In vitro test system for evaluation of immune activation potential of new single‐stranded DNA‐based therapeutics. Issue 4 (12th May 2014)

• Record Type: Journal Article
• Authors: Avci‐Adali, Meltem; Hann, Ludmilla; Michel, Tatjana; Steinle, Heidrun; Stoppelkamp, Sandra; Stang, Katharina; Narita, Miwako; Schlensak, Christian; Wendel, Hans P.
• Is Part Of: Drug testing and analysis. Volume 7:Issue 4(2015:Apr.)
• Page Start: 300
• Page End: 308
• Publication Date: 2014-05-12
• Subjects: Drugs -- Analysis -- Periodicals; Drug testing -- Periodicals; Chemistry, Forensic -- Periodicals; 615.1901
• DOI: 10.1002/dta.1670
• Languages: English
• ISSNs: 1942-7603

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>in vivo</italic>, 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 <italic>in vivo</italic> 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 <italic>in vivo</italic> studies will facilitate the <italic>in vitro</italic> prediction of immune activation potential of aptamers. Copyright © 2014 John Wiley & Sons, Ltd.</p> </abstract>