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

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. METHODS: Rats 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). RESULTS: We 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. CONCLUSION: Peripheral administration of the vector-encoded NR1 shRNAmir is a promising therapy for persistent inflammatory pain.