNA molecules to a new chimeric mRNA. RNA trans-splicing can be used to reprogram 5', 3' or internal mRNA portions [6,8]
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