Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

Mansfield, S. Gary; Kole, Jolanta; Puttaraju, M.; Yang, Chaozhe; García-Blanco, Mariano A.; Cohn, Jonathan; Mitchell, Lloyd G.

doi:10.1038/sj.gt.3301307

Cited by 91 publications

(67 citation statements)

References 39 publications

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“…Previously reported experiments using co-transfecting mini-gene targets and ATMs have also demonstrated high levels of RNA trans-splicing compared to endogenous premRNA trans-splicing, with efficiency ranging from 1 to 80% based on real-time RT-PCR detection. 12,26,27 However, the efficiency of trans-splicing to an endogenous pre-mRNA target expressed in a stable cell line has generally been lower; with reports of between 3 and 7% efficiency when assayed by PCR, 13,26 which is comparable to the maximum trans-splicing efficiency achieved in our study.…”

Section: Exon 15 Mcssupporting

confidence: 69%

“…18 SMaRT has been shown to repair defective pre-mRNA molecules in cultured mammalian cells, xenografts and animal models of human disease including cystic fibrosis, 12 hemophilia A (factor VIII deficiency), 13 X-linked CD40 ligand immunodeficiency, 14 human apolipoprotein A1, 15 DNA protein kinase deficiency, 19 epidermolysis bullosa simplex with muscular dystrophy 20 and spinal muscular atrophy. 21 In DM1, a strategy to remove or correct mutant mRNA is appropriate in view of evidence that the accumulation of mutant mRNA in the nucleus is the primary basis of the disease.…”

Section: Discussionmentioning

confidence: 99%

“…The ATMs were constructed with an antisense-binding region of different lengths complementary to the 3 0 end of intron 14, 3 0 -splicing elements and the coding sequence of DMPK exon 15 fused in reading frame with GFP (see Materials and Methods, and Figure 1a). The construction of the ATMs was based primarily on the approach of Mansfield et al 12 who had designed two types of trans-splicing molecules, carrying linear and safety spacer sequences. Likewise, we constructed four linear ATMs (ATM130, ATM166, ATM214 and ATM250), four safety ATMs (ATM130S, ATM166S, ATM214S and ATM250S) and one negative control ATM without an antisense-binding region.…”

Section: Generation Of Atmsmentioning

confidence: 99%

“…12 Four different lengths of antisense-binding domain regions were designed to target intron 14 and at least one of two predicted U2-binding domains. We predicted two U2-binding domains in DMPK intron 14, namely (CTAAC) located at À138 to À134 bp (PU2) and a reverse sequence complimentary to AGCCAATCGA located at À49 to À39 bp (PU2r) from exon 15.…”

Section: Exon 15 Mcsmentioning

confidence: 99%

“…We examined artificial trans-splicing of corrective molecules as a molecular therapeutic strategy for DM1, namely the technique of spliceosome-mediated RNA transsplicing (SMaRT; Intronn proprietary technology, http://www.intronn.com). [12][13][14][15][16][17] In this study, the goal is to develop a trans-splicing system for DM1 and to investigate the efficiency of artificial trans-splicing molecules (ATMs) to target and correct the DMPK transcript without degradation. Our approach provides intron-specific gene correction at the pre-mRNA level in the nucleus.…”

Section: Introductionmentioning

confidence: 99%

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Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

et al. 2008

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Dystrophia myotonica type 1 (DM1), the most common muscular dystrophy in adults, results from expansion of a CTG repeat in the 3 0 -untranslated region of the dystrophia myotonica protein kinase gene (DMPK). Correction of the mutant DMPK transcript is a potential therapeutic strategy in DM1. We investigated the efficacy of artificial trans-splicing molecules (ATMs) to target and correct DMPK transcripts. ATMs designed to target intron 14 of DMPK pre-mRNA transcripts were tested for their ability to trans-splice the transcripts of a DMPK mini-gene construct and the endogenous DMPK transcripts of human myosarcoma cells (CCL-136). On agarose gel electrophoresis analysis, six of eight ATMs showed trans-splicing efficacy when applied to DMPK mini-gene construct transcripts, of which three were able to trans-splice endogenous DMPK pre-mRNA transcripts in myosarcoma cells, with trans-splicing efficiency ranging from 1.81 to 7.41%. These findings confirm that artificial trans-splicing can repair DMPK pre-mRNA and provide proof-of-principle evidence for this approach as potential therapeutic strategy for DM1.

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Section: Exon 15 Mcssupporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Section: Generation Of Atmsmentioning

confidence: 99%

Section: Exon 15 Mcsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

et al. 2008

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mRNAtrans‐splicing in gene therapy for genetic diseases

et al. 2016

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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.1347For further resources related to this article, please visit the WIREs website.

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Chimeric RNAs in cancer and normal physiology

Chwalenia

Facemire

2017

WIREs RNA

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Traditionally, chimeric RNAs were considered to be exclusive to cancer cells. When occasionally observed in normal samples, they were usually considered to be transcriptional 'noises,' or artifacts due to template switching during the reverse transcription and/or Polymerase chain reaction (PCR) steps of experimentation. However, with the advances being made in next generation sequencing technologies and software tools, as well as the accumulation of new experimental evidences, increasing numbers of chimeric transcripts are being identified in noncancerous tissues and cells. Recent studies have also demonstrated functional relevance, for at least a subset of chimeric RNAs in normal physiology. The advances have resulted in an influx of knowledge; this knowledge indicates that chimeric RNAs are a component of basic biology, and thus challenging traditional dogma. In addition to chromosomal rearrangement, chimeric RNAs can also be formed via different molecular mechanisms including cis-splicing of adjacent genes (cis-SAGe) and trans-splicing, as well as others. Little is known about the details of these noncanonical splicing processes. However, research in this new field promises to not only advance our basic understanding of the human genome and gene regulation, but also lead to improvements in clinical practice, especially in the areas of cancer diagnostics and treatment. WIREs RNA 2017, 8:e1427. doi: 10.1002/wrna.1427 For further resources related to this article, please visit the WIREs website.

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Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

Cited by 91 publications

References 39 publications

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

Correction of dystrophia myotonica type 1 pre-mRNA transcripts by artificial trans-splicing

mRNAtrans‐splicing in gene therapy for genetic diseases

Chimeric RNAs in cancer and normal physiology

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