Volume 19, Issue 5, Pages 860-869 (May 2011) Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder  Carmine Spampanato, Elvira De Leonibus, Paola Dama, Annagiusi Gargiulo, Alessandro Fraldi, Nicolina Cristina Sorrentino, Fabio Russo, Edoardo Nusco, Alberto Auricchio, Enrico M Surace, Andrea Ballabio  Molecular Therapy  Volume 19, Issue 5, Pages 860-869 (May 2011) DOI: 10.1038/mt.2010.299 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Brain parenchyma transduction upon intraventricles injection of adeno-associated virus serotype 2/9 (AAV2/9) in neonatal mice. (a-d) Enhanced green fluorescent protein (EGFP) expression 4 weeks after intraventricles injection of rAAV9 vector-CMV-EGFP-IRES-LUC in wild-type mice. Indirect fluorescence microscopy analysis of brain sections demonstrated EGFP-positive cells in the (a) olfactory bulb, (b) cortex, (c) ventral tegumental area, and (d) cerebellum (the selected regions within the brain parenchyma are shown as squares in e, ×20 magnification). Black arrow shows rAAV9 vector injection site (e) Sagittal section of the brain showing regions in a-d and the five hemi-coronal brain slabs quantified in f. (f) Histogram representing relative expression levels of rAAV9 vector (black bars) compared to rAAV4 (white bars) in the different regions of the brain (1, 2, 3, 4, 5) as showed in e. Levels of luciferase activity, i.e., relative light units, were normalized for β-gal expression to minimize the potential variability in the dose of the vector delivered following intraventricles administration.23 CMV, cytomegalovirus; rAAV9, recombinant adeno-associated virus serotype 9; β-gal, β-galactosidase. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Expression of sulfatase modifying factor 1 (SUMF1) in brain and visceral tissues results in activation of sulfatases. (a) Western blot analysis with anti-FLAG antibodies of multiple sulfatase deficiency (MSD) mice tissues 1 month after adeno-associated virus (AAV) vector administration. Systemic injection resulted in robust expression of SUMF1 in the lung (Lu), muscles (Mu), heart (He) and liver (Li), but lack of expression in the brain (Br). Combined injections enabled the expression also in the brain. (b) Sulfatase activities in MSD tissues 1 month after rAAV9 vector administration. ARSA, ARSC, IDS activities were measured in the extracts of five hemi-coronal brain segments (see above brain diagram, black arrow depicts the site of injection) derived from MSD mice, MSD administered intravenously with AAV vector (Syst.), MSD administered intraventricles with rAAV9 vector (ICLV) and MSD simultaneously administered through the temporal vein and brain lateral ventricles (Comb.). Sulfatase activities are indicated as percentage of wild-type activity. (c) Sulfatase activities in visceral MSD tissues 1 month after rAAV9 vector administration. The error bars indicate the SEM *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. ARSC, arylsulfatase C; ARSA, arylsulfatase A; IDS, iduronate-2-sulfatase; rAAV9, recombinant adeno-associated virus serotype 9. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Comparison of weight and survival benefit in multiple sulfatase deficiency (MSD) mice treated with recombinant adeno-associated virus serotype 9 (rAAV9) vector. (a) Animals systemically injected (MSD Syst.) and the combination group (MSD Comb.) gained weight over time similarly to the wild-type (WT). In contrast, the ICLV group lost weight over time, similarly to MSD mice. (b) The Kaplan–Meier survival curve shows that animals systemically injected and the combination group displayed increased survival rate compared to untreated MSD mice (P < 0.01, in both cases). No significant difference was observed between the ICLV group and untreated MSD mice and between systemically and combined administration (P = 0.241 and P = 0.571, respectively). ICLV, intracerebral lateral ventricles. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Brain and visceral pathology are improved in recombinant adeno-associated virus serotype 9 (rAAV9) vector-SUMF1 combined injected mice. (a) Toluidine blue stained tissue sections of 4-month old multiple sulfatase deficiency (MSD) mice. Extensive vacuolization and morphological alterations in the olfactory bulb, cerebral cortex, and hippocampus were evident in MSD mice as opposed to wild-type mice (WT), and were reduced in mice simultaneously administered through the temporal vein and brain lateral ventricles (Comb.). Higher magnification of single cells within tissues analyzed are highlighted, black arrows indicate vacuolization. Magnification ×100. (b) Alcian blue stained tissue sections of the heart, liver, lung, and muscle of wild-type mice (WT), MSD mice, MSD administered intravenously with AAV vector (Syst.) and MSD simultaneously administered through the temporal vein and brain lateral ventricles (Comb.). Squares represent zoom-in portion of tissues (dotted squares). Black arrows show glycosaminoglycan (GAG) accumulation. Tissue sections were counterstained with Nuclear-Fast red reagent, ×20 magnification. (c) Quantitative analysis of GAG accumulation in treated MSD mice. GAG content in the tissue homogenates (tissues as indicated) from the control (WT), untreated MSD, MSD rAAV9 vector-CMV-SUMF1-injected by temporal vein (Syst.), and MSD rAAV9 vector CMV-SUMF1-injected simultaneously through the temporal vein and brain lateral ventricles (Comb.). The GAG content are indicated as percentage and all of the tissues from treated mice reached normal levels, as seen in the wild-type animals. The error bars indicate the SEM. CMV, cytomegalovirus; SUMF1, sulfatase modifying factor 1. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Decreased inflammation in recombinant adeno-associated virus serotype 9 (rAAV9) vector injected mice. (a-l) Reduction of microglial activation in the brain of rAAV 9 vector-CMV-SUMF1-3XFLAG-treated multiple sulfatase deficiency (MSD) mice 3 months after vector delivery. CD68 immunofluorescence revealed a massive activation of microglia (b and c) in the cerebral cortex, (f and g) striatum and (j and k) hippocampus of untreated MSD mice and MSD administered intravenously with rAAV9 vector (Syst.). No signs of macrophage activation were observed in the brain of wild-type (WT) mice (a, e, and i). Treatment with rAAV9 vector-CMV-SUMF1-3XFLAG resulted in a reduction of microglial activation (CD68-positive cells) in the (d) cerebral cortex, (h) striatum, and (l) hippocampus of MSD treated mice. (m-p) Amelioration of astrogliosis in the brain of rAAV9 vector-CMV-SUMF1-3XFLAG-treated MSD mice 3 months after vector delivery. Glial fibrillary acidic protein (GFAP) immunofluorescence in untreated MSD mice exhibited a diffuse astrogliosis that was particularly evident in the (n) cerebral cortex, compared with the same region of (m) wild-type mice. A reduction in astrogliosis was observed in the cerebral cortex of MSD mice treated with rAAV9 vector-CMV-SUMF1-3XFLAG, (p) simultaneously administered through the temporal vein and brain lateral ventricles, but not in the (o) cortex of systemically injected MSD mice. (q-t) CD68 immunofluorescence revealed a massive activation of macrophages in the liver (r) of untreated MSD mice, as opposed to (q) wild-type mice. Treatment with rAAV9 vector-CMV-SUMF1-3XFLAG resulted in a reduction of macrophage activation in the liver of both MSD mice administered (s) intravenously with rAAV vector and MSD mice simultaneously administered through the (t) temporal vein and brain lateral ventricles. CMV, cytomegalovirus; Comb., rAAV9 vector CMV-SUMF1-injected simultaneously through the temporal vein and brain lateral ventricles; SUMF1, sulfatase modifying factor 1. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Combined recombinant adeno-associated virus serotype 9 (rAAV9) vector systemic and intrabrain injection reverses multiple sulfatase deficiency (MSD) behavioral defects. (a) Maximal walking speed in the open field (5 minutes recording) was dramatically impaired in 3–4 months old MSD mice. Systemic or combined injection, but not ICLV injection of rAAV9 vector rescued this behavioral defect. (b) The percentage time spent in the target annulus measured in the water maze on the probe trial when the platform was removed (day 7); MSD and MSD-systemically injected animals were dramatically impaired in searching the platform in the correct annulus. On the contrary, the ICLV and Comb groups were not affected as compared to wild-type (WT) animals. (c) Track plot samples of animals belonging to the different experimental groups during the water maze probe trial (60 seconds). WT animals focused their searching activity in the annulus (black arrow) where the platform was located during training; MSD and MSD-systemically injected animals displayed a random searching strategy, while MSD-Comb. animal behaved similarly to WT animals. Mean ± SEM; *P < 0.05 versus WT. Comb., rAAV9 vector CMV-SUMF1-injected simultaneously through the temporal vein and brain lateral ventricles; ICLV, intracerebral lateral ventricles. Molecular Therapy 2011 19, 860-869DOI: (10.1038/mt.2010.299) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions