title: In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.
abstract: We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.
authors.affiliation: Department of Anesthesiology, University of California San Diego, San Diego, California, United States of America. q1xu@ucsd.edu
authors.forename: Qinghao
authors.initials: Q
authors.lastname: Xu
authors.forename: Beverly
authors.initials: B
authors.lastname: Chou
authors.forename: Bethany
authors.initials: B
authors.lastname: Fitzsimmons
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyanohara
authors.forename: Veronica
authors.initials: V
authors.lastname: Shubayev
authors.forename: Camila
authors.initials: C
authors.lastname: Santucci
authors.forename: Michael
authors.initials: M
authors.lastname: Hefferan
authors.forename: Martin
authors.initials: M
authors.lastname: Marsala
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Hua
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism* / Q000821:virology*; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000633:toxicity; D007278:Injections, Spinal; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D059225:Nociception; D034741:RNA, Small Interfering / Q000235:genetics / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D058570:TOR Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases