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Trans-splicing improvement by the combined application of antisense strategies.

Koller U, Hainzl S, Kocher T, Hüttner C, Klausegger A, Gruber C, Mayr E, Wally V, Bauer JW, Murauer EM - Int J Mol Sci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. u.koller@salk.at.

ABSTRACT
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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Comparison of AS RNA-mediated influence on trans-splicing efficiency. (A) Triple transfections of COL7A1-MG, RTM_31 and the most promising AS RNA (AS RNA-13) increased the amount of GFP expressing cells upon accurate trans-splicing from 24% to 34%. Mean + SD of five different experiments is shown. An unpaired T-test (two-tailed) was performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA) to prove the statistical significance between AS RNA-13 and the control pcDNA (**p value = 0.0073); (B) Co-transfection of RTM_31 and individual AS RNAs of the library into a COL7A1-MG expressing target cell line leads to a variable trans-splicing rate. AS RNA-13 showed the most promising effect, capable of increasing the trans-splicing efficiency of the RTM 2–3-fold on the protein level (measured by flow cytometry). AS RNA-13 was compared to the control (pcDNA). The mean + SD of 4 different experiments is shown. Unpaired, two-tailed t-test (*p value = 0.0298) confirmed statistical the significant increase in AS RNA-13 compared to the control pcDNA; and (C) Binding position of RTM_31 (red) and individual AS RNAs within the COL7A1 target region.
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ijms-16-01179-f002: Comparison of AS RNA-mediated influence on trans-splicing efficiency. (A) Triple transfections of COL7A1-MG, RTM_31 and the most promising AS RNA (AS RNA-13) increased the amount of GFP expressing cells upon accurate trans-splicing from 24% to 34%. Mean + SD of five different experiments is shown. An unpaired T-test (two-tailed) was performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA) to prove the statistical significance between AS RNA-13 and the control pcDNA (**p value = 0.0073); (B) Co-transfection of RTM_31 and individual AS RNAs of the library into a COL7A1-MG expressing target cell line leads to a variable trans-splicing rate. AS RNA-13 showed the most promising effect, capable of increasing the trans-splicing efficiency of the RTM 2–3-fold on the protein level (measured by flow cytometry). AS RNA-13 was compared to the control (pcDNA). The mean + SD of 4 different experiments is shown. Unpaired, two-tailed t-test (*p value = 0.0298) confirmed statistical the significant increase in AS RNA-13 compared to the control pcDNA; and (C) Binding position of RTM_31 (red) and individual AS RNAs within the COL7A1 target region.

Mentions: Analogue to the BD selection, the fluorescence-based RTM screening procedure was performed in order to evaluate the efficiency of AS RNAs capable of masking competitive cis-splicing elements on the COL7A1-MG. In brief, the PCR amplified intron 102/exon 103 COL7A1 region was fragmented by sonication and cloned into the pcDNA 4.0 vector. A powerful AS RNA will lead to an enhanced trans-splicing efficiency and increased full-length GFP expression in transfected HEK293 cells. In total, 86 different potential AS RNAs from the library were analyzed by sequencing. Eight individual AS RNAs (Figure 2) with antisense sequences for intron 102/exon 103 of COL7A1 were selected for initial experiments. Triple-transfections of COL7A1-MG (0.5 µg), RTM_31 and selected AS RNAs in a relation of 1:6 (0.5 µg RTM: 3 µg AS RNA) into HEK293 cells resulted in a diverse level of GFP expression (the average amount of GFP expressing cells ranged from 18% to 34%) in comparison to cells transfected with RTM_31, COL7A1-MG and empty pcDNA 4.0 expression plasmids (Figure 2A). The most efficient AS RNA (AS RNA-13 with a binding length of 362 nucleotides) increased the level of GFP expressing cells from 24% (control) to over 34%. Endogenous experiments using a COL7A1-MG-expressing HEK293 target cell line confirmed the results of the triple transfection experiments. AS RNA-13 was able to increase the average % of GFP-expressing cells upon RTM_31 and AS RNA treatment from ~11% (pcDNA4.0) to over 26% (Figure 2B).AS RNA-13 probably blocks all potential splicing enhancer and splicing elements downstream of the RTM_31 binding region and is therefore a promising candidate for future experiments. Additionally, RTM_31 and AS RNA-13 have no overlapping binding sites within the target region, so we expect no significant binding competition and steric interferences between both molecules (Figure 2C).


Trans-splicing improvement by the combined application of antisense strategies.

Koller U, Hainzl S, Kocher T, Hüttner C, Klausegger A, Gruber C, Mayr E, Wally V, Bauer JW, Murauer EM - Int J Mol Sci (2015)

Comparison of AS RNA-mediated influence on trans-splicing efficiency. (A) Triple transfections of COL7A1-MG, RTM_31 and the most promising AS RNA (AS RNA-13) increased the amount of GFP expressing cells upon accurate trans-splicing from 24% to 34%. Mean + SD of five different experiments is shown. An unpaired T-test (two-tailed) was performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA) to prove the statistical significance between AS RNA-13 and the control pcDNA (**p value = 0.0073); (B) Co-transfection of RTM_31 and individual AS RNAs of the library into a COL7A1-MG expressing target cell line leads to a variable trans-splicing rate. AS RNA-13 showed the most promising effect, capable of increasing the trans-splicing efficiency of the RTM 2–3-fold on the protein level (measured by flow cytometry). AS RNA-13 was compared to the control (pcDNA). The mean + SD of 4 different experiments is shown. Unpaired, two-tailed t-test (*p value = 0.0298) confirmed statistical the significant increase in AS RNA-13 compared to the control pcDNA; and (C) Binding position of RTM_31 (red) and individual AS RNAs within the COL7A1 target region.
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Related In: Results  -  Collection

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Show All Figures
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ijms-16-01179-f002: Comparison of AS RNA-mediated influence on trans-splicing efficiency. (A) Triple transfections of COL7A1-MG, RTM_31 and the most promising AS RNA (AS RNA-13) increased the amount of GFP expressing cells upon accurate trans-splicing from 24% to 34%. Mean + SD of five different experiments is shown. An unpaired T-test (two-tailed) was performed with GraphPad Prism software (GraphPad Software, San Diego, CA, USA) to prove the statistical significance between AS RNA-13 and the control pcDNA (**p value = 0.0073); (B) Co-transfection of RTM_31 and individual AS RNAs of the library into a COL7A1-MG expressing target cell line leads to a variable trans-splicing rate. AS RNA-13 showed the most promising effect, capable of increasing the trans-splicing efficiency of the RTM 2–3-fold on the protein level (measured by flow cytometry). AS RNA-13 was compared to the control (pcDNA). The mean + SD of 4 different experiments is shown. Unpaired, two-tailed t-test (*p value = 0.0298) confirmed statistical the significant increase in AS RNA-13 compared to the control pcDNA; and (C) Binding position of RTM_31 (red) and individual AS RNAs within the COL7A1 target region.
Mentions: Analogue to the BD selection, the fluorescence-based RTM screening procedure was performed in order to evaluate the efficiency of AS RNAs capable of masking competitive cis-splicing elements on the COL7A1-MG. In brief, the PCR amplified intron 102/exon 103 COL7A1 region was fragmented by sonication and cloned into the pcDNA 4.0 vector. A powerful AS RNA will lead to an enhanced trans-splicing efficiency and increased full-length GFP expression in transfected HEK293 cells. In total, 86 different potential AS RNAs from the library were analyzed by sequencing. Eight individual AS RNAs (Figure 2) with antisense sequences for intron 102/exon 103 of COL7A1 were selected for initial experiments. Triple-transfections of COL7A1-MG (0.5 µg), RTM_31 and selected AS RNAs in a relation of 1:6 (0.5 µg RTM: 3 µg AS RNA) into HEK293 cells resulted in a diverse level of GFP expression (the average amount of GFP expressing cells ranged from 18% to 34%) in comparison to cells transfected with RTM_31, COL7A1-MG and empty pcDNA 4.0 expression plasmids (Figure 2A). The most efficient AS RNA (AS RNA-13 with a binding length of 362 nucleotides) increased the level of GFP expressing cells from 24% (control) to over 34%. Endogenous experiments using a COL7A1-MG-expressing HEK293 target cell line confirmed the results of the triple transfection experiments. AS RNA-13 was able to increase the average % of GFP-expressing cells upon RTM_31 and AS RNA treatment from ~11% (pcDNA4.0) to over 26% (Figure 2B).AS RNA-13 probably blocks all potential splicing enhancer and splicing elements downstream of the RTM_31 binding region and is therefore a promising candidate for future experiments. Additionally, RTM_31 and AS RNA-13 have no overlapping binding sites within the target region, so we expect no significant binding competition and steric interferences between both molecules (Figure 2C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. u.koller@salk.at.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus