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

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.     

In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors.

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme alpha-L-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.     

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor

BACKGROUND: One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. METHODS: In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. RESULTS: A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). CONCLUSIONS: MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.     

Quantitative Analysis of α-L-Iduronidase Expression in Immunocompetent Mice Treated with the Sleeping Beauty Transposon System

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models.     

Three novel α-L-iduronidase mutations in 10 unrelated Chinese mucopolysaccharidosis type I families

Mucopolysaccharidosis type I (MPS I) arises from a deficiency in the α-L-iduronidase (IDUA) enzyme. Although the clinical spectrum in MPS I patients is continuous, it was possible to recognize 3 phenotypes reflecting the severity of symptoms, viz., the Hurler, Scheie and Hurler/Scheie syndromes. In this study, 10 unrelated Chinese MPS I families (nine Hurler and one Hurler/Scheie) were investigated, and 16 mutant alleles were identified. Three novel mutations in IDUA genes, one missense p.R363H (c.1088G > A) and two splice-site mutations (c.1190-1G > A and c.792+1G > T), were found. Notably, 45% (nine out of 20) and 30% (six out of 20) of the mutant alleles in the 10 families studied were c.1190-1G > A and c.792+1G > T, respectively. The novel missense mutation p.R363H was transiently expressed in CHO cells, and showed retention of 2.3% IDUA activity. Neither p.W402X nor p.Q70X associated with the Hurler phenotype, or even p.R89Q associated with the Scheie phenotype, was found in this group. Finally, it was noted that the Chinese MPS I patients proved to be characterized with a unique set of IDUA gene mutations, not only entirely different from those encountered among Europeans and Americans, but also apparently not even the same as those found in other Asian countries.     

Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system

The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems. The stages of this protocol are preparation of DNA-PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA-PEI complexes to analysis of transient transgene expression.     

Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice

Background aimsMucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-l-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I.MethodsWe produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice.ResultsAn increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme.ConclusionsThe results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.     

Characterization of the defective beta-glucuronidase activity in canine mucopolysaccharidosis type VII

Canine mucopolysaccharidosis type VII results from deficient activity of lysosomal beta-glucuronidase. Residual enzymatic activity (0.2-1.7% of normal) was detected in tissue homogenates from affected dogs. In contrast, serum and urine from affected animals had up to 15% residual activity. To further characterize the nature of the defective enzyme, hepatic beta-glucuronidase was partially purified from normal and MPS VII dogs for determination of their physical and kinetic properties. About 65% of the total beta-glucuronidase in normal canine liver required detergent for solubilization (i.e., membrane-associated), whereas only 22% of the residual activity in canine MPS VII liver was membrane-associated. Compared to the normal hepatic enzyme, the Km towards 4-methylumbelliferyl-beta-glucuronide was markedly increased in MPS VII dogs (i.e., 0.48 versus greater than 2.5 mmol/l). In contrast, the thermo-, cryo-, and pH stability properties, as well as the pH optimum (approximately 4.6), were essentially unaffected. In addition, the canine MPS VII hepatic residual activity was unresponsive to sulfhydryl reducing reagents and divalent cations, despite the fact that incubation of normal canine beta-glucuronidase with dithiothreitol and magnesium and/or calcium enhanced the activity more than 15-fold.     

Use of lipoplex-induced nuclear factor-kappaB activation to enhance transgene expression by lipoplex in mouse lung

BACKGROUND: Although lipofection-induced TNF-alpha can activate nuclear factor kappaB (NF-kappaB), which, in turn, increases the transgene expression from plasmid DNA in which any NF-kappaB responsive element is incorporated, no attempts have been made to use such biological responses as NF-kappaB activation against a vector to enhance vector-mediated gene transfer. METHODS: A lipoplex composed of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium and cholesterol liposome and plasmid DNA encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pCMV-Luc) was intravenously injected into mice. Luciferase activity as well as NF-kappaB activation in the lung were evaluated. Then, a novel plasmid DNA, pCMV-kappaB-Luc, was constructed by inserting 5 repeats of NF-kappaB-binding sequences into the pCMV-Luc. RESULTS: NF-kappaB in the lung was activated by injection of the lipoplex and its nuclear localization was observed. An injection of lipopolysaccharide 30 min prior to the lipofection further activated NF-kappaB. At the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and NF-kappaB activity. Based on these findings, we tried to enhance the lipoplex-based transgene expression by using NF-kappaB activation. The lipoplex consisting of pCMV-kappaB-Luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pCMV-Luc. CONCLUSIONS: We demonstrated that NF-kappaB activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting NF-kappaB-binding sequences into plasmid DNA. These findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.     

Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β-Glucuronidase Gene Transfer

Abstract: The inherited deficiency of β-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal β-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.     

Evidence of lysosomal membrane permeabilization in mucopolysaccharidosis type I: Rupture of calcium and proton homeostasis

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α‐iduronidase (IDUA), which leads to intralysosomal accumulation of glysosaminoglycans. Patients with MPS I present a wide range of clinical manifestations, but the mechanisms by which these alterations occur are still not fully understood. Genotype–phenotype correlations have not been well established for MPS I; hence, it is likely that secondary and tertiary alterations in cellular metabolism and signaling may contribute to the physiopathology of the disease. The aim of this study was to analyze Ca²⁺ and H⁺ homeostasis, lysosomal leakage of cysteine proteases, and apoptosis in a murine model of MPS I. After exposition to specific drugs, cells from Idua?/? mice were shown to release more Ca²⁺ from the lysosomes and endoplasmic reticulum than Idua+/+ control mice, suggesting a higher intraorganelle store of this ion. A lower content of H⁺ in the lysosomes and in the cytosol was found in cells from Idua?/? mice, suggesting an alteration of pH homeostasis. In addition, Idua?/? cells presented a higher activity of cysteine proteases in the cytosol and an increased rate of apoptotic cells when compared to the control group, indicating that lysosomal membrane permeabilization might occur in this model. Altogether, our results suggest that secondary alterations—as changes in Ca²⁺ and H⁺ homeostasis and lysosomal membrane permeabilization—may contribute for cellular damage and death in the physiopathology of MPS I. J. Cell. Physiol. 223: 335–342, 2010. © 2010 Wiley‐Liss, Inc.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Usefulness of double gene construct for rapid identification of transgenic mice exhibiting tissue-specific gene expression.

Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.     

The role of IFN-gamma and Toll-like receptors in nephropathy induced by Toxoplasma gondii infection

The pathologic links between Toxoplasma gondii infections and renal diseases have not yet been established. Gamma interferon (IFN-gamma) and Toll-like receptors (TLRs) are involved in the host defense mechanism against T. gondii infection. The role of IFN-gamma and TLRs in renal function of T. gondii -infected mice was studied using wild type (WT), TLR2-deficient and TLR4-deficient mice perorally infected with cysts of an avirulent cyst-forming Fukaya strain of T. gondii. T. gondii was abundant in kidneys in IFN-gamma KO (GKO) mice as determined by a quantitative competitive-polymerase chain reaction (QC-PCR). But, T. gondii was not detected in kidneys in WT, TLR2-deficient and TLR4-deficient mice. Interestingly, renal function of TLR2-deficient and TLR4-deficient mice was damaged as evaluated by serum creatinine, serum blood urea nitrogen (BUN), and urine albumin/creatinine ratio (ACR), whereas renal function of GKO and WT mice was not damaged. Histopathology of TLR2-deficient mice exhibited glomerular and extracellular matrix swelling with advancing glomerular tissue proliferation, thickened Bowman's capsules and vacuolization of tubules. Renal immunofluorescence study of T. gondii -infected TLR2-deficient mice displayed positive staining of the glomerular basement membrane, mesangial areas and peritubular capillaries. The damage of kidney from TLR4-deficient mice was less severe compared to TLR2-deficient mice, and histopathological damage of kidney was not observed in WT and GKO mice. These results indicate that TLR2, but not IFN-gamma, plays a role in the protection of the renal function against T. gondii infection.     

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Gastrointestinal pathology in a mouse model of mucopolysaccharidosis type IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in sulphamidase (NS), a lysosomal enzyme required for the degradation of heparan sulphate glycosaminoglycans (gags). The MPS IIIA mouse is a naturally occurring model that accurately reflects the human pathology and disease course. It displays primarily central nervous system pathology accompanied by widespread accumulation of gag in somatic tissues. MPS IIIA mice exhibit greater bodyweight gain than normal littermates and attain a higher mature bodyweight. In this study, gastrointestinal morphology and function was characterised in the IIIA mouse. Stomach and duodenum weight increased in MPS IIIA mice and duodenum length also increased. An increased submucosal thickness was observed in MPS IIIA intestine compared to normal mice and lysosomal storage of gag was observed in this region. Storage was also observed in the lamina propria of the villus tip. All other morphometric measurements including villus height and crypt depth fell within the normal range. The gastric emptying half‐life of solid and liquid meals decreased with age in normal mice whereas the T_½ of solid meals did not alter with age in MPS IIA mice such that they were elevated above normal by 38 weeks of age. Sucrase activity was higher than normal in MPS IIIA at all ages tested. These abnormalities in GI structure and function observed in MPS IIIA may contribute to weight gain in this disorder. J. Cell. Physiol. 219: 259–264, 2009. © 2009 Wiley‐Liss, Inc.