Volume 6, Issue 2, Pages 179-189 (August 2002) Demonstration of Feasibility of In Vivo Gene Therapy for Gaucher Disease Using a Chemically Induced Mouse Model  John Marshall, Kerry Anne McEachern, Julie A.Cavanagh Kyros, Jennifer B Nietupski, Tracey L Budzinski, Robin J Ziegler, Nelson S Yew, Jennifer Sullivan, Abraham Scaria, Nico van Rooijen, John A Barranger, Seng H Cheng  Molecular Therapy  Volume 6, Issue 2, Pages 179-189 (August 2002) DOI: 10.1006/mthe.2002.0650 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Localization of GL-1 in the induced Gaucher mouse liver (A). BALB/c mice were treated with CBE (delivered intraperitoneally) and GL-1-containing liposomes (delivered intravenously) and 24 hours later, administered with either clodronate liposomes or PBS. After an additional 24 hours, the livers were harvested, and the levels of GL-1 in the liver were determined. Treatment with clodronate liposomes results in depletion of the Kupffer cells. The data shown are for individual tissue GL-1 values (E) and the mean (A) ± SEM (n = 6). Unpaired t-test P values are indicated for the corresponding groups. (B, C, D) Localization of a fluorescent analog of GL-1. BALB/c mice were injected intravenously with GL-1-containing liposomes containing 1% of the fluorescent analog, GL-1B (BODIPY FL C12-glucocerebroside). After 24 hours the livers were harvested and processed for histological staining. (B) The 5 μm sections were incubated with a rat anti-mouse F4/80 (macrophage marker) antibody and detected with an AlexaFluor488 goat anti-rat IgG secondary antibody. (C) GL-1B fluorescence was visualized using a rhodamine filter. The image in (D) is an overlay of images in (B) and (C), showing co-localization of GL-1B and macrophages. Arrows depict the co-localization of GL-1B in two separate macrophages. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Effect of adding exogenous recombinant glucocerebrosidase on GL-1 levels in the induced Gaucher mouse liver. BALB/c mice were treated with CBE (delivered intraperitoneally) and GL-1-containing liposomes (delivered intravenously) and 24 hours later administered either 40 U/kg Cerezyme or PBS. After an additional 24 hours the livers were harvested and the levels of GL-1 determined. The data shown are for individual tissue GL-1 values (○) and the mean (▴) ± SEM (n = 5). Unpaired t-test P values are indicated for the corresponding groups. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Localization of systemically delivered recombinant human glucocerebrosidase in the liver. Fischer 344 rats were administered intravenously with 80 U/kg Cerezyme (A–F) or with 80 U/kg unmodified glucocerebrosidase (G–I). The livers were harvested 1 hour later and processed for histological staining. Liver sections were incubated with two anti-human glucocerebrosidase monoclonal antibodies (1B5 and 8E4) and detected with an AlexaFluor568 goat anti-mouse IgG1 secondary antibody (A, D, G). The same sections were also incubated with either a mouse anti-rat CD11b (macrophage marker) antibody (B, H), or a sheep anti-rat von Willebrand factor (endothelial cell marker) antibody (E). These were detected using an Alexafluor488 goat anti-mouse IgG2a and an AlexaFluor488 donkey anti-sheep secondary antibody, respectively. Images in (C), (F), and (I) are overlays of the corresponding images from (A), (D), and (G) with (B), (E) and (H), respectively. Arrows indicate examples of cells that show human glucocerebrosidase localized in either the macrophages (C, I) or endothelial cells (F). Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Tissue expression levels of glucocerebrosidase following systemic delivery of the recombinant adenoviral vector Ad2/CMV-GC BALB/c mice received an intravenous injection of 5 × 1012 particles/kg of Ad2/CMV-GC Serum, liver, spleen, and lungs were analyzed for glucocerebrosidase activity 2 days post-injection. Levels of the enzyme in naïve BALB/c mice tissues were also assayed. The dashed line indicates the steady state concentration of Cerezyme attained in the serum of Gaucher patients during a therapeutic infusion. Data shown are expressed as mean ± SEM (n = 8). Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Localization of recombinant human glucocerebrosidase in the liver following systemic delivery of Ad2/CMV-GC. Fischer 344 rats were administered intravenously with either 8 × 1012 particles/kg (A–C) or 2 × 1012 particles/kg (D–F) Ad2/CMV-GC. The livers were processed for histological staining2 days post-injection. Liver sections were incubated with two anti-human glucocerebrosidase monoclonal antibodies (1B5 and 8E4) and detected with an Alexafluor568 goat anti-mouse IgG1 secondary antibody (A, D). The same sections were also incubated with a mouse anti-rat CD11b (macrophage marker) antibody (B, E), and detected using AlexaFluor488 goat anti-mouse IgG2a. The image in (C) is an overlay of the images in (A) and (B), and the image in (F) is an overlay of the images in (D) and (E). Arrows indicate examples of cells showing both human glucocerebrosidase and macrophage staining. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Effect of intravenous administration of Ad2/CMV-CC on liver GL-1 levels in the induced Gaucher mouse. BALB/c mice were treated with CBE (delivered intraperitoneally) and GL-1-containing liposomes (delivered intravenously) and 24 hours later administered either 5 × 106, 1.5 × 106, or 0.5 × 1012 particles/kg Ad2/CMV-CC. The animals were killed, and the levels of GL-1 in their livers were assayed 2 days later. The data shown are for individual tissue GL-1 values (E) and the mean (F) ± SEM (n = 6). Unpaired t-test P values are indicated for the corresponding groups. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 7 Plasmid DNA-mediated transduction of the livers of induced Gaucher mice. (A) Tissue expression levels of glucocerebrosidase following hydrodynamic delivery of the plasmid pGZB-sGC. BALB/c mice were treated with CBE (delivered intraperitoneally) and GL-1-containing liposomes (delivered intravenously) and then 24 hours later, injected hydrodynamically with 10 μg of pGZB-sGC. The levels of glucocerebrosidase were assayed in the serum and liver 2 days post-injection. The levels of glucocerebrosidase in naïve mice and in mice injected with pGZB-agal (encoding human a-galactosidase A) were also determined. Data shown are expressed as mean ± SEM (n = 8). (B) Effect of hydrodynamic administration of pGZB-sGC on GL-1 levels in the induced Gaucher mouse. Mice were treated as described above. The levels of GL-1 in the livers were assayed 2 days later. The data shown are for individual tissue GL-1 values (○) and the mean (▴) ± SEM (n = 8). Unpaired t-test P values are indicated for the corresponding groups. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 8 Effect of intranasal delivery of Ad2/CMV-CC on liver GL-1 levels in the induced Gaucher mouse. BALB/c mice were treated with CBE (delivered intraperitoneally) and GL-1-containing liposomes (delivered intravenously) and 24 hours later administered 5 × 1011 particles/kg Ad2/CMV-CC either intravenously (i.v.) or intranasally (i.n.). The animals were killed and the levels of GL-1 in the livers were assayed 2 days later. The data shown are for individual tissue GL-1 values (O) and the mean (▴) ± SEM (n = 6). Unpaired t-test P values are indicated for the corresponding groups. Molecular Therapy 2002 6, 179-189DOI: (10.1006/mthe.2002.0650) Copyright © 2002 American Society for Gene Therapy Terms and Conditions