ges in DRG neurons can prevent trophic changes and alleviate facilitated pain states. Thus, selective gene knockdown in DRG neurons can be achieved by intrathecal application of antisense oligodeoxynucleotides or siRNAs. Although antisense oligos directed against some pro-nociceptive molecules in DRG displayed analgesia, the SU11274 cost utility of antisense oligos and synthetic siRNA are limited by several factors including toxicity and short-lasting effect. Alternatively, siRNA can be derived from a short hairpin precursor that is expressed from a viral vector. Recently it has been reported that several serotypes of adeno-associated virus are efficient in transducing DRG neurons in rodents. However, the transduction efficiency and tropism in the DRG vary considerably, depending upon the routes of administration, animal species and viral serotypes. AAV5 vectors directly injected into rat DRGs resulted in transduction in up to 90% of the neurons, including most nociceptors. In contrast, following IT injection in mouse, the same serotype targeted large-diameter DRG neurons, while excluding the isolectin-B4 binding, non-peptidergic nociceptors. AAV6 transduces both neurons and satellite cells in rats following direct DRG injection but preferentially transduces neurons following mouse sciatic nerve or IT injection. Understanding the vector tropisms is important for studies aiming to target certain subsets of DRG neurons. The in vivo efficacy of AAV-mediated RNA interference in the nervous system in general has been extensively studied but information regarding the DRG is still limited. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22190001 A recent paper reported that AAV5 encoding a short-hairpin RNA produced a significant loss of neuropilin2 mRNA in the rat DRG following a direct injection. While the result is interesting, neuropilin2 is not expressed in all DRG neuron populations. It is therefore In Vivo DRG Gene Knockdown Mediated by AAV5 difficult to determine the cell types that can be targeted by AAVencoded siRNA. Further, direct injection of DRG requires an invasive surgical procedure that is time-consuming and may itself cause inflammation and pain. Intrathecal administration as a less invasive approach produces satisfactory viral transduction but the capacity of IT vector to deliver siRNA has not been evaluated. In the current study, we aimed to investigate: 1) the transduction efficacy and tropism of an AAV5 vector in rat DRG and spinal cord after an intrathecal delivery, and 2) the efficiency of gene expression knockdown in 
rat DRG by an siRNA when delivered by the AAV5 vector. We choose mTOR as the target gene, for its ubiquitous expression in DRG neurons and for its reported role in peripheral nociception. Results GFP expression in the DRG and spinal cord following intrathecal AAV5 administration To study the transduction efficacy and tropism of AAV5 in rats, we first constructed a vector encoding GFP driven by a cytomegalovirus promoter. Ten microliters of virus was administered in adult rats through an intrathecal catheter, with the tip ending at the spinal level of L3L4. The GFP expression began to be visualized in the DRG and spinal cord at 1 week following the vector administration, however the intensity of GFP immunoreactivity at this time point was very weak, and the distribution pattern was not analyzed. A profound GFP expression was observed after 2 weeks in the lumbar DRG neurons, while in the thoracic and cervical DRGs GFP was not detectable. We have analyzed a total of 3781 neuron
