Functional inhibition of chemokine receptor CCR2 by dicer-substrate-siRNA prevents pain development.
BACKGROUND
Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund's adjuvant.


RESULTS
To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2'-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund's adjuvant-induced inflammatory chronic pain model.


CONCLUSIONS
Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain.
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada Philippe.Sarret@USherbrooke.ca.
Valérie
V
Bégin-Lavallée
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Élora
É
Midavaine
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Marc-André
MA
Dansereau
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Pascal
P
Tétreault
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Jean-Michel
JM
Longpré
Integrated DNA Technologies Inc, Coralville, IA, USA.
Ashley M
AM
Jacobi
Integrated DNA Technologies Inc, Coralville, IA, USA.
Scott D
SD
Rose
Integrated DNA Technologies Inc, Coralville, IA, USA.
Mark A
MA
Behlke
Department of Anesthesiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Nicolas
N
Beaudet
Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Philippe
P
Sarret
D000818:Animals; D048430:Cell Shape; D005453:Fluorescence; D005727:Ganglia, Spinal / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D006930:Hyperalgesia / Q000150:complications / Q000378:metabolism; D007249:Inflammation / Q000150:complications / Q000473:pathology; D008297:Male; D009457:Neuroglia / Q000378:metabolism; D010146:Pain / Q000150:complications / Q000378:metabolism* / Q000517:prevention & control*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D054390:Receptors, CCR2 / Q000037:antagonists & inhibitors* / Q000235:genetics; D015203:Reproducibility of Results; D043244:Ribonuclease III / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism; D013379:Substrate Specificity
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D054390:Receptors, CCR2; D043244:Ribonuclease III
CCL2; DsiRNA; MCP-1; Transductin; chemokines; gene silencing; mechanical allodynia