Intrathecal shRNA-AAV9 Inhibits Target Protein Expression in the Spinal Cord and Dorsal Root Ganglia of Adult Mice Takashi Hirai, 1 Mitsuhiro Enomoto, 1 Akira Machida, 2 Mariko Yamamoto, 2 Hiroya Kuwahara, 2 Mio Tajiri, 2 Yukihiko Hirai, 3 Shinichi Sotome, 1 Hidehiro Mizusawa, 2 Kenichi Shinomiya, 1 Atsushi Okawa, 1 and Takanori Yokota 2 Abstract Gene therapy for neurological diseases requires efficient gene delivery to target tissues in the central and pe- ripheral nervous systems. Although adeno-associated virus is one of the most promising vectors for clinical use against neurological diseases, it is difficult to get it across the blood–brain barrier. A clinically practical approach to using a vector based on adeno-associated virus to decrease the expression of a specific gene in both the central and the peripheral nervous system has yet to be established. Here, we analyzed whether upper lumbar intrathecal administration of a therapeutic vector incorporating adeno-associated virus and short-hairpin RNA against su- peroxide dismutase-1 bypassed the blood–brain barrier to target the spinal cord and dorsal root ganglia. The therapeutic vector effectively suppressed mRNA and protein expression of endogenous superoxide dismutase-1 in the lumbar spinal cord and dorsal root ganglia. Moreover, neither neurological side effects nor toxicity due to the incorporated short-hairpin RNA occurred after the injection. We propose that this approach could be developed into novel therapies for motor neuron diseases and chronic pain conditions, such as complex regional pain syndrome, through silencing of the genes responsible for pathologies in the spinal cord and dorsal root ganglia. Introduction T he spinal cord is an important organ for sensory and motor signal processing and is an important anatomical target for neurological disorders, including inflammatory and demyelinating diseases, neurodegenerative diseases, traumatic injury, and neuropathic pain. The delivery of drugs to the spinal cord via systemic administration, such as oral ingestion, intravenous injection, and dermal application, encounters several challenges. Various types of gene therapy vectors have been developed for targeting the central nervous system (CNS). Intravenous injection of a vector based on adeno-asso- ciated virus (AAV) can deliver target genes to multiple organs, including the liver and skeletal and cardiac muscle (Mitchell et al., 2000; Gregorevic et al., 2004; Bish et al., 2008). However, delivery of systemically administered AAV to the CNS via the blood–brain barrier has not yet been established, and to avoid systemic side effects, selective administration to the brain or spinal cord is required. Gene therapy trials for Parkinson’s disease (Marks et al., 2008), Canavan (Janson et al., 2002), and Batten disease (Worgall et al., 2008) have successfully involved direct brain injection of AAV vectors, but such an invasive method is limited in its application in common clinical practice. RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes through the posttran- scriptional silencing of gene expression (Fire et al., 1998; Dorn et al., 2004). The RNAi pathway is initiated by the enzyme Dicer, which cleaves long, double-stranded RNAs into short (21- to 23-nucleotide) interfering RNA molecules (siRNAs) that mediate sequence-specific gene silencing (Mikami and Yang, 2005; Li et al., 2008). Intraventricular (Senn et al., 2005; Senechal et al., 2007) and intrathecal administration (Luo et al., 2005) of naked or lipid-encapsulated siRNA (Uno et al., 2011) has been used to target the CNS. However, because they still show low transduction efficiencies, insufficient in- hibition of gene expression, and short duration of therapeutic effects, these methods are unsuitable for treating chronic neurological disorders (Hassani et al., 2005). To address these 1 Department of Orthopedic Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo 113–8519, Japan. 2 Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Tokyo 113–8519, Japan. 3 Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo 113–0022, Japan. HUMAN GENE THERAPY METHODS 23:119–127 (April 2012) ª Mary Ann Liebert, Inc. DOI: 10.1089/hgtb.2012.035 119