Intranasal delivery of an antisense oligonucleotide to the RNA-binding protein HuR relieves nerve injury-induced neuropathic pain

Abstract

Neuropathic pain remains an undertreated condition and there is a medical need to develop effective treatments. Accumulating evidence indicates that posttranscriptional regulation of gene expression is involved in neuropathic pain; however, RNA processing is not clearly investigated. Our study investigated the role of HuR, an RNA binding protein, in promoting neuropathic pain and trauma-induced microglia activation in the spared nerve injury mouse model. To this aim, an antisense oligonucleotide (ASO) knockdown of HuR gene expression was used. Antisense oligonucleotides poorly cross the blood–brain barrier and an intranasal (i.n.) administration was used to achieve central nervous system penetration through a noninvasive delivery. The efficacy of i.n. ASO administration was compared to an intrathecal (i.t.) delivery. I.n. administered ASO reduced spinal HuR protein and relieved pain hypersensitivity with a similar efficacy to i.t. administration. Immunofluorescence studies showed that HuR was expressed in activated microglia, colocalized with p38 and, partially, with extracellular signal-regulated kinase (ERK)1/2 within the spinal cord dorsal horn. An anti-HuR ASO inhibited the activation of spinal microglia by reducing the levels of proinflammatory cytokines, inducible nitric oxide synthase, the activation of nuclear factor-κB (NF-κB), and suppressed the spared nerve injury–induced overphosphorylation of spinal p38, ERK1/2 and c-Jun-N-terminal kinase (JNK)-1. In addition, HuR silencing increased the expression of the anti-inflammatory cytokine IL-10, promoting the shift of microglial M1 to M2 phenotype. Targeting HuR by i.n. anti-HuR ASO might represent a noninvasive promising perspective for neuropathic pain management by its powerful inhibition of microglia-mediated spinal neuroinflammation and promotion of an anti-inflammatory and neuroprotectant response.

Targeting the RNA-Binding Protein HuR as Potential Thera-Peutic Approach for Neurological Disorders: Focus on Amyo-Trophic Lateral Sclerosis (ALS), Spinal Muscle Atrophy (SMA) and Multiple Sclerosis

“…Another important effect was that anti-HuR ASO administration reduced the mechanical allodynia produced by the EAE model. This effect was also observed in a peripheral model of neuropathic pain, in which HuR colocalized with microglia cells and induced a shift from a pro-inflammatory to an anti-inflammatory state [ 91 ]. HuR is ubiquitously expressed in PBMC, and its activity has been observed in Th17 cells, which are deeply involved in the encephatoligenic process promoted by MS in the CNS [ 92 ].…”

Translational Approaches for Brain Delivery of Biologics via Cerebrospinal Fluid

“…In a more recent example, a study compared IT‐ICV and IN delivery of anti‐HuR ASO in mice. 48 The effect was comparable between the two routes with similar extents of spinal HuR protein reduction and inhibition of microglial‐mediated spinal neuroinflammation suggesting the potential for effective access of nucleotide therapeutics by the IN route.…”

“…Successful delivery of siRNA in different cell types of the prefrontal cortex (cytoplasm of neurons and glial cells) was noted along with functional benefit post repeat dosing with minimal off‐target effects in the lung. In a more recent example, a study compared IT‐ICV and IN delivery of anti‐HuR ASO in mice 48 . The effect was comparable between the two routes with similar extents of spinal HuR protein reduction and inhibition of microglial‐mediated spinal neuroinflammation suggesting the potential for effective access of nucleotide therapeutics by the IN route.…”

“…Although the primary route of delivery for ASOs used clinically has been to the CSF given IT‐L, recently, there have been a few noteworthy preclinical examples of successful delivery of nucleotide therapeutics using delivery systems, such as nanoplexes and targeted conjugates to the brain via IN administration 26,34,46–48 . In one case, an anti‐SNCA ASO was selectively targeted to monoamine neurons in mice using an indatraline‐ASO conjugate 47 .…”

“…Although the primary route of delivery for ASOs used clinically has been to the CSF given IT‐L, recently, there have been a few noteworthy preclinical examples of successful delivery of nucleotide therapeutics using delivery systems, such as nanoplexes and targeted conjugates to the brain via IN administration. 26 , 34 , 46 , 47 , 48 In one case, an anti‐SNCA ASO was selectively targeted to monoamine neurons in mice using an indatraline‐ASO conjugate. 47 Confocal fluorescence microscopy demonstrated that functional monoamine transporters served to enable selective oligonucleotide uptake and internalization in monoamine neurons along with target mRNA and protein knockdown.…”