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.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.

N-methyl D-aspartate receptor subtype 2B antagonist, Ro 25-6981, attenuates neuropathic pain by inhibiting postsynaptic density 95 expression

“…It has been shown that intrathecal delivery of an NMDA subtype 2B (NR2B)-mimetic peptide attenuates both neuronal hyperexcitability and abnormal pain-related behaviors by perturbing PSD-95-NR2B interactions 11 , 12 , suggesting that both proteins play an essential role in the generation of central sensitization. On the other hand, central sensitization is considered to contribute to the formation and development of chronic pain states 13 – 15 . However, the role and the underlying mechanism of the relationship between PSD-95 and NR2B in the development or maintenance of neuropathic pain are largely unknown.…”

An Intra-brainstem Circuit for Pain-induced Inhibition of Itch

“…The advent of fluorescence sensors enabling the study of neuropeptides (Wang et al, 2023) in live behaving mice can allow us to determine how and when neuropeptides Substance P (product of tac1 gene) and Grp are released where the Tacr1 and Grpr are expressed in PBN and RVM. AAV-mediated delivery of silencing shRNAs (Liu et al, 2017; Rohn et al, 2024) targeting Tacr1 and Grpr can reveal the potential roles of these GPCRs in pain and itch. Notably, based on the results, the Tacr1 receptor and Tacr1-expressing neurons in the PBN and RVM can be targeted to modulate pain and itch.…”

<p>MicroRNA And Circular RNA Expression In Affected Skin Of Patients With Postherpetic Neuralgia</p>

“…Animal studies suggested that interfering with the miRNA expression in the skin affects the prognosis of diseases. For example, interfering NR1 subunit of the NMDA receptor by intradermally injecting lentiviral vector-encoded miRNA-based shRNA reduced inflammatory pain in rats 38. Intradermally injecting miR-129 and/or miR-335 agomirs into the wound edges accelerated wound healing in rats with diabetes 39.…”