Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII  Brian A. Karolewski, John H. Wolfe  Molecular Therapy  Volume 14, Issue 1, Pages 14-24 (July 2006) DOI: 10.1016/j.ymthe.2006.02.012 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 GUSB enzyme activity and mRNA expression in the 1-month-old brain of normal C3H/HeOuJ mice after an in utero (E15.5) brain injection with AAV1 or AAV2. Biologically active GUSB was detected by enzyme histochemistry (A–C), and virally encoded mRNA was detected by in situ hybridization (D–F) using an antisense riboprobe against the human GUSB complementary DNA. A parenchymal injection of 1 μl of 1.4 × 1012 genome equivalents (GE)/ml of AAV1 produced localized GUSB activity (A) and mRNA expression (D) in the rostral mesencephalon. A parenchymal injection with 1 μl of 1.4 × 1012 GE/ml AAV2 (highest titer available) produced localized GUSB activity (B) and mRNA expression (E) in the septostriatal brain region. GUSB enzyme activity (C) and mRNA expression (F) were not detected after an intraventricular fetal brain injection with 1 μl of 1.4 × 1012 GE/ml AAV2. Scale bar = 4.3 mm. Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Fetal (E15.5) intraventricular brain injection with 1 μl of 3.3 × 1013 GE/ml of AAV1 resulted in GUSB enzyme activity and mRNA expression throughout the normal mouse brain at 1 month postpartum. Biologically active GUSB was detected by enzyme histochemistry (A–E), and virally encoded mRNA was detected by in situ hybridization (F–J) using an antisense riboprobe against the human GUSB cDNA. The location from Bregma of each depicted section is graphically represented on the sagittal drawings of the mouse brain (K–O) (adapted from ref [50]). An injection into the lateral cerebral ventricle with AAV1 produced widespread enzyme activity throughout the brain (A–E) as demonstrated in the olfactory bulb (Bregma +4.28) caudally to the cerebellum (Bregma −6.10). The high level of GUSB activity resulted from extensive and symmetrical widespread vector transduction in the brain (F–J). Major areas of vector transduction were seen in the mitral cell layer of the olfactory bulb (F), cortex (G–I), lateral septum (G), stratum pyramidale and granule cell layer of the hippocampus (H, I), superior colliculus (I), inferior colliculus (J), and brain stem (J). Scale bar: 4.0 mm (A–J). Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 A fetal (E15.5) intraventricular brain injection with 1 μl of 3.3 × 1013 GE/ml of AAV1 resulted in long-term and widespread transduction in the brain of normal mice 1 year postpartum. Biologically active GUSB was detected by enzyme histochemistry (A, C–F, K–N), and virally encoded mRNA was detected by in situ hybridization (B, G–J, O–R) using an antisense riboprobe against the human GUSB cDNA. Symmetrical vector transduction and GUSB activity were observed throughout the brain, as depicted in the septostrial brain region (A, B). GUSB activity and expression were maintained for 1 year in the hippocampus (C, G), hypothalamus (D, H), thalamus (E, I), superior (K, O) and inferior (L, P) colliculi, and choroid plexus (N, R). GUSB activity was maintained in the cerebellum (M) despite a decrease in vector expression in the Purkinje cell layer (Q). The ganglion cell layer of the retina had enzyme-positive cells (F) in the absence of GUSB mRNA (J). Scale bar = 2.8 mm (A, B); 500 μm (C–E, G–I, K–R), 250 μm (F, J). Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Lack of storage lesions in MPS VII brains, 1 year postpartum, after a fetal (E15.5) intraventricular brain injection with AAV1. C3H/HeOuJ normal control (A–C), AAV1-treated MPS VII (1 μl of 3.3 × 1013 GE/ml) (D–F), AAV2-treated MPS VII (1 μl of 1.4 × 1012 GE/ml of AAV2) (G–I), uninjected MPS VII (J–L) brains. The pyriform cortex (A, D, G, J), stratum pyramidale layer of the hippocampus (B, E, H, K), and Purkinje cell layer of the cerebellum (C, F, I, L) are shown. Arrows point to neurons with storage vacuoles. Scale bar = 60 μm. Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Lack of storage lesions in the MPS VII spinal cord, 1 year postpartum, after a fetal (E15.5) intraventricular brain injection with AAV1. Biologically active GUSB was detected by enzyme histochemistry (A), and virally encoded mRNA was detected by in situ hybridization (B) using an antisense riboprobe against the human GUSB cDNA. GUSB enzyme activity (A) and vector transduction (B) were observed in the dorsal horn of the spinal cord. Storage lesions were not observed in the C3H/HeOuJ normal control (C) or AAV1-treated MPS VII (1 μl of 3.3 × 1013 GE/ml) (D) spinal cords. Storage lesions were detected in the AAV2-treated MPS VII (1 μl of 1.4 × 1012 GE/ml of AAV1) (E), and uninjected MPS VII (F) spinal cords. Arrows point to neurons with lysosomal storage vacuoles. Scale bar = 850 μm (A, B), 60 μm (C–F). Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 The Kaplan-Meier survival plot for MPS VII, normal, and treated mice at 12 months of age. The survival probability of MPS VII mice (n = 10) treated in utero (E15.5) with AAV1 (1 μl of 3.3 × 1013 GE/ml) was compared to normal C3H/HeOuJ (n = 73) and MPS VII (n = 91) mice. The lifespan of treated mice was similar to normal mice (p = 0.725), and significantly better than MPS VII mice (P = 0.0176). Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Analysis of vector genome in the spleen, liver, lung, and testicles of treated MPS VII mice. MPS VII fetuses received injections into the lateral cerebral ventricle with 1 μl of 3.3 × 1013 GE/ml of AAV1 on day 15 of gestation. Spleen (SP), liver (LV), lung (LN), and gonads (G) were collected at 12 months of age and frozen. DNA was isolated and analyzed using a nested PCR reaction. Vector genome was not present in the organs of the treated mice. As a control for the presence of DNA, a portion of the mouse GUSB gene was amplified producing the expected 300-bp fragment. AAV1-injected muscle (M) and water (W) were used as controls in this experiment. Molecular Therapy 2006 14, 14-24DOI: (10.1016/j.ymthe.2006.02.012) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions