Volume 22, Issue 12, Pages 2018-2027 (December 2014) Intrathecal Gene Therapy Corrects CNS Pathology in a Feline Model of Mucopolysaccharidosis I  Christian Hinderer, Peter Bell, Brittney L Gurda, Qiang Wang, Jean-Pierre Louboutin, Yanqing Zhu, Jessica Bagel, Patricia O'Donnell, Tracey Sikora, Therese Ruane, Ping Wang, Mark E Haskins, James M Wilson  Molecular Therapy  Volume 22, Issue 12, Pages 2018-2027 (December 2014) DOI: 10.1038/mt.2014.135 Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 IDUA expression in CSF and serum following IT AAV9 delivery. Five MPS I cats were treated with an intracisternal injection of an AAV9 vector (1012 GC/kg) expressing feline IDUA from a CB (gray symbols) or CMV (black symbols) promoter. CSF and serum were serially collected from the treated animals as well as three untreated MPS I cats. IDUA activity was measured using the fluorogenic substrate 4MU-iduronide in CSF (a) and serum (b). All values are the mean of duplicate assays. Normal serum and CSF activity (dashed line) are the mean values from two wild-type animals. Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Normalization of CSF hexosaminidase activity despite a transgene-specific antibody response. CSF samples collected before vector administration and at the end of the study were tested for the presence of antibodies against IDUA by indirect ELISA (a). Titers are based on a standard curve of a serially diluted positive sample, which was arbitrarily assigned a titer of 1:1,000. (b) Total Hex activity was measured in CSF using the fluorogenic substrate MUG. Normal activity (dashed line) is the mean of two heterozygous control samples. The untreated MPS I level (dotted line) is the mean of untreated controls. Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Global CNS transduction and biochemical correction following IT gene transfer. Vector genomes were quantified in DNA extracted from CNS and peripheral tissues of treated cats by TaqMan PCR (a). Tissue samples from MPS I cats and heterozygous controls were collected from sites throughout the CNS for measurement of IDUA activity (b) and Hex activity (c). Treated animals are stratified into those with normal CSF IDUA activity (8911, 8932, 9058) and those with low CSF activity (8982, 9050). All enzyme activities are normalized to protein concentration. Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Reversal of brain ganglioside storage. Immunostaining for the ganglioside GM3 was performed on tissue sections from four brain regions in untreated MPS I cats (a–d), treated cats with high CSF IDUA activity (e–h) and low CSF activity (i–l), as well as normal controls (m–p). Scale bar = 500 µm. Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Correction of neuronal storage lesions. Sections from the cerebral cortex were stained with H&E (a,f,k,p). Arrows indicate the distended neuron cell bodies typical of MPS I. The treated cats were stratified into those with low CSF IDUA activity and those with high activity. Cholesterol accumulation was evaluated in matched brain sections by filipin staining in untreated (b,c), treated (g,h,l,m) and normal control (q,r) cats. Immunostaining for the lysosomal integral membrane protein LIMP2 was performed on corresponding sections from each animal (d,e,i,j,n,o,s,t). Scale bar = 500 µm (4×), 200 µm (10×), or 100 µm (H&E). Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Reduced GAG storage in the cerebral vasculature and meninges. Cortical brain sections were stained for GAGs using Alcian blue. Low magnification images (a,d,g,j) show the cerebral cortex with the associated meninges. High magnification images show representative cortical blood vessels (b,e,h,k) and segments of meninges (c,f,i,l). Scale bars = 500 µm (left column) and 100 µm (middle and right columns). Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Correction of somatic lesions. Sections from heart, lung, liver, spleen, and kidney were stained with Alcian blue. Animals are stratified according to antibody response and circulating enzyme activity as in Figure 6. Scale bar = 100 µm. Molecular Therapy 2014 22, 2018-2027DOI: (10.1038/mt.2014.135) Copyright © 2014 American Society of Gene & Cell Therapy Terms and Conditions