The Experts below are selected from a list of 7992 Experts worldwide ranked by ideXlab platform
Johann W Bauer - One of the best experts on this subject based on the ideXlab platform.
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RNA Trans-Splicing Modulation via Antisense Molecule Interference.
International journal of molecular sciences, 2018Co-Authors: Bernadette Liemberger, Eva M Murauer, Verena Wally, Johann W Bauer, Josefina Piñón Hofbauer, Claudia Arzt, S. Hainzl, Thomas Kocher, Julia Reichelt, Ulrich KollerAbstract:In recent years, RNA Trans-Splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher Trans-Splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA Trans-Splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the Trans-Splicing efficiency. Thus, we assume that besides the RNA Trans-Splicing molecule, short antisense molecules can act as Splicing modulators, thereby increasing the Trans-Splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.
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A Gene Gun-mediated Nonviral RNA Trans-Splicing Strategy for Col7a1 Repair
Elsevier, 2016Co-Authors: Patricia Peking, Ulrich Koller, S. Hainzl, Thomas Kocher, Julia Reichelt, Sophie Kitzmueller, Elisabeth Mayr, Alexander Nyström, Thomas Lener, Johann W BauerAbstract: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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Trans-Splicing Improvement by the Combined Application of Antisense Strategies
International Journal of Molecular Sciences, 2015Co-Authors: Ulrich Koller, Verena Wally, Johann W Bauer, S. Hainzl, Thomas Kocher, Elisabeth Mayr, Clemens Hüttner, Alfred Klausegger, Christina Gruber, Eva M MurauerAbstract:Spliceosome-mediated RNA Trans-Splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA Trans-Splicing molecules (RTMs) are designed to induce a specific Trans-Splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM Trans-Splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the Trans-Splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-Transfections of the RTM and a selected AS RNA, interfering with competitive Splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA Trans-Splicing efficiency. Thereby, accurate Trans-Splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high Trans-Splicing efficiency is required.
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the design and optimization of rna Trans Splicing molecules for skin cancer therapy
Molecular Oncology, 2013Co-Authors: Christina Gruber, Eva M Murauer, Ulrich Koller, S. Hainzl, Thomas Kocher, Clemens Hüttner, Andrew P. South, Helmut Hintner, Johann W BauerAbstract:Targeting tumor marker genes by RNA Trans-Splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA Trans-Splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific Trans-Splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their Trans-Splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a Trans-Splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.
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The design and optimization of RNA Trans‐Splicing molecules for skin cancer therapy
Molecular oncology, 2013Co-Authors: Christina Gruber, Eva M Murauer, Ulrich Koller, S. Hainzl, Thomas Kocher, Clemens Hüttner, Andrew P. South, Helmut Hintner, Johann W BauerAbstract:Targeting tumor marker genes by RNA Trans-Splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA Trans-Splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific Trans-Splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their Trans-Splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a Trans-Splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.
Timothy W. Nilsen - One of the best experts on this subject based on the ideXlab platform.
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new components of the spliced leader rnp required for nematode Trans Splicing
Nature, 2002Co-Authors: John A. Denker, Patricia A. Maroney, David M Zuckerman, Timothy W. NilsenAbstract:Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition Trans-Splicing1,2. In this unusual RNA processing reaction, a short common 5′ exon, the SL, is affixed to the 5′-most exon of multiple pre-mRNAs. The nematode SL is derived from a Trans-Splicing-specific ∼100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP)5. Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (Mr 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL Trans-Splicing; however, neither protein is required either for conventional cis-Splicing or for bimolecular (Trans-) Splicing of fragmented cis constructs. The Mr 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL Trans-Splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a Trans-Splicing specific network of protein–protein interactions analogous to the U1 snRNP–SF1/BBP–U2AF complex that comprises the cross-intron bridge in cis-Splicing.
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Evolutionary origin of SL-addition Trans-Splicing: still an enigma
Trends in genetics : TIG, 2001Co-Authors: Timothy W. NilsenAbstract:Abstract Spliced-leader (SL) Trans -Splicing is an essential step in pre-mRNA maturation in a variety of lower eukaryotic organisms. However, this processing pathway is absent in mammals, insects, yeast and plants. The patchy phylogenetic distribution of SL Trans -Splicing is consistent with either ‘multiple gain' or ‘multiple loss' evolutionary scenarios. Recent studies show that two additional metazoan phyla carry out SL Trans -Splicing, significantly increasing its phylogenetic range. However, it remains unclear whether this unusual type of Splicing is an ancestral or an acquired trait.
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Trans-Splicing: An update
Molecular and biochemical parasitology, 1995Co-Authors: Timothy W. NilsenAbstract:5′-end maturation of messenger RNAs via acquisition of a Trans-spliced leader sequence occurs in several primitive eukaryotes, some of which are parasitic. This type of Trans-Splicing proceeds though a two-step reaction pathway directly analogous to that of cis-Splicing and like cis-Splicing it requires multiple U snRNP cofactors. This minireview attempts to provide a brief synopsis of our current understanding of the evolution and biological significance of Trans-Splicing. Progress in deciphering the mechanism of Trans-Splicing, particularly as it relates to current models of cis-Splicing, is also discussed.
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Trans-Splicing of Nematode Premessenger RNA
Annual review of microbiology, 1993Co-Authors: Timothy W. NilsenAbstract:In nematodes, many mRNAs contain a common 5' terminal 22-nt sequence. This sequence, the spliced leader (SL), is acquired from a small (approximately 100 nt) SL RNA via Trans-Splicing. Parallel in vitro and in vivo experiments have begun to clarify both the mechanism and biological role of Trans-Splicing. In vitro analysis (in cell free extracts) has shown that Trans-Splicing is remarkably similar to the snRNP mediated removal of intervening sequences from pre-mRNAs (cis-Splicing). Additionally, this analysis has suggested a mechanism that may explain how the two substrates of Trans-Splicing (the SL RNA and pre-mRNA) efficiently associate with one another in the absence of sequence complementarity. In vivo experiments suggest that a major biological function of Trans-Splicing in nematodes may be to process polycistronic Transcription units. Results obtained from the study of both parasitic and free-living species are discussed, and Trans-Splicing in nematodes is compared and contrasted to the analogous process in trypanosomatid protozoans.
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Trans-Splicing in protozoa and helminths.
Infectious agents and disease, 1992Co-Authors: Timothy W. NilsenAbstract:Trans-Splicing is defined as the process whereby exons derived from two separately Transcribed RNAs are joined together. In one type of Trans-Splicing, nuclear pre-mRNAs acquire their 5' terminal exon (the spliced leader) from a small spliced leader RNA (SL RNA) via an RNA processing reaction that is directly analagous to the removal of intervening sequences (cis-Splicing). Such leader-addition by Trans-Splicing has been extensively studied in trypanosomatid protozoans and in nematodes. This review summarizes recent advances in research on Trans-Splicing in these two systems. Progress in elucidating functionally significant sequence elements within SL RNAs and progress in understanding the mechanism and biological role of Trans-Splicing is discussed.
Christian L. Lorson - One of the best experts on this subject based on the ideXlab platform.
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Optimization of Trans-Splicing for Huntington's Disease RNA Therapy
Frontiers in neuroscience, 2017Co-Authors: Hansjörg Rindt, Christian L. Lorson, Colton M Tom, Virginia B. MattisAbstract:Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin (HTT) gene. We have previously demonstrated that spliceosome-mediated Trans-Splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here the targeted tethering efficacy of the pre-mRNA Trans-Splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3’ end of HTT intron 1 or the intron 1 branch point were shown Trans-splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the Trans-Splicing efficacy from 1–5% to 10-15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mtHTT allele in the context of HD.
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Optimization of SMN Trans-Splicing Through the Analysis of SMN Introns
Journal of Molecular Neuroscience, 2012Co-Authors: Monir Shababi, Christian L. LorsonAbstract:Spinal muscular atrophy (SMA), a neurodegenerative disease, is the leading genetic cause of infantile death and is caused by the loss of survival motor neuron 1 ( SMN1 ). Humans carry a duplicated copy gene, SMN2 , which produces very low levels of functional protein due to an alternative Splicing event. This Splicing difference is the reason that SMN2 cannot prevent SMA development when SMN1 is deleted. SMN2 generates a Transcript lacking exon 7 and consequently gives rise to an unstable truncated SMN protein that cannot protect from SMA. To increase full-length SMN protein, we utilize a strategy referred to as Trans -Splicing. This strategy relies upon pre-mRNA Splicing occurring between two separate molecules: (1) the endogenous target RNA and (2) the therapeutic RNA that provides the correct RNA sequence via a Trans -Splicing event. The initial Trans -Splicing RNA targeted intron 6 and replaced exon 7 with the SMN1 exon 7 in SMN2 pre-mRNA. To determine the most efficient intron for SMN Trans -Splicing event, a panel of Trans -Splicing RNA molecules was constructed. Each Trans -Splicing RNA molecule targets a specific intron within the SMN2 pre-mRNA and based on the target intron, replaces the downstream exons including exon 7. These constructs were examined by RT-PCR, immunofluorescence, and Western blotting. We have identified intron 3 as the most efficient intron to support Trans -Splicing in cellular assays. The intron 3 Trans -Splicing construct targets intron 3 and replaces exons 4–7 and was distinguished based on its ability to produce the highest level of the Trans -spliced RNA and full-length SMN protein in SMA patient fibroblasts. The efficiency of the intron 3 construct was further improved by addition of an antisense that blocks the 3′ splice site at the intron 4/exon 5 junction. Most importantly, intracerebroventricular injection of the Int3 construct into SMNΔ7 mice elevated the SMN protein levels in the central nervous system. This research demonstrates an alternative platform to correct genetic defects, including SMN expression and examines the molecular basis for Trans -Splicing.
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development of a single vector system that enhances Trans Splicing of smn2 Transcripts
PLOS ONE, 2008Co-Authors: Tristan H Coady, Monir Shababi, Travis D Baughan, Marco A Passini, Christian L. LorsonAbstract:RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA Splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative Splicing. We previously reported a Trans-Splicing RNA (tsRNA) that re-directed SMN2 Splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of Trans-Splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the Trans-Splicing mechanism and a means of significantly enhancing Trans-Splicing activity in vivo.
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restoration of smn function delivery of a Trans Splicing rna re directs smn2 pre mrna Splicing
Molecular Therapy, 2007Co-Authors: Tristan H Coady, Monir Shababi, Gregory E Tullis, Christian L. LorsonAbstract:Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients; however SMN2 produces low levels of functional protein due to alternative Splicing. Recently a therapeutic approach has been developed referred to as Trans-Splicing. Conceptually, this strategy relies upon pre-messenger RNA (pre-mRNA) Splicing occurring between two separate molecules: (i) the endogenous target RNA and (ii) the therapeutic RNA that provides the correct RNA sequence via a Trans-Splicing event. SMN Trans-Splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct Splicing from a SMN2 mini-gene as well as from endogenous Transcripts. Subsequently, recombinant adeno-associated viral vectors were developed that expressed and delivered Trans-Splicing RNAs to SMA patient fibroblasts. In the severe SMA patient fibroblasts, SMN2 Splicing was redirected via Trans-Splicing to produce increased levels of full-length SMN mRNA and total SMN protein levels. Finally, small nuclear ribonucleoprotein (snRNP) assembly, a critical function of SMN, was restored to SMN-deficient SMA fibroblasts following treatment with the Trans-Splicing vector. Together these results demonstrate that the alternatively spliced SMN2 exon 7 is a tractable target for replacement by Trans-Splicing.
Lloyd G Mitchell - One of the best experts on this subject based on the ideXlab platform.
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rna Trans Splicing targeting endogenous β globin pre messenger rna in human erythroid cells
Human Gene Therapy Methods, 2017Co-Authors: Naoya Uchida, Charlotte Platner, Josiah Ballantine, Matthew M. Hsieh, Brian Mozer, Kareem Washington, Luke P Skala, Lydia Raines, Anna Shvygin, Lloyd G MitchellAbstract:Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by Trans-Splicing is a potential therapeutic strategy. Therefore, this study sought to develop Trans-Splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA Trans-Splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and Trans-Splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral Transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended Trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-Splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for Trans-Splicing. Detectable levels of Trans-Splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future Trans-Splicing directed gene therapy.
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440. Optimization of RNA Trans-Splicing for the β-Globin Gene; Detecting Trans-Splicing Events Targeting Endogenous β-Globin Pre-mRNA in Human Erythroid Cells
Molecular Therapy, 2015Co-Authors: Naoya Uchida, Lloyd G Mitchell, Charlotte Platner, Josiah Ballantine, Matthew M. Hsieh, Brian Mozer, Kareem Washington, John F. TisdaleAbstract:Sickle cell disease is caused by a point mutation in exon 1 of the β-globin gene (3 exons and 2 introns). RNA Splicing between two distinct pieces of pre-mRNA, known as Trans-Splicing, represents a potential therapeutic strategy allowing replacement of the mutated exon 1 with a normal exon. Induction of exogenous mRNA Splicing using spliceosome-mediated Trans-Splicing requires an RNA Trans-Splicing molecule (RTM) which imitates endogenous cis-Splicing elements. However, clinical application of Trans-Splicing for globin disorders will require high efficiency, thus we sought to optimize the efficiency of Trans-Splicing targeting the β-globin gene to replace the exon 1 among human erythroid cells.To optimize the binding domain targeting the β-globin gene, the randomized binding domains of 20-600b were generated by sonicating the β-globin gene, and these were inserted into RTM-expressing plasmids downstream of the 5’ half of GFP and a 5’ splice site. In addition, we designed a target plasmid which encodes the β-globin gene and an artificial 3’ splice site connected to 3’ site of the other half of GFP. In Transfection of both RTM and target plasmids, we selected 6 candidates from several thousand RTMs, which produced the brightest GFP-positive cells (maximal Trans-Splicing), and interestingly, all 6 binding domains targeted the β-globin intron 2.To evaluate efficiency of Trans-Splicing for human endogenous β-globin RNA, we constructed lentiviral vectors encoding RTMs which contain the γ-globin cDNA connected to a 5’ splice site and 3 candidates (1E1; 0.6kb, 2D10; 0.7kb, and 13-1; 0.8kb) of β-globin binding domains (selected from the 6 candidates). Human CD34+ cells were Transduced with these RTM-encoding lentiviral vectors, and these cells were differentiated to erythroid cells in vitro. Trans-Splicing was detected by RT-PCR and sequencing in RTM 2D10 and 13-1 but not in RTM 1E1, suggesting that longer binding domains improve Trans-Splicing efficiency. To elongate the binding domain in RTM 13-1, we designed RTM BGin2 (containing 5’ splice site of β-globin intron 2; 0.9kb) and BGex1-in2 (containing whole β-globin sequence except exon 3; 1.3kb). In both RTM BGin2 and BGex1-in2, ~4-fold higher Trans-Splicing was detected by RT-qPCR than RTM 13-1, which resulted in ~0.07% Trans-Splicing efficiency as compared to endogenous human β-globin RNA, implying that the 5’ splice site of intron 2 should be included as a binding domain for efficient Trans-Splicing.In summary, we developed a screening system to select more efficient binding domains for Trans-Splicing. For the first time, we obtained detectable levels of spliceosome-mediated Trans-Splicing (~0.07%) for endogenous β-globin RNA in human erythroid cells which were Transduced with RTM-expressing lentiviral vectors. Further optimization is required to develop Trans-Splicing based gene therapy.
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5 Trans Splicing repair of the plec1 gene
Journal of Investigative Dermatology, 2008Co-Authors: Verena Wally, Ulrich Koller, Lloyd G Mitchell, Alfred Klausegger, Helmut Hintner, Hanns Lochmuller, Sabine Krause, Gerhard Wiche, Johann W BauerAbstract:The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA Trans-Splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient Transfection of EBS-MD fibroblasts with a 5′ pre-Trans-Splicing molecule encoding wild-type exons 2–9 led to specific replacement of the mutated 5′ portion of the endogenous PLEC1 Transcript through Trans-Splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were Transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.
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712. High Capacity Screen To Select Optimal Pre-Trans-Splicing Molecules for Trans-Splicing Applications|[ast]|
Molecular Therapy, 2004Co-Authors: Madaiah Puttaraju, Lloyd G Mitchell, Yanping Yang, Jonathan E. Spindler, Jun Wang, Colette A. Cote, Garry P. Nolan, S. Gary Mansfield, Edward OttoAbstract:Spliceosome Mediated RNA Trans-Splicing (SMaRT™) is a platform technology to reprogram genes at the pre-mRNA level. Applications include RNA therapy and real-time molecular imaging, with each application dependent upon the nature of the sequences encoded by the pre-Trans-Splicing molecule (PTM). SMaRT utilizes native spliceosomes to carry out Trans-Splicing reactions between two RNA molecules: a pre-mRNA target present in the cell and a delivered PTM.
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messenger rna repair and restoration of protein function by spliceosome mediated rna Trans Splicing
Molecular Therapy, 2001Co-Authors: Madaiah Puttaraju, Lloyd G Mitchell, Janet Dipasquale, Carl C Baker, Mariano A GarciablancoAbstract:The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA Trans-Splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective Transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-Trans-Splicing RNA molecules efficiently and specifically repaired mutated lacZ Transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA Trans-Splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-Trans-Splicing molecules.
Eva M Murauer - One of the best experts on this subject based on the ideXlab platform.
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RNA Trans-Splicing Modulation via Antisense Molecule Interference.
International journal of molecular sciences, 2018Co-Authors: Bernadette Liemberger, Eva M Murauer, Verena Wally, Johann W Bauer, Josefina Piñón Hofbauer, Claudia Arzt, S. Hainzl, Thomas Kocher, Julia Reichelt, Ulrich KollerAbstract:In recent years, RNA Trans-Splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher Trans-Splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA Trans-Splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the Trans-Splicing efficiency. Thus, we assume that besides the RNA Trans-Splicing molecule, short antisense molecules can act as Splicing modulators, thereby increasing the Trans-Splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.
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Trans-Splicing Improvement by the Combined Application of Antisense Strategies
International Journal of Molecular Sciences, 2015Co-Authors: Ulrich Koller, Verena Wally, Johann W Bauer, S. Hainzl, Thomas Kocher, Elisabeth Mayr, Clemens Hüttner, Alfred Klausegger, Christina Gruber, Eva M MurauerAbstract:Spliceosome-mediated RNA Trans-Splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA Trans-Splicing molecules (RTMs) are designed to induce a specific Trans-Splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM Trans-Splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the Trans-Splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-Transfections of the RTM and a selected AS RNA, interfering with competitive Splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA Trans-Splicing efficiency. Thereby, accurate Trans-Splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high Trans-Splicing efficiency is required.
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the design and optimization of rna Trans Splicing molecules for skin cancer therapy
Molecular Oncology, 2013Co-Authors: Christina Gruber, Eva M Murauer, Ulrich Koller, S. Hainzl, Thomas Kocher, Clemens Hüttner, Andrew P. South, Helmut Hintner, Johann W BauerAbstract:Targeting tumor marker genes by RNA Trans-Splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA Trans-Splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific Trans-Splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their Trans-Splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a Trans-Splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.
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The design and optimization of RNA Trans‐Splicing molecules for skin cancer therapy
Molecular oncology, 2013Co-Authors: Christina Gruber, Eva M Murauer, Ulrich Koller, S. Hainzl, Thomas Kocher, Clemens Hüttner, Andrew P. South, Helmut Hintner, Johann W BauerAbstract:Targeting tumor marker genes by RNA Trans-Splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA Trans-Splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific Trans-Splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their Trans-Splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a Trans-Splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.
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RNA Trans -Splicing for Genodermatoses
Methods of Molecular Biology, 2012Co-Authors: Johann W Bauer, Eva M Murauer, Verena Wally, Ulrich KollerAbstract:Spliceosome-mediated RNA Trans-Splicing (SMaRT) is a tool that facilitates the recombination of two distinct pre-mRNA molecules. Its application for gene therapeutic purposes has been hindered by laborious procedures to identify gene-specific molecules. We have established a screening method for the identification of highly functional RNA Trans-Splicing molecules based on fluorescence reporters, facilitating the generation of most potent therapeutic molecules for the correction of any gene of interest.
