Mucopolysaccharidosis Type VII Research Articles

Clinical course of sly syndrome (mucopolysaccharidosis type VII)

BackgroundMucopolysaccharidosis VII (MPS VII) is an ultra-rare disease characterised by the deficiency of β-glucuronidase (GUS). Patients’ phenotypes vary from severe forms with hydrops fetalis, skeletal dysplasia and mental retardation to...

Evidence of early bone response after initiation of enzyme replacement therapy in a 3year old patient with MPS VII

Evidence of early bone response after initiation of enzyme replacement therapy in a 3year old patient with MPS VII

Reduced chondrocyte proliferation and hypertrophy contribute to delayed endochondral bone formation in murine mucopolysaccharidosis VII

Reduced chondrocyte proliferation and hypertrophy contribute to delayed endochondral bone formation in murine mucopolysaccharidosis VII

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.

Systems biology study of mucopolysaccharidosis using a human metabolic reconstruction network.

Mucopolysaccharidosis (MPS) is a group of lysosomal storage diseases (LSD), characterized by the deficiency of a lysosomal enzyme responsible for the degradation of glycosaminoglycans (GAG). This deficiency leads to the lysosomal accumulation of partially degraded GAG. Nevertheless, deficiency of a single lysosomal enzyme has been associated with impairment in other cell mechanism, such as apoptosis and redox balance. Although GAG analysis represents the main biomarker for MPS diagnosis, it has several limitations that can lead to a misdiagnosis, whereby the identification of new biomarkers represents an important issue for MPS. In this study, we used a system biology approach, through the use of a genome-scale human metabolic reconstruction to understand the effect of metabolism alterations in cell homeostasis and to identify potential new biomarkers in MPS. In-silico MPS models were generated by silencing of MPS-related enzymes, and were analyzed through a flux balance and variability analysis. We found that MPS models used approximately 2286 reactions to satisfy the objective function. Impaired reactions were mainly involved in cellular respiration, mitochondrial process, amino acid and lipid metabolism, and ion exchange. Metabolic changes were similar for MPS I and II, and MPS III A to C; while the remaining MPS showed unique metabolic profiles. Eight and thirteen potential high-confidence biomarkers were identified for MPS IVB and VII, respectively, which were associated with the secondary pathologic process of LSD. In vivo evaluation of predicted intermediate confidence biomarkers (β-hexosaminidase and β-glucoronidase) for MPS IVA and VI correlated with the in-silico prediction. These results show the potential of a computational human metabolic reconstruction to understand the molecular mechanisms this group of diseases, which can be used to identify new biomarkers for MPS.

33. Ex Vivo Gene Therapy for Lysosomal Storage Disease in the CNS: Patient iPSC-Derived Neural Stem Cells Correct Pathology in the MPS VII Mouse Brain

Neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) may offer an immunologically compatible delivery vehicle for therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using patient-derived cells in a well-characterized mouse model of the lysosomal storage disease, MPS VII. Fibroblasts from a patient with MPS VII were reprogrammed into iPSCs, and differentiated into multi-potent NSCs. Patient-derived NSCs were able to engraft across the rostrocaudal axis following transplantation in neonatal NOD/SCID mice but were present in very low densities, similar to that seen with primary NSCs. We used a PiggyBac-based transposon vector to deliver a functional version of the missing GUSB gene to patient NSCs, which then expressed normal levels of enzymatically active GUSB. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult MPS VII mice reversed neuropathology in a zone surrounding the grafts, while control contralateral grafts did not. The patient-derived iPSC-NSCs had similar engraftment properties as iPSC-NSCs from a normal human subject, and both lines engrafted similarly in normal and MPS VII NOD-SCID mouse brains. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues.

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage.

Microwave assisted CLARITY for whole organ surveillance

CO RR EC TE D P RO OF transposon system for gene therapy of both MPS I and MPS VII in adult mice. Although more than 99% of transgene expression comes from the liver following hydrodynamic delivery, restoration of deficient enzyme activity in other organs can be achieved through metabolic cross-correction whereby either IDUA or GUSB enters the circulation and is distributed to other tissues. However, the efficacy of hydrodynamic delivery to treat animals larger than mice has been discouraging. We have tested the use of balloon-catheters for intravascular infusion and expression of SB transposons in the liver of dogs as a large animal model for human therapy. Ballooncatheters were introduced under fluoroscopic guidance through either the jugular vein or the femoral vein and positioned in the inferior vena cava for occlusion of venous outflow of the retrograde infusion through the left hepatic vein. Infusions were 10 s in duration and delivered a volume of DNA solution equal to three times the estimated blood volume of the left side of the liver. Using canine secreted alkaline phosphatase (cSEAP) as a reporter we have developed effective protocols for infusion of transposons into the liver of dogs as a scale-up for preclinical gene therapy studies in large animal models of human disease. We tested our protocol on MPS VIIdeficient young dogs by hydrodynamically infusing two plasmids, (1) with a T2-SB transposon containing a canine GUSB gene under transcriptional direction of a CAGS or Liver Specific Promoter (LSP) and (2) with the SB100 transposase regulated by the CMV early promoter (Figure). Duration of sustained GUSB expression in the two dogs and transient increases in the levels of specific liver enzymes will be presented.

Chondroitin 6-sulfate as a novel biomarker for mucopolysaccharidosis IVA and VII

Chondroitin 6-sulfate as a novel biomarker for mucopolysaccharidosis IVA and VII

Partial rescue of mucopolysaccharidosis type VII mice with a lifelong engraftment of allogeneic stem cells in utero.

In utero hematopoietic cell transplantation (IUHCT) has been performed in Mucopolysaccharidosis Type VII (MPSVII) mice, but a lifelong engraftment of allogeneic donor cells has not been achieved. In this study, we sought to confirm a lifelong engraftment of allogeneic donor cells immunologically matched to the mother and to achieve partial rescue of phenotypes in the original MPSVII strain through IUHCT by intravenous injection. We performed in vitro fertilization in a MPSVII murine model and transferred affected embryos to ICR/B6-GFP surrogate mothers in cases where fetuses receiving IUHCT were all homozygous. Lineage-depleted cells from ICR/B6-GFP mice were injected intravenously at E14.5. Chimerism was confirmed by flow cytometry at 4 weeks after birth, and β-glucuronidase activity in serum and several phenotypes were assessed at 8 weeks of age or later. Donor cells in chimeric mice from ICR/B6-GFP mothers were detected at death, and were confirmed in several tissues including the brains of sacrificed chimeric mice. Although the serum enzyme activity of chimeric mice was extremely low, the engraftment rate of donor cells correlated with enzyme activity. Furthermore, improvement of bone structure and rescue of reproductive ability were confirmed in our limited preclinical study. We confirmed the lifelong engraftment of donor cells in an original immunocompetent MPSVII murine model using intravenous IUHCT with cells immunologically matched to the mother without myeloablation, and the improvement of several phenotypes.

Performance of the dimethylmethylene blue test for the diagnosis of mucopolysaccharidoses

The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders resulting from inherited defects in the breakdown of glycosaminoglycans (GAG). Affected children develop progressive connective tissue changes and/or progressive psychomotor retardation. Treatment is available for some disorders and is most effective when started early. Early manifestations are non-specific and diagnosis is usually reliant on the finding of an increased GAG excretion performed as part of a metabolic screen, using a dye binding assay with dimethylmethylene blue (DMB). Some reports have questioned the reliability of this technique for detecting MPS disorders. We have performed this test since 1997 and present a review of the performance of the assay in our hands. Results Over 70,000 urines have been tested with 54 confirmed positive cases (14 MPS I, 14 MPS II, 21 MPS III, 2 MPS IV, 2 MPS VI and 1 MPS VII). One MPS III patient had a result at the upper limit of normal and one adult patient with MPS IV had a result in the normal range. The remainder were all clearly abnormal. Conclusion The DMB test is a reliable first line investigation for MPS but negative results should be reported with a comment regarding discussion with the laboratory if there are strong clinical suspicions of an MPS disorder.

Bilateral single-site intracerebral injection of a nonpathogenic herpes simplex virus-1 vector decreases anxiogenic behavior in MPS VII mice

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.

The effect of Tlr4 and/or C3 deficiency and of neonatal gene therapy on skeletal disease in mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder caused by the deficiency of the enzyme β-glucuronidase (Gusb(-/-)) and results in glycosaminoglycan (GAG) accumulation. Skeletal abnormalities include stunted long bones and bone degeneration. GAGs have been hypothesized to activate toll-like receptor 4 (Tlr4) signaling and the complement pathway, resulting in upregulation of inflammatory cytokines that suppress growth and cause degeneration of the bone. Gusb(-/-) mice were bred with Tlr4- and complement component 3 (C3)-deficient mice, and the skeletal manifestations of the doubly- and triply-deficient mice were compared to those of purebred Gusb(-/-) mice. Radiographs showed that purebred Gusb(-/-) mice had shorter tibias and femurs, and wider femurs, compared to normal mice. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice. The glenoid cavity and humerus were scored on a scale from 0 (normal) to +3 (severely abnormal) for dysplasia and bone irregularities, and the joint space was measured. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice, and their joint space remained abnormally wide. Gusb(-/-) mice treated neonatally with an intravenous retroviral vector (RV) had thinner femurs, longer legs, and a narrowed joint space compared with untreated purebred Gusb(-/-) mice, but no improvement in glenohumeral degeneration. We conclude that Tlr4- and/or C3-deficiency fail to ameliorate skeletal abnormalities, and other pathways may be involved. RV treatment improves some but not all aspects of bone disease. Radiographs may be an efficient method for future evaluation, as they readily show glenohumeral joint abnormalities.

First human treatment with investigational rhGUS enzyme replacement therapy in an advanced stage MPS VII patient

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is a very rare lysosomal storage disease caused by a deficiency of the enzyme β-glucuronidase (GUS), which is required for the degradation of three glycosaminoglycans (GAGs): dermatan sulfate, heparan sulfate, and chondroitin sulfate. Progressive accumulation of these GAGs in lysosomes leads to increasing dysfunction in numerous tissues and organs. Enzyme replacement therapy (ERT) has been used successfully for other MPS disorders, but there is no approved treatment for MPS VII. Here we describe the first human treatment with recombinant human GUS (rhGUS), an investigational therapy for MPS VII, in a 12-year old boy with advanced stage MPS VII. Despite a tracheostomy, nocturnal continuous positive airway pressure, and oxygen therapy, significant pulmonary restriction and obstruction led to oxygen dependence and end-tidal carbon dioxide (ETCO2) levels in the 60–80mmHg range, eventually approaching respiratory failure (ETCO2 of 100mmHg) and the need for full-time ventilation. Since no additional medical measures could improve his function, we implemented experimental ERT by infusing rhGUS at 2mg/kg over 4h every 2weeks for 24weeks. Safety was evaluated by standard assessments and observance for any infusion associated reactions (IARs). Urinary GAG (uGAG) levels, pulmonary function, oxygen dependence, CO2 levels, cardiac valve function, liver and spleen size, and growth velocity were assessed to evaluate response to therapy. rhGUS infusions were well tolerated. No serious adverse events (SAEs) or IARs were observed. After initiation of rhGUS infusions, the patient's uGAG excretion decreased by more than 50%. Liver and spleen size were reduced within 2weeks of the first infusion and reached normal size by 24weeks. Pulmonary function appeared to improve during the course of treatment based on reduced changes in ETCO2 after off-ventilator challenges and a reduced oxygen requirement. The patient regained the ability to eat orally, gained weight, and his energy and activity levels increased. Over 24weeks, treatment with every-other-week infusions of rhGUS was well tolerated with no SAEs, IARs, or hypersensitivity reactions and was associated with measurable improvement in objective clinical measures and quality of life.

Lentiviral-mediated gene therapy results in sustained expression of β-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII.

A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.

Umbilical Cord Mesenchymal Stem Cells Suppress Host Rejection: The Role Of The Glycocalyx

Umbilical cord mesenchymal stem cells (UMSCs) have unique immunosuppressive properties enabling them to evade host rejection and making them valuable tools for cell therapy. We previously showed that human UMSCs survive xenograft transplantation and successfully correct the corneal clouding defects associated with the mouse model for the congenital metabolic disorder mucopolysaccharidosis VII. However, the precise mechanism by which UMSCs suppress the immune system remains elusive. This study aimed to determine the key components involved in the ability of the UMSCs to modulate the inflammatory system and to identify the inflammatory cells that are regulated by the UMSCs. Our results show that human UMSCs transplanted into the mouse stroma 24 h after an alkali burn suppress the severe inflammatory response and enable the recovery of corneal transparency within 2 weeks. Furthermore, we demonstrated in vitro that UMSCs inhibit the adhesion and invasion of inflammatory cells and also the polarization of M1 macrophages. UMSCs also induced the maturation of T-regulatory cells and led to inflammatory cell death. Moreover, UMSCs exposed to inflammatory cells synthesize a rich extracellular glycocalyx composed of the chondroitin sulfate-proteoglycan versican bound to a heavy chain (HC)-modified hyaluronan (HA) matrix (HC-HA). This matrix also contains TNFα-stimulated gene 6 (TSG6), the enzyme that transfers HCs to HA, and pentraxin-3, which further stabilizes the matrix. Our results, both in vivo and in vitro, show that this glycocalyx confers the ability for UMSCs to survive the host immune system and to regulate the inflammatory cells.

Aspects neurologiques des mucopolysaccharidoses

Mucopolysaccharidoses (MPS) are progressive multisystem lysosomal storage diseases caused by defective catabolism of complex molecules, namely the glycosaminoglycans and their consequent accumulation in tissues. Of the 7 clinical types representing 11 different enzyme deficiencies, only MPS III (Sanfilippo syndromes A, B, C and D) and severe forms of MPS I (Hurler's syndrome), MPS II (severe Hunter syndrome) and MPS VII (severe Sly syndrome) give rise to progressive cerebral disease. Neurosensory complications (hearing impairment, retinopathy and optic atrophy) can also occur in some types. Carpal (or even tarsal) tunnel syndrome is a common complication of the MPS, except for MPS III and MPS IV. Hydrocephalus due to abnormal circulation or resorption of cerebrospinal fluid, chronic spinal compression may occur as complications of MPS I, II and VII and also in MPS IV and VI. Atlanto-axial instability may be observed in type IV and I.

Corrective GUSB Transfer to the Canine Mucopolysaccharidosis VII Brain

Severe deficiency in lysosomal β-glucuronidase (β-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in β-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, β-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector

Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.

Central Nervous System Delivery of Helper-Dependent Canine Adenovirus Corrects Neuropathology and Behavior in Mucopolysaccharidosis Type VII Mice

Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing β-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in β-glucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of β-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease.