Restoration of Cytoskeleton Homeostasis After Gigaxonin Gene Transfer for Giant Axonal Neuropathy Silke Mussche, 1 Bart Devreese, 2 Sahana Nagabhushan Kalburgi, 3 Lavanya Bachaboina, 3, * Jonathan C. Fox, 3 Hung-Jui Shih, 3 Rudy Van Coster, 1 R. Jude Samulski, 3 and Steven J. Gray 3 Abstract Giant axonal neuropathy (GAN) is caused by loss of function of the gigaxonin protein. On a cellular level GAN is characterized by intermediate filament (IF) aggregation, leading to a progressive and fatal peripheral neurop- athy in humans. This study sought to determine if re-introduction of the GAN gene into GAN-deficient cells and mice would restore proper cytoskeleton IF homeostasis. Treatment of primary skin fibroblast cultures from three different GAN patients with an adeno-associated virus type 2 (AAV2) vector containing a normal human GAN transgene significantly reduced the number of cells displaying vimentin IF aggregates. A proteomic analysis of these treated cells was also performed, wherein the abundance of 32 of 780 identified proteins significantly changed in response to gigaxonin gene transfer. While 29 of these responding proteins have not been directly described in association with gigaxonin, three were previously identified as being disregulated in GAN and were now shifted toward normal levels. To assess the potential application of this approach in vivo and even- tually in humans, GAN mice received an intracisternal injection of an AAV9/GAN vector to globally deliver the GAN gene to the brainstem and spinal cord. The treated mice showed a nearly complete clearance of peripherin IF accumulations at 3 weeks post-injection. These studies demonstrate that gigaxonin gene transfer can reverse the cellular IF aggregate pathology associated with GAN. Introduction G iant axonal neuropathy (GAN, OMIM #256850) is a rare chronic neurodegenerative disease characterized by enlarged axons with disordered microtubules and interme- diate filaments, which is fatal by the third decade of life. The disease pathology is due to homozygous loss-of-function mutations in the GAN gene, which encodes the protein giga- xonin. Onset of symptoms is usually 3–4 years of age, with a slightly awkward gait. By the end of the second decade of life, patients normally are wheelchair bound with limited use of the arms and little to no use of their legs. Death normally occurs in the second or third decade of life. Peripheral nerve biopsies from humans show enlarged axons with densely packed and disorganized microtubules and intermediate fil- aments (IFs) (Demir et al., 2005; Nalini et al., 2008). The dys- function and degeneration of peripheral nerves is attributed to this pathology. These same studies showed white matter abnormalities in the brain by magnetic resonance imaging, indicating an involvement of the central nervous system. This peripheral nerve pathology (enlarged axons densely filled with neurofilaments) serves as a basis for diagnosis, which is then confirmed by sequencing the GAN gene. There are no statistics on the incidence of the disease, but it is considered extremely rare. Giant axonal neuropathy was characterized over 25 years ago as an inborn error in intermediate filament organization (Pena et al. 1983), which was later found to be from muta- tions in the gigaxonin gene and resulting loss of functional gigaxonin protein (Bomont et al., 2000). More than 40 mu- tations have been identified in GAN patients, including de- letions, insertions, missense and nonsense mutations, which lead to loss of function of gigaxonin (Bomont et al., 2000, 2003; Bruno et al., 2004; Demir et al., 2005; Houlden et al., 2007; Koop et al., 2007; Kuhlenba ¨ umer et al., 2002; Leung et al., 2007). Gigaxonin is a broadly expressed Cul3 ubiquitin ligase adaptor protein, normally present at extremely low levels throughout the brain (Cleveland et al., 2009). Known direct targets of gigaxonin are MAP8, MAP1B, and TCB-C, which are involved with IF regulation (Allen et al., 2005; 1 Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, Ghent 9000, Belgium. 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent 9000, Belgium. 3 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. *Current address: USA Mitchell Cancer Institute, 1660 Springhill Avenue, Mobile, AL 36604. HUMAN GENE THERAPY 24:209–219 (February 2013) ª Mary Ann Liebert, Inc. DOI: 10.1089/hum.2012.107 209