Volume 3, Issue 3, Pages 351-358 (March 2001) Intracranial Injection of Recombinant Adeno-associated Virus Improves Cognitive Function in a Murine Model of Mucopolysaccharidosis Type VII  W.Anthony Frisella, Lynn H. O'Connor, Carole A. Vogler, Marie Roberts, Steve Walkley, Beth Levy, Thomas M. Daly, Mark S. Sands  Molecular Therapy  Volume 3, Issue 3, Pages 351-358 (March 2001) DOI: 10.1006/mthe.2001.0274 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 GUSB activity approaches or exceeds normal levels in MPS VII mice after intracranial injections of AAV at birth. Eighteen newborn MPS VII mice were injected with 1.5 × 105 infectious units of AAVβGEnh (17) in both the anterior cortex and the hippocampus of each hemisphere (6 × 105 total IU). Three injected mice each (asterisks) were sacrificed at 2, 4, 8, 12, 14, and 18 weeks of age and the GUSB-specific activity in one hemisphere was measured. The specific activity in the brains of age-matched normal mice (filled squares) is shown for comparison. Each point represents the average and 1 standard deviation from the mean. Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 GUSB activity and the recombinant AAV genome are concentrated near the injection sites in MPS VII mice that received intracranial injections of AAV at birth. Histochemical staining demonstrates a concentration of GUSB activity (red) near the injection sites in the anterior cortex and hippocampus (A). Quantitative biochemical assays performed on homogenates from 1-mm coronal sections show that increased GUSB-specific activities are observed in the cortex and hippocampus (B). The data represent the means and 1 standard deviation from assays performed on sections from three AAV-injected mice. Relatively more PCR product from the AAV-encoded human GUSB cDNA (240, hcDNA) is observed in sections from the anterior cortex and hippocampus compared to other regions of the brain (C). The PCR product (454, mGene) from the murine GUSB gene served as an internal control. The numbers (1–10) correspond to the specific 1-mm section from the injected brains. Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 GUSB protein is localized to both neuronal and nonneuronal cells in the brains of AAV-injected MPS VII mice. Anti-NeuN (A) and anti-human GUSB (B) antibodies label the same cell (C) in the cortex of AAV-injected MPS VII mice. Similarly, colocalization (F) of the astrocyte-specific marker GFAP (D) and human GUSB (E) is also observed in the cortex. Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Lysosomal storage is reduced in many regions of the brain at 8 weeks following AAV-mediated gene transfer performed in newborn MPS VII mice. Distended lysosomes (white arrows) can be seen in the frontal cortex, parietal cortex, hippocampus, and cerebellum of untreated age-matched MPS VII (mps/mps) mice. Histopathologic evidence of lysosomal storage (distended lysosomes) is prevented in the frontal and parietal cortex and the hippocampus of AAV-treated MPS VII (mps/mps + AAV) mice. There were comparable amounts of lysosomal storage (white arrows) in cells of the cerebellum from both treated and untreated MPS VII mice. Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 The secondary accumulation of GM2 gangliosides in the brains of MPS VII mice is prevented by AAV-mediated gene therapy at birth. Immunohistochemical analysis shows extensive accumulation of GM2 ganglioside (brown stain) in neuronal and nonneuronal cells of the brain from an untreated MPS VII mouse (A). The amount of GM2 ganglioside is dramatically reduced in the brains of AAV-treated MPS VII mice (B). Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 AAV-treated MPS VII mice perform significantly better than untreated mutant animals in some phases of the Morris Water Maze test. AAV-treated MPS VII mice (asterisk) perform similar to normal (filled square) and significantly (P = 0.0151) better than untreated mutants (filled circle) in the Acquisition (A), Relearning (B), and Relocation (C) phases of the test. The average performance of AAV-treated MPS VII mice was better than that of untreated MPS VII mice in the Probe (D) phase of the test. However, the difference was not statistically significant (P < 0.16). Molecular Therapy 2001 3, 351-358DOI: (10.1006/mthe.2001.0274) Copyright © 2001 American Society for Gene Therapy Terms and Conditions