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  • Gene silencing and antitumoral effects of Eg5 or Ran siRNA oligoaminoamide polyplexes.

Gene silencing and antitumoral effects of Eg5 or Ran siRNA oligoaminoamide polyplexes.

Drug delivery and translational research (2015-03-20)
Daniel Edinger, Raphaela Kläger, Christina Troiber, Christian Dohmen, Ernst Wagner
ABSTRACT

Two antitumoral siRNAs (directed against target genes Eg5 and Ran) complexed with one of three sequence-defined cationic oligomers were compared in gene silencing in vitro and antitumoral in vivo efficacy upon intratumoral injection. Two lipo-oligomers (T-shape 49, i-shape 229) and the three-arm oligomer 386 were chosen because of their high efficiency in previous marker gene silencing screens. The oligomers showed very similar target-specific gene knockdown in murine neuroblastoma cells. Silencing persisted only for a short period (maximum on day 1 at mRNA and day 2 at protein level) triggering siRNA specific in vitro tumor cell killing. The fastest onset of protein knockdown and strongest antitumoral effect was mediated by oligomer 386. Tumor growth reduction in vivo was evaluated in the subcutaneous Neuro2A mouse model. Intratumoral injections of either Eg5 or Ran siRNA/oligomer 49 polyplexes led to reduced tumor growth and prolonged survival of mice compared to control siRNA and buffer treatment. Target knockdown was evidenced in tumors by mitotic Aster formation for Eg5 knockdown and apoptotic TUNEL stain for Ran knockdown. Ran siRNA displayed better antitumoral efficacy and was chosen for in vivo comparison of the oligomers. A very clear order of antitumoral activity (oligomer 386 > 49 > 229) was observed. In summary, the similar in vitro gene silencing efficiencies on mRNA level by the tested oligomers did not correlate with the observed therapeutic effects in vivo. Oligomer 386 with the fastest onset of protein knockdown and best in vitro cell killing mediated the best in vivo antitumoral efficacy.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
MISSION® esiRNA, targeting human KIF11
Sigma-Aldrich
Propidium iodide solution
Sigma-Aldrich
MISSION® esiRNA, targeting mouse Kif11
Sigma-Aldrich
Tris(tert-butoxy)silanol, 99.999%
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Citrate Concentrated Solution, BioReagent, suitable for coagulation assays, 4 % (w/v)
Sigma-Aldrich
Citrate Concentrated Solution, BioUltra, for molecular biology, 1 M in H2O