Trans-Splicing Improvement by the Combined Application  of Antisense Strategies

Koller, Ulrich; Hainzl, Stefan; Köcher, Thomas; Hüttner, Clemens; Klausegger, Alfred; Gruber, Christina; Mayr, Elisabeth; Wally, Verena; Bauer, Johann; Murauer, Eva M.

doi:10.3390/ijms16011179

Cited by 18 publications

(24 citation statements)

References 25 publications

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“…First, a pool of highly variable binding domains (BD) was generated through amplifying the COL7A1 gene region spanning from intron 75 to exon 80 and subsequent fragmentation via sonification. The resulting BD fragments ranging from 50 to 300 nucleotides were cloned into a previously described 3′ RTM screening vector [16,17,22] (Figure 1A). In total, 24 3′ RTMs with BDs in antisense orientation to the COL7A1 target region intron 75–exon 80 were included in co-transfection studies in HEK293 cells.…”

Section: Resultsmentioning

confidence: 99%

“…The stable COL7A1 -dTS-MG expressing cell line was created according to a previously published protocol [16]. …”

Section: Methodsmentioning

confidence: 99%

“…For the severe blistering skin disease epidermolysis bullosa, we have previously described several trans -splicing strategies where we replaced either 5′ or 3′ regions of a transcript, in order to correct EB-associated mutations in PLEC , KRT14 , COL17A1 and COL7A1 at pre-mRNA level [9,10,15,16,17,18]. These promising results provided us with substantial information on the optimal design of RNA trans -splicing molecules in order to execute a robust trans -splicing process.…”

Section: Introductionmentioning

confidence: 99%

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Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

Hüttner

Murauer

Hainzl

et al. 2016

IJMS

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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.

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Section: Resultsmentioning

confidence: 99%

“…The stable COL7A1 -dTS-MG expressing cell line was created according to a previously published protocol [16]. …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

Hüttner

Murauer

Hainzl

et al. 2016

IJMS

Self Cite

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“…Various experiments have shown that minor variations in length, sequence or localization of the target-binding position influence the splicing characteristics of a given RTM. 20, 21, 22, 40 The rational design of RTMs is difficult since up to now only few guidelines for generating potential RTMs exist. Consequently, we have recently developed an RTM reporter-based screening system that accelerates and facilitates RTM construction for any target pre-mRNA region of choice in a co-transfection assay.…”

Section: Discussionmentioning

confidence: 99%

Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations

et al. 2016

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RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3′ half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.

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“…Another example of using SMaRT to reprogram mRNA is the correction of the Tau isoform imbalance that is characteristic of FTDP-17 and tauopathies [67,68,69]. Recently, the trans -splicing system has been optimized through the combination of the trans-splicing RNA and antisense RNA interfering with competitive splicing elements on the pre-mRNA [70,71]. A comprehensive review detailing the use of SMaRT in gene therapy for genetic diseases has been recently published [63].…”

Section: Therapeutic Approachesmentioning

confidence: 99%

Targeting Splicing in the Treatment of Human Disease

Suñé-Pou

Prieto-Sánchez

Boyero-Corral

et al. 2017

Genes

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The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event.

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Trans-Splicing Improvement by the Combined Application of Antisense Strategies

Cited by 18 publications

References 25 publications

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations

Targeting Splicing in the Treatment of Human Disease

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