Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release.

Wanzhi Wang,Jianghui Meng,Jiafu Wang,Yanqing Li,Yinghao Zhang,Yang Liu,Shanghai Xue,Weiwei Chen,Miaomiao Kong,Yu Dou,Xiaolong Dai

doi:10.3390/ijms22168826

Abstract

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.

Highlights

It is estimated that ~1.5 billion people worldwide suffer from chronic pain [1], which causes a major healthcare challenge and huge societal burden
Western blotting results indicated human Pirt promoter was able to drive the expression of light-chain protease of botulinum neurotoxin A (LCA) gene as revealed by the cleavage of synaptosomal associated protein 25-kDa (SNAP-25) in rDRGs (Figure 3C) and rat TGNs (rTGNs) (Figure 3D)
We have demonstrated that LentiPirtLCA can dose-dependently cleave SNAP-25 in infected murine dorsal root ganglionic neurons (mDRGs) (Figure 2B,D)

Summary

Introduction

It is estimated that ~1.5 billion people worldwide suffer from chronic pain [1], which causes a major healthcare challenge and huge societal burden. We are in urgent need of new therapies for long-term pain management and gene transfer of BoNT LC protease into peripheral sensory neurons provides a novel means to alleviate chronic pain. To avoid the unwanted transfer of the LC of BoNT/A (LCA) into motor neurons to prevent muscle paralysis side effect, it is ideally to use a sensory neuronal promoter to drive the transgene expression. Pirt gene promoter fulfils the criteria for selective expression of analgesic transgene in the sensory neurons for management peripheral derived pain. Viral-mediated LCA expression lowered the mRNA levels of certain neuropeptide, receptor and transducer genes in proinflammatory cytokines stimulated sensory neurons. LentiPirtLCA-induced persistent cleavage of SNAP-25 and inhibition of neuropeptide release in the infected sensory neurons with potential to yield long-lasting relief of chronic pain in patients

Results

Discussion

Animals

Design and Production of Lentiviral Vectors

Detection of Lentiviral Mediated GFP Expression and SNAP-25 Cleavage

Immunofluorescence Staining

Statistical Analysis