An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons.
We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect (vehicle), and delivered as repeated daily bolus doses (0.5 microg to 4 microg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala(2), Glu(4)]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.
Luo
Miaw-Chyi
MC
Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA. mluo@u.arizona.edu
Zhang
Dong-Qin
DQ
Ma
Shou-Wu
SW
Huang
Yuan-Yuan
YY
Shuster
Sam J
SJ
Porreca
Frank
F
Lai
Josephine
J
D000818:Animals; D002460:Cell Line; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D007278:Injections, Spinal; D008159:Lumbar Vertebrae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009271:Naltrexone / Q000031:analogs & derivatives / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009619:Nociceptors / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010147:Pain Measurement; D017933:Peripheral Nervous System / Q000166:cytology* / Q000378:metabolism*; D020671:Posterior Horn Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000378:metabolism; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism*
C481052:Alexa fluor 546; D009842:Oligopeptides; D011806:Quinolinium Compounds; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; C060209:deltorphin II, Ala(2)-; D009271:Naltrexone; C055382:naltrindole