title: Identification of potentially effective antisense oligodeoxyribonucleotide (ODN) sequences for inhibiting plasminogen activator inhibitor-2 (PAI-2) production by monocytes.
abstract: 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).
  
  
  CONCLUSIONS
  Use of the RRL screening system allowed the selection of a novel antisense sequence, which significantly reduced PAI-2 production in monocytes.
authors.affiliation: Academic Dept of Surgery, GKT Medical School, Kings College, St Thomas' Hospital, London, UK.
authors.forename: Julia
authors.initials: J
authors.lastname: Humphries
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Burnand
authors.forename: Philip
authors.initials: P
authors.lastname: Cunningham
authors.forename: Adrian
authors.initials: A
authors.lastname: Brock
authors.forename: Nigel
authors.initials: N
authors.lastname: Westwood
authors.forename: Alberto
authors.initials: A
authors.lastname: Smith
mesh_terms: D002474:Cell-Free System; D004353:Drug Evaluation, Preclinical; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology*; D017396:Plasminogen Activator Inhibitor 2 / Q000096:biosynthesis / Q000235:genetics*; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
chemical_list: D020319:Oligodeoxyribonucleotides, Antisense; D017396:Plasminogen Activator Inhibitor 2; D015842:Serine Proteinase Inhibitors