Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice

Abstract

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.

FIG. 1

Anti-β-glucuronidase antibody and β-glucuronidase activity levels in plasma. MPS type VII mice were injected with Adβgluc on day 0 and with MR-1 or control immunoglobulin on days −1, 0, 1, 2, 4, 6, 9, and 12. Plasma samples obtained at the indicated time points were analyzed for anti-β-glucuronidase IgG (A) and for β-glucuronidase activity (B). Anti-β-glucuronidase IgG concentrations are expressed as units per milliliter of plasma, which refers to the β-glucuronidase-capturing capacity of the plasma IgG. n was 3 to 5 for Adβgluc+control immunoglobulin-injected ice; n was 5 or 6 for Adβgluc+MR-1-treated mice. Error bars represent standard errors of the means.

FIG. 2

Histochemical detection of β-glucuronidase in liver and kidney sections 16 weeks after Adβgluc injection. MPS type VII mice were injected with Adβgluc, with or without MR-1 treatment. Sixteen weeks after Adβgluc injection, 10-μm-thick cryosections from liver (A, B, C, and D) and kidney (E, F, G, and H) tissues were histochemically stained for β-glucuronidase. A red precipitation product is formed in the presence of β-glucuronidase. No staining is observed in sections from negative control (naive) MPS type VII mice (panels A and E), while sections from Adβgluc+control immunoglobulin-injected mice (panels B and F) and Adβgluc+MR-1-injected mice (panels C and G) have intensely positive cells. The positive activity in sections from normal C57BL/6 mice is shown for comparison (panels D and H). Bar, 50 μm.

FIG. 3

Effects of Adβgluc-mediated gene transfer on storage bodies in liver and kidney of MPS type VII mice. Thin sections (0.5-μm thickness) from livers and kidneys of MPS type VII mice sacrificed 16 weeks after Adβgluc injection were analyzed for distended lysosomes, identified as unstained intracellular vacuoles. The liver from an age-matched untreated MPS type VII mouse is notably affected (A), with greatly enlarged lysosomes in Kupffer cells (closed arrow) and numerous distended lysosomes in hepatocytes (open arrow). Adβgluc injection without (B) or with (C) MR-1 treatment reduced the size and number of vacuoles to levels of normal (C57BL/6) mice (D). For an untreated MPS type VII mouse, glomerular and cortical tubule cell lysosomal accumulations in the kidney are readily apparent (E). Injection of Adβgluc without MR-1 treatment (F) is only partially effective, while Adβgluc+MR-1 injection (G) restores the phenotype to near normal (H). Bar, 10 μm.

FIG. 4

Adβgluc-mediated gene transfer to brain results in long-term β-glucuronidase expression and improvements in storage disease. Sixteen weeks after adenovirus vector injection, 10-μm-thick coronal cryosections of the brain were histochemically stained for β-glucuronidase activity, and 0.5-μm-thick sections of plastic-embedded brain were analyzed for distended lysosomes. Histochemistry (A) shows intense β-glucuronidase activity in the injected hemisphere and moderate activity in the noninjected cortex and striatum. Thin sections from the striatum (B) and cortex (C) of age-matched Adβgal-injected MPS type VII mice show numerous distended lysosomes within cells. In sections from Adβgluc-injected mice, cells with reduced storage are present in the injected striatum (D), noninjected striatum (E), and noninjected cortex (F). Bar, 10 μm for panels B through F.