Abstract
The application of short-interfering RNA (siRNA) to induce RNA interference (RNAi) has emerged as a powerful genetic tool to silence gene expression and provides intriguing possibilities for therapeutic strategies to enhance axonal regeneration in the CNS. We have demonstrated that transfection of dorsal root ganglion (DRG) neurons with siRNA targeted against RhoA GTPase significantly overcame CNS myelin-mediated inhibition of neurite outgrowth in vitro1. However, before nucleic acid medicines based on RNAi are developed further careful design of siRNA sequences are needed to ensure that drugs based on this technology are safe. In particular, a greater understanding of the global biological impact of siRNA on neuronal homeostasis is mandatory.
The aim of this study was to analyse the biological impact of anti-RhoA siRNA on DRG cultures prepared from adult Sprague-Dawley rats. Spotted rat microarrays containing 27,648 oligonucleotides showed that following transfection with anti-RhoA siRNA, the expression of 177 genes was altered by ≥ 2-fold (p0.05). Expression of 44 genes was down-regulated, including transcripts encoding for RhoA and related cytoskeletal rearrangement and cell motility proteins, such as transgelin. In contrast, many up-regulated transcripts encoded proteins involved in a cytokine response to anti-RhoA siRNA, including interferon beta, GTP-binding protein MX1 and 2’-5’-oligoadenylate synthetase 1. Evaluation of different strategies to ablate stress responses with anti-RhoA siRNA demonstrated that (i) interferon responses varied significantly when different anti-RhoA siRNA sequences obtained from the literature were evaluated, and (ii) StealthTM modification of anti-RhoA siRNA totally ablated the interferon response in DRG cultures.
These results demonstrate that microarray profiling can identify both predicted and novel molecular interactions with RhoA following mRNA knockdown, hence, defining the biological impact of siRNA. We predict that studies such as these will facilitate the in vivo development of effective nucleic acid medicines for axon regeneration based on knockdown of mRNA.
1Ahmed, Z., et al. (2006). Brain: 129:1517-33.
The aim of this study was to analyse the biological impact of anti-RhoA siRNA on DRG cultures prepared from adult Sprague-Dawley rats. Spotted rat microarrays containing 27,648 oligonucleotides showed that following transfection with anti-RhoA siRNA, the expression of 177 genes was altered by ≥ 2-fold (p0.05). Expression of 44 genes was down-regulated, including transcripts encoding for RhoA and related cytoskeletal rearrangement and cell motility proteins, such as transgelin. In contrast, many up-regulated transcripts encoded proteins involved in a cytokine response to anti-RhoA siRNA, including interferon beta, GTP-binding protein MX1 and 2’-5’-oligoadenylate synthetase 1. Evaluation of different strategies to ablate stress responses with anti-RhoA siRNA demonstrated that (i) interferon responses varied significantly when different anti-RhoA siRNA sequences obtained from the literature were evaluated, and (ii) StealthTM modification of anti-RhoA siRNA totally ablated the interferon response in DRG cultures.
These results demonstrate that microarray profiling can identify both predicted and novel molecular interactions with RhoA following mRNA knockdown, hence, defining the biological impact of siRNA. We predict that studies such as these will facilitate the in vivo development of effective nucleic acid medicines for axon regeneration based on knockdown of mRNA.
1Ahmed, Z., et al. (2006). Brain: 129:1517-33.
| Original language | English |
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| Pages | 29 |
| Number of pages | 1 |
| Publication status | Published - 1 Jan 2006 |
| Event | European Society of Gene Therapy Annual Meeting - Duration: 1 Jan 2006 → … |
Conference
| Conference | European Society of Gene Therapy Annual Meeting |
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| Period | 1/01/06 → … |