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Mark S. Sands - One of the best experts on this subject based on the ideXlab platform.

  • Lentiviral-Transduced Human Mesenchymal Stem Cells Persistently Express Therapeutic Levels of Enzyme in a Xenotransplantation Model of Human Disease
    Stem Cells, 2008
    Co-Authors: Todd E. Meyerrose, Carole Vogler, Marie S. Roberts, Louisa Wirthlin, Jan A. Nolta, Kevin K Ohlemiller, Mark S. Sands
    Abstract:

    Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.

  • Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity.
    Stem Cells, 2008
    Co-Authors: David A. Hess, Mark S. Sands, Timothy P. Craft, Louisa Wirthlin, Michael H. Creer, Sarah A. Hohm, Ping Zhou, William C. Eades, Jan A. Nolta
    Abstract:

    Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell-based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into β-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII mice, we characterized the distribution of lineage-depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true nonhematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of nonhematopoietic cells. However, relying solely on continued expression of cell surface markers, as used in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. Disclosure of potential conflicts of interest is found at the end of this article.

  • Clinical response to persistent, low-level β-glucuronidase expression in the murine model of mucopolysaccharidosis type VII
    Journal of Inherited Metabolic Disease, 2007
    Co-Authors: Anthony Donsante, Carole Vogler, Beth Levy, Mark S. Sands
    Abstract:

    Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by β-glucuronidase (GUSB) deficiency. This disease exhibits a broad spectrum of clinical signs including skeletal dysplasia, retinal degeneration, cognitive deficits and hearing impairment. Sustained, high-level expression of GUSB significantly improves the clinical course of the disease in the murine model of MPS VII. Low levels of enzyme expression (1–5% of normal) can significantly reduce the biochemical and histopathological manifestations of MPS VII. However, it has not been clear from previous studies whether persistent, low levels of circulating GUSB lead to significant improvements in the clinical presentation of this disease. We generated a rAAV2 vector that mediates persistent, low-level GUSB expression in the liver. Liver and serum levels of GUSB were maintained at ∼5% and ∼2.5% of normal, respectively, while other tissue ranged from background levels to 0.9%. This level of activity significantly reduced the secondary elevations of α-galactosidase and the levels of glycosaminoglycans in multiple tissues. Interestingly, this level of GUSB was also sufficient to reduce lysosomal storage in neurons in the brain. Although there were small but statistically significant improvements in retinal function, auditory function, skeletal dysplasia, and reproduction in rAAV-treated MPS VII mice, the clinical deficits were still profound and there was no improvement in lifespan. These data suggest that circulating levels of GUSB greater than 2.5% will be required to achieve substantial clinical improvements in MPS VII.

  • 237. Evaluation of Low-Level, Constitutive Expression of |[beta]|-glucuronidase on the Clinical Manifestations of Mucopolysaccharidosis Type VII
    Molecular Therapy, 2005
    Co-Authors: Anthony Donsante, Carole Vogler, Beth Levy, Mark S. Sands
    Abstract:

    Mucopolysaccharidosis Type VII (MPS VII) is caused by a deficiency in the acid hydrolase, |[beta]|-glucuronidase (GUSB). In the absence of this enzyme, glycosaminoglycans (GAGs) cannot be fully degraded and accumulate within the lysosomes of many cell types. Biochemically, MPS VII is characterized by elevated GAG levels and secondary elevations in other lysosomal enzymes, such as |[alpha]|-galactosidase (|[alpha]|-gal). Clinical symptoms include hearing and vision loss, skeletal dysplasia, mental retardation, and shortened life span. A mouse model of MPS VII exhibits many of these abnormalities, as well as reproductive and immune defects, and has been used extensively to evaluate new therapies. Previous studies have suggested that constitutive, low levels of GUSB (0.5|[ndash]|5% of normal) are sufficient to significantly correct this disease. At these levels, the biochemical symptoms show considerable improvement. However, the main goal of therapy is to alleviate the clinical symptoms of MPS VII. The relationship between biochemical and clinical improvement has not been carefully examined. Previous studies have demonstrated that adeno-associated virus-based (AAV) vectors delivered at birth and expressing high levels of GUSB can vastly improve the clinical manifestations of MPS VII. In order to understand how lower levels of expression impact disease progression, we constructed an AAV vector that drives expression of human GUSB in a liver-specific manner via the control of the human |[alpha]|-1 antitrypsin promoter. MPS VII mice, identified at birth, were given 2|[times]|109 IU of AAV intravenously. AAV-treated MPS VII mice had 4% normal levels of GUSB in the liver and maintained serum levels at 2% normal. GUSB activities in the spleen, kidney, lung and brain were 0.5%, 0.1%, 0.3%, 0.7%, respectively. At these levels, significant reductions in both |[alpha]|-gal levels (liver: MPS VII- 244|[plusmn]|3 U/mg; AAV-treated- 91|[plusmn]|29 U/mg; spleen: MPS VII- 186|[plusmn]|5 U/mg; AAV-treated- 121|[plusmn]|60; brain: MPS VII- 113|[plusmn]|11 U/mg; AAV-treated- 52|[plusmn]|2 U/mg) and in GAG levels (liver: MPS VII- 14|[plusmn]|0.6 |[mu]|g/mg; AAV-treated- 2.3|[plusmn]|0.8 |[mu]|g/mg) were observed. However, the clinical impact of our therapy does not mirror the biochemical improvements. AAV-treated MPS VII mice show no significant increase in longevity and the hearing loss shows only a small but significant improvement in the middle of the hearing range (20 kHz: wild type- 28|[plusmn]|10 dB; MPS VII- 82|[plusmn]|8 dB; AAV-treated- 62|[plusmn]|9 dB, p < 0.0002). Matings between AAV-treated MPS VII mice frequently resulted in pregnancies (83%), but many of the dams have fatal complications during labor. Retinal deficits show the best response to the therapy (wild type- 862|[plusmn]|254 |[mu]|V; MPS VII- 254|[plusmn]|114 |[mu]|V; AAV-treated- 575|[plusmn]|37 |[mu]|V, p

  • 243. Transplantation of Neonatal NOD/SCID/MPSVII Mice with Human Mesenchymal Stem Cells
    Molecular Therapy, 2005
    Co-Authors: Phillip E. Herrbrich, Mark S. Sands, A. Alex Hofling, Todd E. Meyerrose, Jan A. Nolta
    Abstract:

    In this study we assessed the transfer and expression of the gene for beta-glucuronidase (GUSB) into multiple tissues by infusing normal human mesenchymal stem cells (MSC) into unconditioned neonatal NOD/SCID/MPSVII (GUSB null) mice. In this study, pups were transplanted before 3 days of age using the facial vein injection strategy. We hypothesized that neonatal pups would provide a rapidly growing environment for engraftment and expansion of transplanted normal human MSC into different tissues. The Sands laboratory has used the MPSVII mouse for many years to study disease progression, including neural defects, and to study correction of the disease by gene replacement therapies. In order to study cellular therapies using normal human cells, the murine MPSVII mutation was backcrossed for ten generations onto the NOD/SCID strain. Human umbilical cord blood CD34+ cells engraft in the resultant NOD/SCID/GUSB null strain to levels of 86% marrow replacement, similar to the NOD/SCID parent (Hofling 2004). The tissues of the mice can be homogenized and subjected to a GUSB enzyme assay, and plotted against a standard curve composed of normal human and GUSB null mouse cells, to quantitate the percentage of human cells that had been residing in the tissue of interest at the time of harvest. There is also a FACS|[ndash]|based assay to detect levels of the GUSB protein, and to allow sorting of donor-derived cells from any tissue after transplantation. The aspect of the NOD/SCID/GUSB null strain that makes it so particularly well suited for the study of human stem cell therapy is the clear-cut enzymatic stain for normal, GUSB positive human cells transplanted into the mice. On tissue slides prepared from mice that had been transplanted at birth with human mesenchymal stem cells, the histochemical stain for GUSB revealed normal cells in multiple tissues. Almost all tissues surveyed (N=19) demonstrated the presence of human MSC by staining for GUSB. The stain is quite specific, and although the released enzyme can be taken up by neighboring cells, it appears to be in very low abundance or, more likely, in a processed form where it is no longer detectable by the histochemical analysis. Thus, the individual transplanted human cells stand out vividly against the background murine tissues. In summary, the use of the NOD/SCID/MPSVII neonatal injection model has allowed accurate tracking and quantitation of human MSC into multiple tissues of mice injected by facial vein within three days of birth. This study may set the stage for neonatal gene/cell therapy for newborns with lysosomal storage diseases.

John H. Wolfe - One of the best experts on this subject based on the ideXlab platform.

  • Bilateral single-site intracerebral injection of a nonpathogenic herpes simplex virus-1 vector decreases anxiogenic behavior in MPS VII mice
    Molecular Therapy - Methods & Clinical Development, 2015
    Co-Authors: Wenpei Liu, John H. Wolfe, Gerald D. Griffin, Trena Clarke, Michael K. Parente, Rita J. Valentino, Nigel W. Fraser
    Abstract:

    Genetic diseases of the brain usually have pathologic lesions distributed throughout, thus requiring global correction. Herpes simplex virus-1 (HSV-1) vectors may be especially useful for gene delivery in these disorders since they can spread trans-synaptically along neuronal pathways to distal sites from a localized injection. We have previously shown that a nonpathogenic HSV-1 (strain 1716), which is deleted in the ICP34.5 gene, and expressing the lysosomal enzyme β-glucuronidase (GUSB) from the latency-associated transcript (LAT) promoter, spreads within the brains of GUSB-deficient mucopolysaccharidosis VII mice to reverse the pathognomonic storage lesions throughout the diseased brain. In this study, we tested the ability of the 1716 LAT-GUSB vector to improve behavioral deficits. The treatment significantly decreased anxiogenic behaviors associated with the mutation, as indicated by open-field behavior and decreased neophobia in a novel object-recognition task. The treated mice also exhibited an improvement in cognitive function associated with the cerebral cortex in a familiar object test. The results indicate the functional therapeutic potential of the 1716 LAT-GUSB vector.

  • Widespread Correction of Lysosomal Storage in the Mucopolysaccharidosis Type VII Mouse Brain with a Herpes Simplex Virus Type 1 Vector Expressing β-Glucuronidase
    Molecular Therapy, 2006
    Co-Authors: Bradford K. Berges, John H. Wolfe, Srikanth Yellayi, Brian A. Karolewski, Richard R. Miselis, Nigel W. Fraser
    Abstract:

    We have inoculated a herpes simplex virus type 1 (HSV-1) vector into a variety of sites in the mouse brain and assayed the regions of latency and expression of a β-glucuronidase (GUSB) cDNA from the latency-associated transcript promoter. Injection sites used were somatosensory cortex, visual cortex, striatum, dorsal hippocampus, and CSF spaces. Latent vector was detected in regions at a distance from the respective injection sites, consistent with axonal transport of vector. Regions of GUSB activity varied by injection site and included cerebral cortex, striatum, thalamus, hypothalamus, substantia nigra, hippocampus, midbrain, pons, medulla, cerebellum, and spinal cord. After a single injection, GUSB enzymatic activity reached wild-type levels in several brain regions. GUSB was found in some areas without any detectable vector, indicative of axonal transport of GUSB enzyme. GUSB-deficient mice, which have the lysosomal storage disease mucopolysaccharidosis (MPS) VII, have lysosomal storage lesions in cells throughout the brain. Adult MPS VII mice treated by injection of vector into a single site on each side of the brain had correction of storage lesions in a large volume of brain. The potential for long-term, widespread correction of lysosomal storage diseases with HSV-1 vectors is discussed.

  • Stable levels of long-term transgene expression driven by the latency-associated transcript promoter in a herpes simplex virus type 1 vector.
    Molecular Therapy, 2005
    Co-Authors: Bradford K. Berges, John H. Wolfe, Nigel W. Fraser
    Abstract:

    Abstract Previous gene transfer studies of the herpes simplex virus type 1 (HSV-1) using the latency-associated transcript (LAT) promoter have reported a decrease in transgene expression in the brain over time, but the extent of this decrease has not been measured and it is unknown if expression eventually stabilizes. We examined LAT promoter-mediated transgene expression in the mouse brain for 1 year following intracranial injection with a HSV-1 vector expressing human β-glucuronidase (GUSB). The vector genome copy number remained stable from 2 to 52 weeks. Quantitative reverse transcriptase PCR detected a peak of LAT intron expression at 2 weeks (corresponding to the end of the acute phase of viral infection), followed by stable expression during latency (13–52 weeks). The number of GUSB-positive cells also had a peak in the acute phase and then was stable during latency (13–52 weeks). GUSB enzymatic activity was maintained at 11% of normal at 6 and 12 months, indicating that the LAT promoter is capable of driving stable transgene expression in the brain.

  • 510. Neonatal Gene Transfer with Non-Human Primate AAV Vector Serotypes Results in Widespread Transduction and Gene Activity in the Mouse Brain|[ast]|
    Molecular Therapy, 2005
    Co-Authors: Brian A. Karolewski, Hennessy Howell, John H. Wolfe
    Abstract:

    Mucopolysaccharidosis (MPS) VII is a heritable lysosomal storage disease caused by the deficiency of Beta-glucuronidase (GUSB). MPS VII is a chronic and progressive multiorgan disorder with signs of pathology presenting early in life. Most therapeutic approaches for MPS VII have focused on adult treatments. Although these approaches can reverse storage lesions, the central nervous system (CNS) is more difficult to treat. Our lab has previously shown complementary patterns of transduction between AAV1 and after neonatal injection into the lateral cerebral ventricles (Passini et al., 2003; Journal of Virology). Using an AAV2 ITR backbone, we investigated neonatal gene transfer to the CNS of normal and MPS VII mice with capsid proteins from AAV7, AAV8, and AAV9. We injected 1.8 |[times]| 10^10 genomes of vector into the lateral cerebral ventricles (2 |[mu]|l each) of normal and MPS VII mice at birth (P O.5). At one month postinjection, each of the AAV serotypes demonstrated extensive and widespread GUSB activity and mRNA expression in the brain, but variations in transduction patterns related to substructures of the brain were observed. AAV 2/7 and AAV 2/8 transduced more cells and expressed more GUSB compared to AAV 2/9. Significant amounts of GUSB were present in the ganglion cells of the retina and spinal cord for each of the tested AAV serotypes. Evaluations of AAV gene transduction patterns and lysosomal storage correction for the eye, brain and spinal cord are ongoing. These results suggest that AAV7, AAV8, and AAV9 are suitable vectors for targeting the CNS, and may be useful for CNS gene therapy.

  • 129. Development of Vectors Utilizing Strong Neuronal Cell Type Specific Promoters for High Level Expression of Beta Glucuronidase in the Central Nervous System
    Molecular Therapy, 2005
    Co-Authors: Tupur Husain, John H. Wolfe
    Abstract:

    Recombinant adeno-associated viruses (AAV) are promising vectors for delivery to the central nervous system, e.g. in lysosomal storage disorder such as GUSB-deficient MPS VII. The cellular tropism of AAV gene delivery varies depending on the serotype used. In lysosomal enzyme deficient cells, the transferred normal enzyme is secreted and can be endocytosed by adjacent cells (sphere of correction). Although treatments have been shown to work in mouse brains, enzyme delivery will need to be significantly increased to produce the same effect in larger brains such as humans. Our laboratory has shown that the level of GUSB expression and secretion is proportional to increased transcription from vectors. Various promoters have been known to modulate different levels of transcription in the CNS. If vector-mediated gene delivery could produce higher levels of enzyme secretion from a minimal number of injections, the sphere of correction could be increased for enzyme diffusion, axonal transport, and migrating cross-correcting cells. We have designed a eukaryotic minigene vector driven by the natural promoter. The vector contains the first two introns in addition to all the exon sequences of hGUSB. Mouse brains were stereotactically injected into the left midbrain region with 2.5 |[times]| 1012 viral genome equivalents per each of four injection sites. The major structures injected were the cortex, striatum, hippocampus and thalamus. The pattern of transduction at 2 months post-injection was assessed by in situ hybridization which shows similar patterns of transduction to a control vector without introns in the cortex, striatum, hippocampus and thalamus. Quantitative measurements for transcription are in progress. The enzyme positive cells were detected at the sites of injection and further extended beyond the regions of transduced cells when rostral and caudal sections were examined. GUSB enzyme in the hippocampus was also detected in the contralateral region of the hippocampus. Experiments are also in progress to test the neuron-specific enolase (NSE) and latency associated (LAT) promoters. The NSE promoter has been known to confer high levels of gene expression when used in recombinant AAV2 vectors in transduced neurons, and has been found to express therapeutic levels of enzyme in other MPS models. In herpesviruses, the LAT promoter is the only viral gene sequence expressed in life-long latent infections in post-mitotic neurons. It has also been shown that significantly more GUSB activity is detected when the GUSB cDNA is inserted near the LAT promoter as opposed to other lower transcriptional level configurations. Evaluation of the effects of the NSE promoter and HSV-LAT promoter in the transcriptional high level configuration for GUSB are currently in progress for comparison with the hGUSB minigene and cDNA vector controls.

Carole Vogler - One of the best experts on this subject based on the ideXlab platform.

  • Lentiviral-Transduced Human Mesenchymal Stem Cells Persistently Express Therapeutic Levels of Enzyme in a Xenotransplantation Model of Human Disease
    Stem Cells, 2008
    Co-Authors: Todd E. Meyerrose, Carole Vogler, Marie S. Roberts, Louisa Wirthlin, Jan A. Nolta, Kevin K Ohlemiller, Mark S. Sands
    Abstract:

    Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.

  • Clinical response to persistent, low-level β-glucuronidase expression in the murine model of mucopolysaccharidosis type VII
    Journal of Inherited Metabolic Disease, 2007
    Co-Authors: Anthony Donsante, Carole Vogler, Beth Levy, Mark S. Sands
    Abstract:

    Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by β-glucuronidase (GUSB) deficiency. This disease exhibits a broad spectrum of clinical signs including skeletal dysplasia, retinal degeneration, cognitive deficits and hearing impairment. Sustained, high-level expression of GUSB significantly improves the clinical course of the disease in the murine model of MPS VII. Low levels of enzyme expression (1–5% of normal) can significantly reduce the biochemical and histopathological manifestations of MPS VII. However, it has not been clear from previous studies whether persistent, low levels of circulating GUSB lead to significant improvements in the clinical presentation of this disease. We generated a rAAV2 vector that mediates persistent, low-level GUSB expression in the liver. Liver and serum levels of GUSB were maintained at ∼5% and ∼2.5% of normal, respectively, while other tissue ranged from background levels to 0.9%. This level of activity significantly reduced the secondary elevations of α-galactosidase and the levels of glycosaminoglycans in multiple tissues. Interestingly, this level of GUSB was also sufficient to reduce lysosomal storage in neurons in the brain. Although there were small but statistically significant improvements in retinal function, auditory function, skeletal dysplasia, and reproduction in rAAV-treated MPS VII mice, the clinical deficits were still profound and there was no improvement in lifespan. These data suggest that circulating levels of GUSB greater than 2.5% will be required to achieve substantial clinical improvements in MPS VII.

  • 237. Evaluation of Low-Level, Constitutive Expression of |[beta]|-glucuronidase on the Clinical Manifestations of Mucopolysaccharidosis Type VII
    Molecular Therapy, 2005
    Co-Authors: Anthony Donsante, Carole Vogler, Beth Levy, Mark S. Sands
    Abstract:

    Mucopolysaccharidosis Type VII (MPS VII) is caused by a deficiency in the acid hydrolase, |[beta]|-glucuronidase (GUSB). In the absence of this enzyme, glycosaminoglycans (GAGs) cannot be fully degraded and accumulate within the lysosomes of many cell types. Biochemically, MPS VII is characterized by elevated GAG levels and secondary elevations in other lysosomal enzymes, such as |[alpha]|-galactosidase (|[alpha]|-gal). Clinical symptoms include hearing and vision loss, skeletal dysplasia, mental retardation, and shortened life span. A mouse model of MPS VII exhibits many of these abnormalities, as well as reproductive and immune defects, and has been used extensively to evaluate new therapies. Previous studies have suggested that constitutive, low levels of GUSB (0.5|[ndash]|5% of normal) are sufficient to significantly correct this disease. At these levels, the biochemical symptoms show considerable improvement. However, the main goal of therapy is to alleviate the clinical symptoms of MPS VII. The relationship between biochemical and clinical improvement has not been carefully examined. Previous studies have demonstrated that adeno-associated virus-based (AAV) vectors delivered at birth and expressing high levels of GUSB can vastly improve the clinical manifestations of MPS VII. In order to understand how lower levels of expression impact disease progression, we constructed an AAV vector that drives expression of human GUSB in a liver-specific manner via the control of the human |[alpha]|-1 antitrypsin promoter. MPS VII mice, identified at birth, were given 2|[times]|109 IU of AAV intravenously. AAV-treated MPS VII mice had 4% normal levels of GUSB in the liver and maintained serum levels at 2% normal. GUSB activities in the spleen, kidney, lung and brain were 0.5%, 0.1%, 0.3%, 0.7%, respectively. At these levels, significant reductions in both |[alpha]|-gal levels (liver: MPS VII- 244|[plusmn]|3 U/mg; AAV-treated- 91|[plusmn]|29 U/mg; spleen: MPS VII- 186|[plusmn]|5 U/mg; AAV-treated- 121|[plusmn]|60; brain: MPS VII- 113|[plusmn]|11 U/mg; AAV-treated- 52|[plusmn]|2 U/mg) and in GAG levels (liver: MPS VII- 14|[plusmn]|0.6 |[mu]|g/mg; AAV-treated- 2.3|[plusmn]|0.8 |[mu]|g/mg) were observed. However, the clinical impact of our therapy does not mirror the biochemical improvements. AAV-treated MPS VII mice show no significant increase in longevity and the hearing loss shows only a small but significant improvement in the middle of the hearing range (20 kHz: wild type- 28|[plusmn]|10 dB; MPS VII- 82|[plusmn]|8 dB; AAV-treated- 62|[plusmn]|9 dB, p < 0.0002). Matings between AAV-treated MPS VII mice frequently resulted in pregnancies (83%), but many of the dams have fatal complications during labor. Retinal deficits show the best response to the therapy (wild type- 862|[plusmn]|254 |[mu]|V; MPS VII- 254|[plusmn]|114 |[mu]|V; AAV-treated- 575|[plusmn]|37 |[mu]|V, p

  • Electrocardiographic and other cardiac anomalies in β-glucuronidase-null mice corrected by nonablative neonatal marrow transplantation
    Proceedings of the National Academy of Sciences, 2004
    Co-Authors: Adam J.t Schuldt, Mark D. Lessard, Carole Vogler, Nancy Galvin, T. J. Hampton, V. Chu, Jane E. Barker
    Abstract:

    Cardiovascular manifestations of lysosomal storage disease (LSD) are a significant health problem for affected patients. Infantileonset cardiac disease, because of its rapid progression, is usually treated symptomatically. Therapy in older patients includes valve replacement and bone marrow (BM) transplantation, both of which are life threatening in the already debilitated patients. Enzyme replacement therapy has potential benefit but has not yet been demonstrated to provide long-term relief for cardiac disease. Here, we demonstrate prevention of severe cardiac manifestations in β-glucuronidase (GUSB) null mice BM-transplanted i.v. as neonates without myeloablative pretreatment. The mice, a model of mucopolysaccharidosis type VII (MPSVII, Sly syndrome), develop progressive LSD unless provided with GUSB early in life. The BM recipients retained GUSB+ donor cells in the peripheral blood and heart until necropsy at >11 months of age. The enzyme β-hexosamindase increased in tissues of GUSB null MPSVII mice was reduced significantly (P = 0.001) in treated MPSVII hearts. Electrocardiography demonstrated normalization of heart rate, PR, PQ, and QRS intervals in BM recipients. Storage was markedly reduced in the stroma of heart valves, adventitial cells of the aortic root, perivascular and interstitial cells of the myocardium, and interstitial cells of the conduction tissue. Heart/body weight ratio normalized. The aortic root was still grossly distended, and the conductive myocytes retained storage, suggesting neither plays a major role in ECG normalization. We conclude that transplantation of MPSVII neonates without toxic intervention can prevent many of the cardiovascular manifestations of LSD.

  • Human CD34+ hematopoietic progenitor cell-directed lentiviral-mediated gene therapy in a xenotransplantation model of lysosomal storage disease.
    Molecular Therapy, 2004
    Co-Authors: A. Alex Hofling, Carole Vogler, Steven M. Devine, Mark S. Sands
    Abstract:

    As a group, lysosomal storage diseases (LSDs) affect roughly 1 in 6700 live births. Treatment of patients with enzyme replacement therapy or allogeneic bone marrow transplantation is severely limited by cost and clinical complications, respectively. In this study, the efficacy of gene therapy targeted to human hematopoietic progenitor cells was investigated for mucopolysaccharidosis type VII (MPSVII), a LSD caused by β-glucuronidase (GUSB) deficiency. Clinical experience has emphasized the need to evaluate transduction protocols directly with human cells through in vivo assays. Therefore, GUSB-deficient mobilized peripheral blood CD34+ cells from a patient with MPSVII were transduced with a third-generation lentiviral vector encoding human GUSB and then assessed in a xenotransplantation system. In this novel strategy, the xenotransplanted murine recipients were also GUSB-deficient, allowing a detailed evaluation of therapeutic efficacy in a host with MPSVII. Twelve weeks posttransplantation, lymphomyeloid expression of GUSB was detected in 10.8 ± 1.6% of the human cells in the bone marrow with an average of 1 to 2 vector genomes measured per positive cell. The corrected cells distributed widely throughout recipient tissues, resulting in significant therapeutic effects including improvements in biochemical parameters and reduction of the lysosomal distension of several host tissues.

Noriyuki Ishii - One of the best experts on this subject based on the ideXlab platform.

  • Two-Dimensional Crystalline Array Formation of Glucuronide Transporter from Escherichia coli by the Use of Polystyrene Beads for Detergent Removal
    The Journal of Membrane Biology, 2013
    Co-Authors: Noriyuki Ishii
    Abstract:

    n -Dodecyl-β- d -maltoside solubilized glucuronide transporter (GUSB), the product of GUSB gene from Escherichia coli , was treated with Bio-Beads as an agent for removing the detergent from a micellar solution under suitable combination with dimyristoylphosphatidylcholine. Optimizing conditions led to a two-dimensional crystalline array formation of GUSB. The crystalline arrays appear to have a hexagonal lattice with layer group P 6, the unit cell dimensions of a  =  b  = 13.8 nm and γ  = 120°. Each stain-protruding periodic unit showed approximately 11.8 ± 0.3 nm in a diameter in the inverse Fourier-filtered image to have formed with pentameric GUSB (5 × 49.7 kDa).

  • Erratum to: Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli
    The Journal of Membrane Biology, 2011
    Co-Authors: Noriyuki Ishii
    Abstract:

    Original Article has an error in the caption of Figure 2. In the last sentense of the caption, ‘‘heptameric’’ should be read as ‘‘pentameric’’. Corrected caption for figure 2 is given below. Fig. 2 Size-exclusion HPLC elution profile of GUSB with moleculer size marker standards. The GUSB–DDM complex was found to have a Stokes radius of 57 A. Six soluble proteins of known Stokes radius were used for calibration: ferritin (63 A), catalase (52 A), aIdorase (46 A), bovine serum albumin (35 A), ovalbumin (28 A) and chymotrypsinogen (22 A). Inset is a nagatively stained transmission electron microscopic image of the peak fraction. Scale bar, 500 nm. Based on the elution profile and TEM image, GUSB in the presence of DDM most probably stays as a pentameric oligomer.

  • Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli
    Journal of Membrane Biology, 2010
    Co-Authors: Noriyuki Ishii
    Abstract:

    The glucuronide transporter GUSB, the product of the GUSB gene from Escherichia coli , is responsible for detoxification of metabolites. In this study, we successfully expressed GUSB homologously in E. coli and investigated its oligomeric state in n -dodecyl-β- d -maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 ± 2 Å for the purified GUSB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GUSB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GUSB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GUSB with lipids.

  • Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli
    Journal of Membrane Biology, 2010
    Co-Authors: Noriyuki Ishii
    Abstract:

    The glucuronide transporter GUSB, the product of the GUSB gene from Escherichia coli , is responsible for detoxification of metabolites. In this study, we successfully expressed GUSB homologously in E. coli and investigated its oligomeric state in n -dodecyl-β- d -maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 ± 2 Å for the purified GUSB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GUSB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GUSB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GUSB with lipids.

Mark E. Haskins - One of the best experts on this subject based on the ideXlab platform.

  • Mucopolysaccharidosis VII in a Cat Caused by 2 Adjacent Missense Mutations in the GUSB Gene.
    Journal of Veterinary Internal Medicine, 2015
    Co-Authors: P. Wang, Mark E. Haskins, J. Sorenson, S. Strickland, C. Mingus, Urs Giger
    Abstract:

    Background Mucopolysaccharidoses (MPS) are common lysosomal storage disorders causing typically progressive skeletal and ocular abnormalities. Objectives To describe the clinic features, metabolic profile and a unique mutation in a domestic shorthair (DSH) kitten with MPS VII. Animals Affected kitten and 80 healthy cats. Methods Serum lysosomal enzyme activities and urinary glycosaminoglycan (GAG) accumulation were assessed. Exons of the β-glucuronidase gene (GUSB) were sequenced from genomic DNA and genotyping was conducted. Results A 3-month-old DSH cat was presented for stunted growth, paresis, facial dysmorphia, multiple skeletal deformities, and corneal opacities. Evaluation of blood smears disclosed metachromatic granules in leukocytes and a urinary mucopolysaccharide spot test was positive. The proband had no GUSB activity but normal or increased activities for other lysosomal enzymes. Sequencing of the GUSB gene from the proband and comparison to the sequence of 2 healthy cats and the published feline genome sequence demonstrated 2 unique single base transitions (c.1421T>G and c.1424C>T) in exon 9, altering 2 adjacent codons (p.Ser475Ala and p.Arg476Trp). These amino acid changes are in a highly conserved domain of the GUSB protein and nontolerable to maintain function. Moreover, the p.Arg476Trp mutation previously has been identified in human patients. None of the other clinically healthy cats had these mutations. Conclusions and Clinic Importance The diagnostic approach to MPS disorders is delineated. This is only the second mutation known to cause MPS VII in cats. Similarly, 2 different mutations have been described in MPS VII dogs, thereby showing the molecular heterogeneity of MPS VII in companion animals.

  • effect of neonatal gene therapy on lumbar spine disease in mucopolysaccharidosis vii dogs
    Molecular Genetics and Metabolism, 2012
    Co-Authors: Lachlan J Smith, Ping Wang, Mark E. Haskins, Patricia Odonnell, John T Martin, Dawn M Elliott, Katherine P. Ponder
    Abstract:

    Abstract Mucopolysaccharidosis VII (MPS VII) is due to deficient β-glucuronidase (GUSB) activity, which leads to accumulation of chondroitin, heparan, and dermatan sulfate glycosaminoglycans in various tissues including those of the spine. Associated spine disease can be due to abnormalities in the vertebrae, the intervertebral disks, or other spine tissues. The goal of this study was to determine if neonatal gene therapy could prevent lumbar spine disease in MPS VII dogs. MPS VII dogs were injected intravenously with a retroviral vector (RV) expressing canine GUSB at 2 to 3 days after birth, which resulted in transduction of hepatocytes that secreted GUSB into blood. Expression was stable for up to 11 years, and mean survival was increased from 0.4 years in untreated dogs to 6.1 years in treated dogs. Despite a profound positive clinical effect, 6-month-old RV-treated MPS VII dogs still had hypoplastic ventral epiphyses with reduced calcification in the lumbar spine, which resulted in a reduced stiffness and increased range of motion that were not improved relative to untreated MPS VII dogs. At six to 11 years of age, ventral vertebrae remained hypoplastic in RV-treated MPS VII dogs, and there was desiccation of the nucleus pulposus in some disks. Histochemical staining demonstrated that disks did not have detectable GUSB activity despite high serum GUSB activity, which is likely due to poor diffusion into this relatively avascular structure. Thus, neonatal gene therapy cannot prevent lumbar spine disease in MPS VII dogs, which predicts that enzyme replacement therapy (ERT) will similarly be relatively ineffective even if started at birth.

  • expression in blood cells may contribute to biochemical and pathological improvements after neonatal intravenous gene therapy for mucopolysaccharidosis vii in dogs
    Molecular Genetics and Metabolism, 2006
    Co-Authors: Bin Wang, Ping Wang, Mark E. Haskins, Lingfei Xu, Thomas Omalley, Patricia Odonnell, Matthew N Ellinwood, Charles H Vite, Katherine P. Ponder
    Abstract:

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB) that results in accumulation of glycosaminoglycans in many organs. We have previously reported that neonatal intravenous injection of a gamma retroviral vector (RV) expressing canine GUSB resulted in transduction of hepatocytes, high levels of GUSB modified with mannose 6-phosphate in blood, and reduction in disease manifestations in the heart, bone, and eye. However, it was unclear if liver was the only site of expression, and the effect upon other organs was not assessed. We demonstrate here that blood cells from these RV-treated MPS VII dogs had substantial copies of RV DNA, and expressed the RNA at 2% of the level found in liver. Therefore, expression of GUSB in blood cells may synergize with uptake of GUSB from blood to reduce storage in organs. The RV-treated dogs had marked biochemical and pathological evidence of reduction in storage in liver, thymus, spleen, small intestines, and lung, and partial reduction of storage in kidney tubules. The brain had 6% of normal GUSB activity, and biochemical and pathological evidence of reduction in storage in neurons and other cell types. Thus, this neonatal gene therapy approach is effective and might be used in humans if it proves to be safe. Both secretion of enzyme into blood by hepatocytes, and expression in blood cells that migrate into organs, may contribute to correction of disease.

  • 247. Gene Therapy with a Retroviral Vector Expressing Canine |[beta]|-Glucuronidase to Juvenile (7 Week Old ) Dogs Improves the Biochemical Manifestations of MPS VII
    Molecular Therapy, 2005
    Co-Authors: Bin Wang, Ping Wang, Thomas O'malley, Mark E. Haskins, Katherine P. Ponder
    Abstract:

    Top of pageAbstract Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficiency of |[beta]|-glucuronidase (GUSB). Manifestations include bone and joint disease, heart disease, and neurological dysfunction. The MPS VII dog has a missense mutation in GUSB (R166H) that results in clinical manifestations that resemble those in humans. We have previously demonstrated that neonatal gene therapy with 3|[times]|109 TU/kg of a retroviral vector (RV) expressing canine GUSB from the human |[alpha]|1-antitrypsin promoter (hAAT-cGUSB-WPRE) resulted in transduction of hepatocytes and 195+/-36 U/ml of GUSB in serum. This resulted in a marked improvement in cardiac, bone and joint, and other manifestations. However, most patients with MPS VII are not identified at birth, and it will be necessary to determine the effect of transfer into older animals. Seven week-old MPS VII dogs were injected IV with 1|[times]|1010 transducing units (TU)/kg of hAAT-cGUSB-WPRE. Some received a cumulative dose of hepatocyte growth factor (HGF) over 24 hours of 2.5 mg/kg, then were injected with RV at 24, 48, 72, and 96 hours after the first dose of HGF (HGF/RV). Others received RV once a day for 4 days without preceding HGF (RV alone). Dogs that received HGF/RV or RV alone at 7 weeks had transduced hepatocytes and achieved stable expression of GUSB in serum at 185+/-54 U/ml or 51+/-5 U/ml for up to 1 year, respectively, although differences were not statistically significant. The serum GUSB achieved per TU/kg given was 28% and 8%, respectively, of that achieved after neonatal gene transfer, which likely reflects lower levels of hepatocyte replication in juveniles. Some animals were sacrificed at 6 months after gene transfer, and organs analyzed for biochemical correction of disease. The results in both juvenile transfer groups were similar and were pooled for statistical analyses. MPS VII results in elevation of the secondary lysosomal enzyme |[beta]|-hexosaminodase (|[beta]|-hex) and glycosaminoglycan (GAG) levels, and normalization of these occurs with successful therapies. In liver, spleen, jejunum, and lung, GUSB activity was 1% to 15% of normal levels and there was a marked reduction in |[beta]|-hex and GAG levels. In thymus, pancreas, kidney, and muscle, GUSB activity was 0.25% to 1% of normal and there was a marked or partial reduction in |[beta]|-hex and GAG levels. However, brain GUSB activity was only 0.2% of normal and there was little reduction in |[beta]|-hex (GAG was not evaluable as untreated MPS VII dogs do not have elevated brain GAG levels). Despite these biochemical improvements, the mobility of the dogs was only slightly improved and all were unable to walk by 12 months or earlier. We conclude that gene therapy into juvenile dogs results in expression similar to that observed after neonatal gene transfer, and marked biochemical improvements in somatic organs. This is the first demonstration of using juvenile gene therapy to improve disease in a large animal model for LSD. However, there was little biochemical improvement in brain, and the bone disease remained severe.

  • Gene Therapy Ameliorates Cardiovascular Disease in Dogs With Mucopolysaccharidosis VII
    Circulation, 2004
    Co-Authors: Meg M. Sleeper, Thomas O'malley, Katherine P. Ponder, John R Melniczek, Margaret A Weil, N. Matthew Ellinwood, B. Fornasari, C.d. Sammarco, Mark E. Haskins
    Abstract:

    Background— Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (GUSB) activity resulting in defective catabolism of glycosaminoglycans (GAGs). Cardiac disease is a major cause of death in MPS VII because of accumulation of GAGs in cardiovascular cells. Manifestations include cardiomyopathy, mitral and aortic valve thickening, and aortic root dilation and may cause death in the early months of life or may be compatible with a fairly normal lifespan. We previously reported that neonatal administration of a retroviral vector (RV) resulted in transduction of hepatocytes, which secreted GUSB into the blood and could be taken up by cells throughout the body. The goal of this study was to evaluate the effect on cardiac disease. Methods and Results— Six MPS VII dogs were treated intravenously with an RV-expressing canine GUSB. Echocardiographic parameters, cardiovascular lesions, and biochemical parameters of these dogs were compared with those of normal and unt...