Lentiviral vector-encoded microRNA-based shRNA-mediated gene knockdown of N-methyl-D-aspartate receptors in skin reduces pain.

Chien‐Cheng Liu,Kuo‐Chuan Hung,Ping‐Heng Tan,Jiin‐Tsuey Cheng,Yuan‐Yi Chia

doi:10.1002/brb3.587

Abstract

Background and Purpose RNA polymerase II promoters that drive the expression of rationally designed primary microRNA‐based shRNA, for example, shRNAmir, can produce more potent gene knockdown than RNA polymerase III promoters. Antagonists of peripheral N methyl‐D‐aspartate (NMDA) receptors that do not interfere with central glutamate processing would prevent the development of adverse central nervous system effects. Thus, in this study, we examined the effects of gene silencing and antinociception on formalin‐ and Complete Freund's adjuvant (CFA)‐induced pain in rats by subcutaneously injecting a lentiviral vector encoding a shRNAmir that targets the NR1 subunit of the NMDA receptor.MethodsRats received intradermal injections of different doses of NR1 shRNAmir at different time points before injection of formalin. Pain behavior was assessed by monitoring the paw flinch response, paw withdrawal threshold, and thermal withdrawal latency. We then analyzed NR1 messenger RNA and protein expression in skin and the L5 dorsal root ganglion (DRG).ResultsWe found that intradermal injection of 1, 5, and 10 μg of shRNAmir significantly inhibited flinch responses (p < .05). Administration of 5 μg of shRNAmir resulted in the attenuation of CFA‐induced mechanical allodynia, but did not affect the time spent on the rotarod. Real‐time polymerase chain reaction and western blotting revealed that NR1 mRNA and protein levels were significantly lower in all NR1 shRNAmir1 groups than in controls (p < .05). There was a significant reduction in the percentage of NR1‐ and pERK‐positive neurons in the DRG ipsilateral to shRNAmir treated paws (p < .05). The effect of antinociception and inhibition of NR1 expression by NR1 shRNAmir was evident on day 3 and persisted for 7 days after injection of 5 μg of vector.ConclusionPeripheral administration of the vector‐encoded NR1 shRNAmir is a promising therapy for persistent inflammatory pain.

Highlights

MicroRNAs are transcribed as long primary transcripts by the RNase III enzyme, Drosha, and processed into mature miRNAs in the cytoplasm (Cullen, 2004; Elbashir, Lendeckel, & Tuschl, 2001; Lee et al, 2003)
In our previous study (Tan et al, 2010), we demonstrated that local peripheral injection of NR1 small interfering RNAs (siRNAs) induced effective and specific silencing of the N methyl-D-aspartate (NMDA) receptor and that it significantly attenuated formalin-induced nociceptive behaviors without producing significant side effects
We hypothesized that vector-encoded shRNAmir-mediated gene knockdown of NMDA receptors in subcutaneous tissues may provide a longer period of analgesia

Summary

| INTRODUCTION

MicroRNAs (miRNA) are transcribed as long primary transcripts by the RNase III enzyme, Drosha, and processed into mature miRNAs in the cytoplasm (Cullen, 2004; Elbashir, Lendeckel, & Tuschl, 2001; Lee et al, 2003). Cullen and colleagues produced a template of miRNA-30 precursor RNA, in which miRNA-30 was replaced by a target gene sequence and found that it effectively inhibited the expression. We hypothesized that vector-encoded shRNAmir-mediated gene knockdown of NMDA receptors in subcutaneous tissues may provide a longer period of analgesia To test this hypothesis, we examined the gene silencing and antinociception effects of intradermal injections of a shRNAmir that targets the NR1 subunit of the NMDA receptor in rat models of formalin-and Complete Freund’s adjuvant (CFA)-induced nociception

| METHODS

Findings

| DISCUSSION