Lysosomal Storage In Liver Research Articles

425. AAV2-Mediated Expression of Iduronate Sulfatase in Peripheral Tissues and the CNS in Mucopolysaccharidosis II Mouse Model by Combined Intravenous and Intracisternal Vector Delivery

Mucopolysaccharidosis II (MPS II, Hunter syndrome) is an X-linked disease due to the deficiency of the lysosomal enzyme iduronate sulfatase (Id-S). Deficiency of iduronate sulfatase results in lysosomal accumulation of dermatan sulfate and heparan sulfate, with progressive tissue and organ dysfunction, and premature death. No definite treatment is available for MPS II patients. Clinical trials of peripheral administration of recombinant Id-S in MPS II patients are in progress, but CNS disease is not expected to be improved. Therapeutic effects of AAV gene delivery on both somatic and the CNS features in adult MPS II |[ldquo]|knockout|[rdquo]| mice were studied using combined intravenous (IV) and intracisternal (IC) injections after pretreatment with mannitol. An AAV serotype 2 vector, AAV2-CMV-hIdS, was used, containing a human iduronate sulfatase cDNA (hIdS) driven by a cytomegalovirus promoter (CMV). A dose of AAV2-CMV-hIdS viral vector (2|[ndash]|4|[times]|1011 viral particles in 200|[mu]|l) was delivered into adult (4|[ndash]|6 week old) MPS II mice (n=20) by tail vein injection 10 minutes after an IV infusion of mannitol (1|[ndash]|2 mg/gm of body weight). A dose of AAV2-CMV-hIdS (2|[ndash]|4|[times]|1010 viral particles in 20 |[mu]|l) was delivered into the posterior cistern after the tail vein injection of AAV2 vector. Treated MPS II mice were scarified when they developed severe neurological symptoms, such as bladder distension, weight loss and gait abnormalities, at 15 to 23 months of age. Our results demonstrated the correction of GAG storage in multiple organs/tissues after the AAV-mediated gene transfer. Complete correction of GAG accumulation in liver was achieved (P<0.01) and partial correction in spleen, heart, muscle, and lung was observed (P<0.05). No decrease of GAG content was observed in the kidney of the mice after AAV delivery. Id-S enzyme activity was detected in the treated MPS II mouse liver (50%|[ndash]|100% of normal mouse liver). The non-treated MPS II mice have no detectable Id-S activity in any tissue. Histopathology and transmission electron microscopy studies demonstrated clearance of lysosomal storage in liver. Decreased CNS lysosomal storage was shown by histopathology in Purkinje cells and also in the neurons of the hippocampus, thalamus and cerebral cortex after AAV-mediated gene transfer. Id-S enzyme was also detected in the brain of the AAV injected mice (1%|[ndash]|7% of normal mouse brain). Physical appearances of treated MPS II mice, such as facial feature and spinal curvature, were clearly improved. Our results demonstrated that the life span of the MPS II mice after the AAV gene therapy was prolonged (means 17.8 months), compared with the life span of the non-treated group (means 13.4 months) (p<0.01). Our results suggest that IV injection combined with an IC injection of AAV2 vector following mannitol pretreatment is a promising approach for treating both somatic and CNS disease in lysosomal storage disorders.

860. Long-Term Correction of Lysosomal Storage in CNS and Somatic Tissue in Adult MPS II Mice by Intravenous Administration of AAV

A major obstacle in developing therapies for the central nervous system (CNS) disease in mucopolysaccharidoses (MPS) is the presence of the blood-brain barrier (BBB). Therapeutic effects of AAV gene delivery on both somatic and the CNS features in adult MPS II mice were studied using IV injection after pretreatment with mannitol, to disrupt the BBB and facilitate the CNS entry of the vector. An AAV serotype 2 vector, AAV2-CMV-hIdS, was used, containing a human iduronate sulfatase cDNA (hIdS) driven by a human cytomegalovirus promoter (CMV). A single dose of AAV2-CMV-hIdS viral vector (2-4×1011 viral particles in 200 μl) was delivered into adult (4-6 week old) MPS II mice (n=20) by tail vein injection 10 minutes after an IV infusion of mannitol (1-2 mg/gm of body weight). Our results demonstrated correction of GAG storage in multiple organs/tissues. A complete correction of GAG accumulation in liver was achieved (P < 0.01) and partial correction in kidney, heart, intestine and muscles was observed (P < 0.05) at 3, 6, 9 and up to 17 months in one animal. No obvious decrease of GAG content was observed in the spleens of the mice after AAV injection. Iduronate sulfatase enzyme activity was detected in the injected mouse liver samples with complete correction of lysosomal storage, at a level of 10-50% of that in the liver of normal mice. Transmission electron microscopy and histopathology study demonstrated clearance of lysosomal storage in liver. In the brain of injected MPS II mice, a decrease of lysosomal storage was shown by histopathology in Purkinje cells and also in the neurons of the hippocampus, thalamus and cerebral cortex. In addition, physical appearances of IV injected MPS II mice, such as facial feature and spinal curvature, were clearly improved, and the life span of IV injected mice was prolonged, compared to that of non-treated MPS II mice. Our results suggest that IV administration of AAV vector following mannitol pretreatment may be a promising approach for treating both somatic and CNS disease in lysosomal storage disorders.

Evaluation of pathological manifestations of disease in mucopolysaccharidosis VII mice after neonatal hepatic gene therapy.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by beta-glucuronidase (GUSB) deficiency. Intravenous injection of a retroviral vector expressing canine GUSB into neonatal MPS VII mice resulted in transduction of 6 to 35% of hepatocytes, which secreted GUSB into blood. Serum GUSB activity was stable for 6 months at 600 (low expression) to 10,000 (high expression) U/ml, and enzyme was modified appropriately with mannose 6-phosphate. The average serum GUSB activity (3531 U/ml) is the highest long-term expression reported for MPS VII mice after gene therapy. Secreted enzyme was taken up by other tissues, as the average enzyme activity was >13% of normal in somatic organs and 2% of normal in brain. Low expression markedly reduced histopathological evidence of lysosomal storage in liver, spleen, kidney, small intestine, neurons, and glial cells. High expression appeared to be more effective than low expression at reducing lysosomal storage in aorta, heart valves, thymus, bronchial epithelium, cornea, and retinal pigmented epithelium. Future experiments will determine if greater pathological improvements will consistently be observed in retrovirus-treated MPS VII mice with higher serum GUSB activity relative to animals with lower activity and if these result in clinical benefits.

In vivo treatment of hemophilia A and mucopolysaccharidosis type VII using nonprimate lentiviral vectors.

Gene therapy holds great promise for the treatment of a variety of inherited diseases, including hemophilia A and mucopolysaccharidosis type VII (MPS VII). In both these disorders, subnormal levels of replacement protein have therapeutic effects. Thus we hypothesized that transduction of a small proportion of cells by feline immunodeficiency virus (FIV)-based lentiviral vectors might provide sufficient levels of transgene expression for phenotypic correction. We intravenously injected replication-deficient FIV-based vectors encoding either human factor VIII or human beta-glucuronidase into factor VIII-deficient or beta-glucuronidase-deficient mice, respectively. This route of delivery targeted multiple organs, with the liver as the primary transduction site. In the hemophilia A mice, factor VIII expression persisted for the duration of the experiments (approximately 5 months), and recipient mice survived an otherwise lethal bleeding episode (tail-clipping). In mucopolysaccharidosis type VII mice, substantial beta-glucuronidase activity was detected in several tissues and corresponded with marked reduction of lysosomal storage in liver and spleen. These findings indicate that gene transfer with FIV-based lentiviral vectors can permanently introduce transgenes into a sufficient number of hepatocytes for long-term therapeutic effect and suggest potential clinical value of FIV-based lentiviral vectors for treatment of hemophilia A and MPS VII.

Delivery of a Retroviral Vector Expressing Human β-Glucuronidase to the Liver and Spleen Decreases Lysosomal Storage in Mucopolysaccharidosis VII Mice

Mucopolysaccharidosis VII (MPS VII) is caused by beta-glucuronidase (beta-gluc) deficiency and results in lysosomal storage due to the inability to degrade glycosaminoglycans. Transfer of a beta-gluc gene into the liver reduces hepatic pathology as well as storage in other organs via uptake of secreted protein. A Moloney murine leukemia-based retroviral vector expressing the human beta-gluc cDNA was injected intravascularly into MPS VII mice during hepatocyte replication, which was induced with im injection of an adenoviral vector that transiently expressed hepatocyte growth factor (Ad.CMV. HGF). This procedure resulted in transduction of approximately 1% of hepatocytes, 1% of normal liver enzyme activity, and a reduction in lysosomal storage in the liver at 3.5 months. Surprisingly, controls that received retroviral vector without HGF had transduction of nonparenchymal cells in the liver, significant levels of enzyme and RNA in the liver at 2 but not 3.5 months, and reduced lysosomal storage at 3.5 months. Transduction was also achieved in the replicating cells of the spleen, where lysosomal storage was reduced. An approach using a retroviral vector without a growth factor might temporarily reduce lysosomal storage in the liver and spleen in humans. Addition of HGF might be used to augment and prolong gene transfer.

Enzyme replacement in a canine model of Hurler syndrome.

The Hurler syndrome (alpha-L-iduronidase deficiency disease) is a severe lysosomal storage disorder that is potentially amenable to enzyme-replacement therapy. Availability of a canine model of the disease and a sufficient supply of corrective enzyme have permitted a therapeutic trial lasting 3 mo. Recombinant human alpha-L-iduronidase, purified to apparent homogeneity from secretions of a stably transfected Chinese hamster ovary cell line, was administered i.v. to homozygous affected animals in doses of approximately 1 mg. The enzyme rapidly disappeared from the circulation in a biphasic manner, with t1/2 of 0.9 and 19 min, respectively, and was taken up primarily by the liver. Biopsy of the liver before and after a very short trial (seven doses administered over 12 days) showed remarkable resolution of lysosomal storage in both hepatocytes and Kupffer cells. After weekly administration of enzyme to three affected animals over a period of 3 mo, the level of enzyme was about normal in liver and spleen, lower but significant in kidney and lung, and barely detectable (0-5% of normal) in brain, heart valves, myocardium, cartilage, and cornea. Light and electron microscopic examination of numerous tissues showed normalization of lysosomal storage in liver, spleen, and kidney glomeruli, but there was no improvement in brain, heart valves, or cornea. Even though the treated dogs developed complement-activating antibodies against alpha-L-iduronidase, clinical symptoms could be prevented by slow infusion of enzyme and premedication.

Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer.

An inherited deficiency of beta-glucuronidase in humans, mice and dogs causes mucopolysaccharidosis VII (Sly syndrome), a progressive degenerative disease that reduces lifespan (to an average of 5 months in mice) and results from lysosomal storage of undegraded glycosaminoglycans in the spleen, liver, kidney, cornea, brain and skeletal system. Bone marrow transplantation in mutant mice provides a source of normal enzyme ('cross-correction'), which substantially improves the clinical condition and extends the average lifespan to 18 months. Gene therapy by transfer of a beta-glucuronidase gene into mutant haematopoietic stem cells is an alternative approach, but it is not known whether the low expression of vector-transferred genes in vivo would be sufficiently effective. Here we show that retroviral vector-mediated transfer of the gene to mutant stem cells results in long-term expression of low levels of beta-glucuronidase which partially corrects the disease by reducing lysosomal storage in liver and spleen.