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mRNA trans‐splicing in gene therapy for genetic diseases

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Spliceosome‐mediated RNA trans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post‐transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre‐mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487–498. doi: 10.1002/wrna.1347 This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > RNA Editing and Modification
Design of a PTM for 3′ trans‐splicing. To achieve a 3′ replacement by trans‐splicing, the PTM must be composed of, from 5′ to 3′: a binding domain, an artificial intron containing a polypyrimidine tract (PPT), a branch point, a 3′ acceptor site (3′ SS), and the replacement cDNA which contains the coding sequence of the exon(s) to be corrected. The necessary 5′ donor site (5' SS) for the splice reaction is given by the endogenous pre‐mRNA.
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Different modes of action of trans‐splicing. (a) The classical splicing mechanism, or cis‐splicing occurs within a single pre‐mRNA molecule and leads to the elimination of the intronic sequences. (b, d) On the contrary, trans‐splicing occurs between two splice sites located on two different pre‐mRNAs. In SMaRT technology, an exogenous RNA, called pre‐mRNA trans‐splicing molecule (PTM) is used to replace one or several exons of an endogenous pre‐mRNA. Depending of the orientation of the PTM, it is possible to replace 3′‐exon(s) (b), 5′ exon(s) (c), or even internal exon(s) (d). Ex n: exon n; *: point mutation.
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Assessment of a fluorescent reporter system for PTM screening. (a) A fluorescent system was adapted to intron 1 of the rhodopsin gene (RHO), and the EaeI restriction site allowed quantifying trans‐splicing using the same methodology described in Berger et al. To that aim, RHO intron 1 was subcloned between sequences coding respectively the N‐terminal part of CFP (CFP‐Nter) and the C‐terminal part of YFP (YFP‐Cter), while the PTM contained the C‐terminal part of CFP (CFP‐Cter). With this system, cis‐splicing leads to an mRNA encoding a chimeric protein fluorescing minimally in green, and trans‐splicing leads to full‐length CFP, characterized by a bright blue fluorescence. (b) Three binding domains, previously characterized in the context of the full rhodopsin pre‐mRNA (for details, see Berger et al.), were tested with the fluorescent reporter system using the end‐point PCR amplification followed by enzymatic restriction method, and led to the same trans‐splicing rate, thus showing that trans‐splicing efficiency is independent of the genomic context of the binding domain. PTM0: negative control without binding domain. Location of binding domains of PTM1, 6, and 12 on RHO intron 1 have been described previously. Bars represent mean ± SD; n = 6 for fluorescent reporter system; for full‐length rhodopsin, n = 9 for PTM0 and 1, and n = 3 for PTM6 and 12.
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