High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Simplex

Wally, Verena; Koller, Ulrich; Bauer, Johann

doi:10.5772/24149

Cited by 5 publications

(9 citation statements)

References 38 publications

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“…This was achieved via PCR amplification, fragmentation and cloning of the resulting fragments into the 5′ RTM screening vector [22,23]. Functional BDs were identified by sequence analysis of a variety of individual RTM-expressing plasmids (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

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%

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

Hüttner

Murauer

Hainzl

et al. 2016

IJMS

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“…COL7A1 is a large gene with over 9kb and is therefore suitable for this approach, in which only a short RTM has to be designed, harbouring only the first 15 exons of the gene. Using an RTM screening system, described by Wally et al 2011 [89], it should be possible to increase the trans-splicing efficiency of designed RTMs to a level where the phenotype of COL7A1 deficient cells can be converted into wildtype. We analyzed the binding properties of randomly designed RTMs specific for intron 15 of murine COL7A1 and tested the most efficient RTM in COL7A1 deficient keratinocytes for endogenous functionality.…”

Section: Resultsmentioning

confidence: 99%

Gene Therapy for the COL7A1 Gene

Mayr¹,

Koller²,

Bauer³

2013

Gene Therapy - Tools and Potential Applications

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“…In the past, low recombination efficiencies impeded the achievement of a therapeutic effect using the SMaRT technology. Therefore, we exerted the special knowledge from our earlier studies 17,18 putting much effort into the optimization of the repair molecules, to design our RTM. We generated a 5′ RTM containing murine Col7a1 exons 1-15, a 2.1 kb wild-type cDNA transgene, to establish a functional 5′ trans-splicing approach.…”

Section: Discussionmentioning

confidence: 99%

“…Regarding to our previous findings, this BD would block competitive cis-splice site elements within the target region and thus suppress cis-splicing within the endogenous pre-mRNA resulting in an enhanced trans-splicing rate. 3,17,18 To avoid the risk of direct RTM expression, leading to the translation of truncated proteins, we removed two in-frame termination codons within the BD sequence. The potential of the BD to induce specific exon replacement was shown in a previously established screening system.…”

Section: Discussionmentioning

confidence: 99%

“…15,16 The BD was designed to cover the endogenous Col7a1 exon15/intron15 junction, due to the positive impact of RTM interference with competitive target splicing elements on the trans-splicing efficiency. 3,17,18 Nonviral RTM delivery via minicircle plasmids is known to exhibit prolonged transgene expression in vivo and due to its decreased vector size the plasmid uptake of the cells is increased. 19 To deliver the minicircle plasmids into the skin of wild-type mice, we used a gene gun approach, posing a safe, efficient and noninvasive in vivo transfer method of nucleic acids.…”

Section: Introductionmentioning

confidence: 99%

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A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

Peking

Koller

Hainzl

et al. 2016

Molecular Therapy - Nucleic Acids

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RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

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High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Simplex

Cited by 5 publications

References 38 publications

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

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

Gene Therapy for the COL7A1 Gene

A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

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