Design and Evaluation of Small Interfering RNAs That Target Expression of the N-Methyl-d-aspartate Receptor NR1 Subunit Gene in the Spinal Cord Dorsal Horn

Abstract

NR1 is an essential subunit of the N-methyl-D-aspartate (NMDA) receptor, which at the spinal level is involved in injury-induced pain hypersensitivity and morphine tolerance. An in vitro luciferase assay was used to identify candidate and control (inactive) short interfering RNA (siRNA) sequences that are expressed by a recombinant adeno-associated virus (rAAV) plasmid. rAAV vectors targeting the NR1 subunit were prepared that express active or control (mismatch) siRNA sequences and injected into the mouse spinal cord dorsal horn (SCDH). Three weeks after vector administration, green fluorescent protein labeling of the ipsilateral SCDH confirmed the spatial localization of the viral transduction. Active siRNAs resulted in a 60 to 75% knockdown of NR1 mRNA and protein in the area of the virus injection. The spatial knockdown persisted for at least 6 months after a single administration of the vector. Neither the active nor the mismatch siRNAs resulted in cellular toxicity as measured by nuclear staining and cell integrity. The vector-derived knockdown of NR1 expression in SCDH did not alter acute thermal or mechanical stimulus paw-withdrawal thresholds. However, the vector-derived siRNA prevented the mechanical allodynia measured at 24 and 48 h after injection into the paw of the inflammatory agent, Complete Freund's adjuvant. These results demonstrate that vector-derived siRNAs can be used to produce an in vivo spatial knockdown of the expression and function of the NMDA receptor that is confined to the ipsilateral SCDH. Vector-derived siRNAs may have therapeutic potential for the management of injury-induced pain resulting from the activation of NMDA receptors in the SCDH.