title: In vitro test system for evaluation of immune activation potential of new single-stranded DNA-based therapeutics.
abstract: Aptamers are synthetic single-stranded DNA (ssDNA) molecules with the ability to fold into complex three-dimensional structures. They can bind their targets with a high selectivity and affinity, thus they have an enormous potential as therapeutic agents. However, since aptamers are synthetic and especially since certain sequences can increasingly bind to the pattern recognition receptors of the immune cells when applied in vivo, they can induce an immune activation. Here, we established a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) based assay to evaluate aptamers-induced immune activation prior to in vivo studies. Human whole blood or plasmacytoid dendritic cell line (PMDC05) were incubated with CpG, R10-60 aptamer, start library, or a CpG containing aptamer. After 2 and 4 h, cytokine expression was measured using qRT-PCR to determine immune reaction against different aptamers. CpG containing a phosphorothioate backbone led to a significant up-regulation of CCL-7, IFN-1α, IFN-1β in whole blood after 4 h. Compared to the samples without ssDNA, significantly higher TNF-α expression was detected after the R10-60 aptamer incubation for 4 h. The stimulation of PMDC05 cells with different ssDNA enabled more sensitive detection of aptamer sequence specific immune activation. After 4 h, CpG led to a significantly higher expression of CCL-8, CXCL-10, IL-1β, IL-6, IL-8, IFN-1β, and TNF-α. R10-60 aptamer caused a significant up-regulation of IL-1β, IFN-1β, and TNF-α. Negative control aptamers did not induce an immune activation. The use of this assay before starting with in vivo studies will facilitate the in vitro prediction of immune activation potential of aptamers.
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen, Tuebingen, Germany.
authors.forename: Meltem
authors.initials: M
authors.lastname: Avci-Adali
authors.forename: Ludmilla
authors.initials: L
authors.lastname: Hann
authors.forename: Tatjana
authors.initials: T
authors.lastname: Michel
authors.forename: Heidrun
authors.initials: H
authors.lastname: Steinle
authors.forename: Sandra
authors.initials: S
authors.lastname: Stoppelkamp
authors.forename: Katharina
authors.initials: K
authors.lastname: Stang
authors.forename: Miwako
authors.initials: M
authors.lastname: Narita
authors.forename: Christian
authors.initials: C
authors.lastname: Schlensak
authors.forename: Hans P
authors.initials: HP
authors.lastname: Wendel
mesh_terms: D015415:Biomarkers / Q000097:blood; D016207:Cytokines / Q000097:blood / Q000276:immunology; D004277:DNA, Single-Stranded / Q000276:immunology* / Q000627:therapeutic use*; D003713:Dendritic Cells / Q000276:immunology; D006801:Humans; D007109:Immunity / Q000276:immunology*; D066298:In Vitro Techniques; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D016207:Cytokines; D004277:DNA, Single-Stranded
keywords: DNA sensing; aptamers; immune activation