Profiling RNA interference (RNAi)‐mediated toxicity in neural cultures for effective short interfering RNA design

Abstract

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. 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. 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 beta 2, plod2 and mmp3, with implications for interpretating the ability of RhoA siRNA to promote axon regeneration. 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.