scAAV9 Intracisternal Delivery Results in Efficient Gene Transfer to the Central Nervous System of a Feline Model of Motor Neuron Disease Thomas Bucher, 1 Marie-Anne Colle, 2 Erin Wakeling, 3 Laurence Dubreil, 2 John Fyfe, 3 Delphine Briot-Nivard, 1 Maud Maquigneau, 1 Sylvie Raoul, 1 Yan Cherel, 2 Ste ´ phanie Astord, 4 Sandra Duque, 4 Thibaut Marais, 4 Thomas Voit, 4 Philippe Moullier, 1,5 Martine Barkats, 4 and Be ´ atrice Joussemet 1 Abstract On the basis of previous studies suggesting that vascular endothelial growth factor (VEGF) could protect motor neurons from degeneration, adeno-associated virus vectors (serotypes 1 and 9) encoding VEGF (AAV.vegf) were administered in a limb-expression 1 (LIX1)-deficient cat—a large animal model of lower motor neuron disease— using three different delivery routes to the central nervous system. AAV.vegf vectors were injected into the motor cortex via intracerebral administration, into the cisterna magna, or intravenously in young adult cats. Intracerebral injections resulted in detectable transgene DNA and transcripts throughout the spinal cord, confirming anterograde transport of AAV via the corticospinal pathway. However, such strategy led to low levels of VEGF expression in the spinal cord. Similar AAV doses injected intravenously resulted also in poor spinal cord transduction. In contrast, intracisternal delivery of AAV exhibited long-term transduction and high levels of VEGF expression in the entire spinal cord, yet with no detectable therapeutic clinical benefit in LIX1-deficient animals. Altogether, we demonstrate (i) that intracisternal delivery is an effective AAV delivery route resulting in high transduction of the entire spinal cord, associated with little to no off-target gene expression, and (ii) that in a LIX1-deficient cat model, however, VEGF expressed at high levels in the spinal cord has no beneficial impact on the disease course. Introduction T he limb-expression 1 (LIX1)-deficient cat represents a unique large animal model of lower motor neuron (MN) disease close to human spinal muscular atrophy (SMA) type III in severity and time of onset (He et al., 2005; Fyfe et al., 2006). Affected cats exhibit juvenile muscle atrophy, gait ab- normalities, and abnormal electromyograms (EMGs) begin- ning at 2 months of age, with disease progression reaching a plateau in adults, around 8 months of age (Wakeling et al., 2011). Interestingly, motor axonal growth defects precede MN cell loss, as it has been described in other animal models lacking survival motor neuron (SMN) gene ( McWhorter et al., 2003; Rossoll et al., 2003; Kariya et al., 2008). Of note, the LIX1 gene product whose absence is responsible for MN degener- ation in cats possesses an RNA-binding domain at its amino terminus, suggesting its role in RNA metabolism (Giot et al., 2003; Fyfe et al., 2006). Similarly, a relationship between RNA metabolism and MN degeneration has also been described for different proteins implicated in human MN diseases such as in SMA with SMN protein ( Jablonka et al., 2000; Lunn and Wang, 2008) or in amyotrophic lateral slerosis (ALS) (van Blitterswijk and Landers, 2010; Ito and Suzuki, 2011). How- ever, the relation between RNA metabolism and MN cell death is not yet fully understood. In this study, we tested the therapeutic effect of the vas- cular endothelial growth factor (VEGF), a neuroprotective factor, in LIX1-deficient cats using three different gene de- livery routes. Initially described as one of the major regula- tors of blood vessel formation (Ferrara et al., 2003), the VEGF was also shown to induce antiapoptotic and neuroprotective activities both in vitro and in vivo (Greenberg and Jin, 2005; 1 INSERM UMR1089, Institut de Recherche The ´rapeutique 1, Universite ´ de Nantes, 44007 Nantes Cedex 01, France. 2 INRA UMR703, Ecole Nationale Ve ´te ´rinaire, Agroalimentaire et de l’Alimentation Nantes-Atlantique (Oniris), 44307, Nantes, France. 3 Department of Microbiology and Molecular Genetics, Michigan State University, 2209 Biomedical Physical Sciences, Lansing, Michigan, MI 48824–4320. 4 INSERM UMR974, Institut de Myologie, Faculte ´ de Me ´decine, 75013 Paris, France. 5 Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610. HUMAN GENE THERAPY 24:670–682 ( July 2013) ª Mary Ann Liebert, Inc. DOI: 10.1089/hum.2012.218 670