Enhanced Adenoviral Gene Delivery to Motor and Dorsal Root Ganglion Neurons Following Injection Into Demyelinated Peripheral Nerves Yongjie Zhang, 1 Yiyan Zheng, 1 Yi Ping Zhang, 2 Lisa B.E. Shields, 2 Xiaoling Hu, 1 Panpan Yu, 1 Darlene A. Burke, 1 Heming Wang, 3 Cai Jun, 4 Jonathan Byers, 1 Scott R. Whittemore, 1 and Christopher B. Shields 2 * 1 Kentucky Spinal Cord Injury Research Center, Louisville, Kentucky 2 Norton Neuroscience Institute, Norton Healthcare, Louisville, Kentucky 3 Department of Human Anatomy, Nanjing Medical University, Jiangsu Province, China 4 Anatomical Science and Neurobiology, University of Louisville, Louisville, Kentucky Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral par- ticles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% ly- solecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelin- ation, 0.5 ll of Ad5-GFP was injected into both sciatic nerves at the site of previous injection. The effective- ness of gene transfer was evaluated by counting GFP 1 neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15- fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus–axon contact. Increased CXADR expression on the demyelinated axons facilitated axo- plasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treat- ment for amyotrophic lateral sclerosis, pain, and spinal cord injury. V V C 2010 Wiley-Liss, Inc. Key words: peripheral nerve; demyelination; adenovirus; motoneuron; DRG neuron Gene transfer of therapeutic molecules provides promising avenues for the treatment of incurable diseases of the central (CNS) and peripheral (PNS) nervous sys- tens. Gene therapy using recombinant viral vectors has not been translated into clinically applicable therapies because of difficulties in delivery to target neurons (Verma and Somia, 1997; St. George, 2003; Burton et al., 2005; Blits and Bunge, 2006; Jakobsson and Lund- berg, 2006; Mandel et al., 2006; Louboutin et al., 2007; Ryan and Federoff, 2007). Targeted expression of dorsal root ganglia (DRG) and alpha motor neurons (aMN) of the spinal cord by foreign genes has several potential therapeutic applications (Turner et al., 2001; Boulis et al., 2003a,b; Kaspar et al., 2003; Azzouz et al., 2004; Dodge et al., 2005; Gu et al., 2005; Nakajima et al., 2005, 2007). Viral vector injection into the spinal cord is problematic insofar as there is infection of both glia and neurons, with poor neuronal selection (Huber et al., 2000; Abdellatif et al., 2006; Blits and Bunge, 2006). Furthermore, myelitis and tissue necrosis may result from intramedullary injection (Hermens and Verhaagen, 1998; Ruitenberg et al., 2002; Boulis et al., 2002; Abdellatif et al., 2006). Alternatively, viral vectors can infect aMN and DRG neurons via intramuscular (i.m.; Haase et al., 1997; Kaspar et al., 2003; Nakajima et al., 2005) or intra- neural (i.n.) injection (Boulis et al., 1999, 2002). Remote viral vector injections represent a promising method for treating disorders of the spinal cord that avoids manipula- tion of CNS tissue. However, to date, this approach is associated with low infection rates. Intraneural injection The first two authors contributed equally to this work. Contract grant sponsor: Kentucky Spinal Cord and Head Injury Research Trust; Contract grant sponsor: NIH; Contract grant number: RR015576; Contract grant sponsor: Norton Healthcare; Contract grant sponsor: Neurosurgical Institute of Kentucky; Contract grant sponsor: Common- wealth of Kentucky Challenge for Excellence (to C.B.S., S.R.W.). *Correspondence to: Christopher B. Shields, MD, Norton Neuroscience Institute, Norton Healthcare, 210 East Gray Street, Suite 1102, Louis- ville, KY 40202. E-mail: cbshields1@gmail.com Received 20 May 2008; Revised 9 January 2010; Accepted 19 February 2010 Published online 5 April 2010 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.22394 Journal of Neuroscience Research 88:2374–2384 (2010) ' 2010 Wiley-Liss, Inc.