Messenger RNA Repair and Restoration of Protein Function by Spliceosome-Mediated RNA Trans-Splicing

Puttaraju, M.; DiPasquale, Janet; Baker, Carl C.; Mitchell, Lloyd G.; García-Blanco, Mariano A.

doi:10.1006/mthe.2001.0426

Cited by 70 publications

(81 citation statements)

References 22 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: 68%

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: 68%

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

et al. 2008

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“…Trans-splicing observed by using TauPTMnt suggests some degree of complementarity between the 5Ј end of TauPTMnt and sequences in intron 9 and is most likely the consequence of high levels of target and PTM expression, as observed previously in similar experimental paradigms (30,31). No such products were observed The 274-bp product corresponds to endogenous tau3R.…”

Section: Trans-splicing Inclusion Of Exon 10mentioning

confidence: 72%

“…For instance, 24% and 12% transsplicing efficiencies were reported with a collagen XVII minigene target (28) and a CFTR minigene (27), respectively. Trans-splicing represented 12-27% of cis-splicing in cells cotransfected with a minigene comprising lacZ exons separated by a truncated intron from the CFTR gene and a targeted PTM (30).…”

Section: Discussionmentioning

confidence: 99%

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

Rodríguez-Martín

García-Blanco

Mansfield

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3 end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9 -exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10 ؊ tau RNA into exon 10 ؉ tau RNA could be achieved with Ϸ34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.T au is a microtubule-associated protein predominantly expressed in neurons and specifically localized in axons that promotes microtubule polymerization and stabilization (1, 2). Mutations in the MAPT gene, encoding tau, on chromosome 17, cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (3-5). FTDP-17 is characterized by intraneuronal aggregates of tau and, as such, belongs to the group of dementias that includes Alzheimer's disease and collectively referred to as tauopathies.The human MAPT gene is a large gene of Ϸ134 kb comprising 16 exons (6). Alternative splicing of exons 2, 3, and 10 in the tau pre-mRNA results in the expression of six isoforms in the brain. Exon 10 encodes the second of four imperfect microtubulebinding repeats in the C-terminal half of the tau protein.Exclusion or inclusion of exon 10 gives rise to tau isoforms with three (tau3R, exon 10 Ϫ ) or four (tau4R, exon 10 ϩ ) microtubulebinding repeats (7). Furthermore, inclusion or exclusion of exons 2 and 3 produces tau isoforms with zero, one, or two inserts near the N terminus. Only tau3R is expressed during embryogenesis whereas tau3R and tau4R are expressed in approximately equal amounts in adult human brain. Exon 10 splicing is regulated by multiple cis-acting elements comprising exonic and intronic silencers and enhancers (for reviews, see refs. 8 and 9). In particular, intron 10 contains a bipartite element comprising a silencer and a modulator downstream to the 5Ј splice...

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“…The principle behind SMaRT is centered on the use of engineered pre-trans-splicing molecules (PTMs), which can replace the mutated portion of a disease-causing gene with the wild-type sequence. SMaRT can also be used to regulate the trans-splicing and expression of almost any desired gene sequence, such as those encoding reporter or toxic molecules (4,8). Embedded within each PTM are active splicing elements that are recognized by the cell's splicing machinery.…”

mentioning

confidence: 99%

“…These promote the formation of spliceosome complexes that trans-splice the PTM encoded exon(s) into the target transcript rather than allowing cis-splicing within the target pre-mRNA to occur. The specificity of the trans-splicing reaction is conferred primarily by the binding domain of the PTM, which is designed to be complementary to intronic sequences in the target of interest (8).…”

mentioning

confidence: 99%

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing

Bhaumik

Walls

Puttaraju

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.mRNA repair ͉ gene correction ͉ reporter I n the postgenomic era, a great impetus has been generated toward designing therapeutic and diagnostic agents that are able to capitalize on the wealth of genetic information now available. Although proteins are the ultimate effectors of genetic programming, there are several preceding steps in the cascade of gene expression where interventions for therapy or diagnosis are possible. Because of the complex tertiary folding and singular structure of each individual protein, it is unfeasible (at present) to design molecules specific for an arbitrary protein based on sequence alone. However, given that the principles behind targeting arbitrary nucleic acid sequences have been well established (1) and our profound knowledge of the human genomic sequence, it is practicable to design molecules that interact with specific genes at the nucleotide level. By exploiting the WatsonCrick base-pairing nature of nucleic acids, researchers have been able to design sequence-specific molecules for purposes ranging from in vivo antisense therapeutics to in vitro detection (2, 3).The concept of therapeutic intervention at the level of nucleic acids has been advanced recently by spliceosome-mediated RNA trans-splicing (SMaRT) (4, 5). SMaRT has effectively repaired disease-causing mutant genes at the level of RNA splicing for several disorders including hemophilia and cystic fibrosis (6, 7). The principle behind SMaRT is centered on the use of engineered pre-trans-splicing molecules (PTMs), which can replace the mutated portion of a disease-causing gene with the wild-type sequence. SMaRT can also be used to regulate the trans-splicing and expression of almost any desired gene sequence, such as those encoding reporter or toxic molecules (4,8). Embedded within each PTM are active splicing elements that are recognized by the cell's splicing machinery. These promote the formation of spliceosome complexes that trans-splice the PTM encoded exon(s) into the target transcript rather than allowing cis-splicing within the target pre-mRNA to occur. The specificity of the trans-splicing reaction is conferred primarily by the bindin...

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Messenger RNA Repair and Restoration of Protein Function by Spliceosome-Mediated RNA Trans-Splicing

Cited by 70 publications

References 22 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

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing

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