Abstract
RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.
Highlights
RNA trans-splicing technology has been implemented in the treatment of a spectrum of dominant and recessive inherited genetic diseases to repair pathogenic mutations [1]
We screened for binding positions in the target pre-mRNA in order to define optimal binding domains (BDs) to be included in the double trans-splicing RNA trans-splicing molecule (RTM)
In this study we have demonstrated the comprehensible steps of double RNA trans-splicing molecules (dRTM) design, and present challenges along the way to generate a functional RTM for endogenous mRNA repair
Summary
RNA trans-splicing technology has been implemented in the treatment of a spectrum of dominant and recessive inherited genetic diseases to repair pathogenic mutations [1]. The size of the corresponding cDNA of titin (100 kb) and dysferlin (6.2 kb), responsible for titinopathies and dysferlinopathies, respectively, exceed the packaging limits of conventional adeno-associated viral vectors. This engenders SMaRT technology as the method of choice [4]. Avale et al generated a manipulated tau mRNA transcript in vivo by using SMaRT technology This re-engineered tau mRNA included the missing exon 10 and displayed a corrected phenotype, which was previously blocked by mis-splicing leading to the tauopathy frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) [5]. RNA trans-splicing is suitable to correct transcripts in inherited diseases, but can be used in a suicide therapy approach, e.g., to induce toxin-mediated cell death in tumour cells, as demonstrated by our group and others [12,13,14]
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