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Profiling RNA interference (RNAi)‐mediated toxicity in neural cultures for effective short interfering RNA design
Author(s) -
Read Martin L.,
Mir Sohaib,
Spice Rachel,
Seabright Ruth J.,
Suggate Ellen L.,
Ahmed Zubair,
Berry Martin,
Logan Ann
Publication year - 2009
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1321
Subject(s) - rhoa , rna interference , small interfering rna , gene silencing , biology , microbiology and biotechnology , innate immune system , rna , microarray analysis techniques , gene expression , signal transduction , immune system , immunology , gene , genetics
Background A promising strategy to enhance axon regeneration is to employ short interfering (si)RNA targeting either RhoA or p75 NTR , which are components of a signalling cascade triggered by growth inhibitory ligands. However, it is important to profile the biological impact of siRNA on cell homeostasis in order to develop safe and effective therapies. Methods We used microarray and quantitative reverse transcriptase‐polymerase chain reaction techniques to analyse the transcriptional effects of siRNA against p75 NTR and RhoA in neuronal cell line and primary cultures. Results Expression analysis showed that primary rat dorsal root ganglion cells were up to 279‐fold more sensitive than nerve growth factor‐differentiated PC12 cells in detecting innate immune responses to siRNA. The sequence and method of synthesis of siRNA critically influenced the magnitude of the innate immune response. Importantly, siRNA sequences were identified that efficiently silenced RhoA and p75 NTR mRNA with attenuated induction of the interferon‐responsive gene mx1 . Moreover, microarray analysis identified genes related to RhoA function, such as tgf β 2, plod2 and mmp3, with implications for interpretating the ability of RhoA siRNA to promote axon regeneration. Conclusions These findings demonstrate the importance of screening the biological impact of different siRNA sequences not only for their silencing efficacy, but also for potential toxicity. The results of the present study suggest that the toxicity observed was sequence‐dependent because only two out of five siRNA sequences targeting RhoA were identified that did not induce a significant innate immune response. Copyright © 2009 John Wiley & Sons, Ltd.