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Identification of potentially effective antisense oligodeoxyribonucleotide (ODN) sequences for inhibiting plasminogen
Julia Humphries1, Kevin G Burnand, Philip Cunningham
1Academic Dept of Surgery, GKT Medical School, Kings College, St Thomas' Hospital, London, UK.
Objective:
Monocyte fibrinolytic activity may influence thrombus resolution. The balance between uPA and PAI-2 could determine the fibrinolytic activity of the monocyte. Inhibiting PAI-2 production using specific antisense sequences might alter this balance. Selecting effective sequences is a problem as prediction of the secondary structure of target mRNA is difficult. This study reports the modification of a cell free system for rapid antisense screening.
Methods:
Five 18-19 mer oligodeoxynucleotides (ODN), sequences A, B, K, T and Q, and their matched scrambled controls were designed and screened using a modified rabbit reticulocyte lysate transcription and translation system (RRL). Intracellular uptake of ODNs was confirmed by fluorescence microscopy, scanning laser confocal microscopy and fluorimetry. Monocytes were transfected with a liposome/ODN complex using sequences A, B, A + B combined, or T and PAI-2 levels measured by ELISA. Inhibition of PAI-2 production was calculated as a percentage of control levels (baseline and scrambled).
Results:
(i) RRL System--Sequence A was the most effective inhibitor of PAI-2 production in this system (median 63%) compared with sequences, B median 9%, K median 14%, T median 11% and Q median -8% respectively (n = 3). Sequence A was the only sequence, which always inhibited PAI-2. This was confirmed using fluorescently labelled protein (n = 2). (ii) Monocyte transfection--Fluorescence microscopy and fluorimetry showed that intracellular delivery of labelled antisense was only achieved when a liposome was used. Transfection of monocytes extracted from 5 subjects showed that sequence A significantly reduced PAI-2 production (mean % 41.4, sem 9.1) compared with sequences B (mean% 3.4, sem 8.9, p = 0.04), A + B (mean % 0.4, sem 7.8, p = 0.04), and T (mean % 5.4, sem 4.9, p = 0.01). Further studies using sequence A on cells from 10 subjects showed a significant reduction in monocyte PAI-2 production (27.6 ng/ml, sem 3.9) compared with matched scrambled controls (mean 38.3 ng/ml, sem 4.5, p = 0.0112) and baseline (mean 51.4 ng/ml, sem 6.7, p = 0.0009).
Conclusion:
Use of the RRL screening system allowed the selection of a novel antisense sequence, which significantly reduced PAI-2 production in monocytes.
Insights
A novel cell-free system rapidly screened antisense sequences to inhibit plasminogen activator inhibitor-2 (PAI-2) production in monocytes. Sequence A effectively reduced PAI-2 levels, offering a new therapeutic strategy for thrombus resolution.
Area of Science:
- Molecular Biology
- Cell Biology
- Thrombosis Research
Background:
- Monocyte fibrinolytic activity is crucial for thrombus resolution.
- The balance between urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2) dictates monocyte fibrinolytic capacity.
- Developing effective antisense sequences to inhibit PAI-2 production is challenging due to difficulties in predicting mRNA secondary structure.
Purpose of the Study:
- To develop and validate a modified cell-free system for rapid screening of antisense oligodeoxynucleotides (ODNs).
- To identify potent antisense sequences capable of inhibiting PAI-2 production in human monocytes.
- To assess the therapeutic potential of antisense-mediated PAI-2 inhibition in the context of thrombus resolution.
Main Methods:
- Five antisense ODNs (sequences A, B, K, T, Q) and their scrambled controls were screened using a modified rabbit reticulocyte lysate (RRL) transcription/translation system.
- Intracellular ODN uptake in monocytes was confirmed using fluorescence microscopy, scanning laser confocal microscopy, and fluorimetry.
- Monocytes were transfected with liposome/ODN complexes, and PAI-2 levels were quantified by ELISA to determine inhibition percentages.
Main Results:
- The RRL system identified Sequence A as the most effective PAI-2 inhibitor (median 63% inhibition).
- Liposome-mediated delivery was essential for efficient intracellular ODN uptake in monocytes.
- Sequence A significantly reduced PAI-2 production in monocytes (mean 41.4% inhibition) compared to other sequences and controls (p < 0.05).
Conclusions:
- A modified RRL cell-free system enables rapid and effective screening of antisense sequences.
- A novel antisense sequence (Sequence A) was identified that significantly inhibits PAI-2 production in human monocytes.
- This study provides a promising strategy for modulating monocyte fibrinolytic activity and potentially enhancing thrombus resolution.
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