Mucopolysaccharidosis IIIB Mice Research Articles

Activities of (Poly)phenolic Antioxidants and Other Natural Autophagy Modulators in the Treatment of Sanfilippo Disease: Remarkable Efficacy of Resveratrol in Cellular and Animal Models

Sanfilippo disease, caused by mutations in the genes encoding heparan sulfate (HS) (a glycosaminoglycan; GAG) degradation enzymes, is a mucopolysaccharidosis (MPS), which is also known as MPS type III, and is characterized by subtypes A, B, C, and D, depending on identity of the dysfunctional enzyme. The lack of activity or low residual activity of an HS-degrading enzyme leads to excess HS in the cells, impairing the functions of different types of cells, including neurons. The disease usually leads to serious psychomotor dysfunction and death before adulthood. In this work, we show that the use of molecules known as dietary (poly)phenolic antioxidants and other natural compounds known as autophagy activators (genistein, capsaicin, curcumin, resveratrol, trehalose, and calcitriol) leads to accelerated degradation of accumulated HS in the fibroblasts of all subtypes of MPS III. Both the cytotoxicity tests we performed and the available literature data indicated that the use of selected autophagy inducers was safe. Since it showed the highest effectivity in cellular models, resveratrol efficacy was tested in experiments with a mouse model of MPS IIIB. Urinary GAG levels were normalized in MPS IIIB mice treated with 50 mg/kg/day resveratrol for 12 weeks or longer. Behavioral tests indicated complete correction of hyperactivity and anxiety in these animals. Biochemical analyses indicated that administration of resveratrol caused autophagy stimulation through an mTOR-independent pathway in the brains and livers of the MPS IIIB mice. These results indicate the potential use of resveratrol (and possibly other autophagy stimulators) in the treatment of Sanfilippo disease.

Targeted Metabolomic Analysis of a Mucopolysaccharidosis IIIB Mouse Model Reveals an Imbalance of Branched-Chain Amino Acid and Fatty Acid Metabolism.

Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids (BCAAs: Val, Ile, and Leu), aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease.

Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy.

Mucopolysaccharidosis IIIB is a paediatric lysosomal storage disease caused by deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU), involved in the degradation of the glycosaminoglycan heparan sulphate. Absence of NAGLU leads to accumulation of partially degraded heparan sulphate within lysosomes and the extracellular matrix, giving rise to severe CNS degeneration with progressive cognitive impairment and behavioural problems. There are no therapies. Haematopoietic stem cell transplant shows great efficacy in the related disease mucopolysaccharidosis I, where donor-derived monocytes can transmigrate into the brain following bone marrow engraftment, secrete the missing enzyme and cross-correct neighbouring cells. However, little neurological correction is achieved in patients with mucopolysaccharidosis IIIB. We have therefore developed an ex vivo haematopoietic stem cell gene therapy approach in a mouse model of mucopolysaccharidosis IIIB, using a high-titre lentiviral vector and the myeloid-specific CD11b promoter, driving the expression of NAGLU (LV.NAGLU). To understand the mechanism of correction we also compared this with a poorly secreted version of NAGLU containing a C-terminal fusion to IGFII (LV.NAGLU-IGFII). Mucopolysaccharidosis IIIB haematopoietic stem cells were transduced with vector, transplanted into myeloablated mucopolysaccharidosis IIIB mice and compared at 8 months of age with mice receiving a wild-type transplant. As the disease is characterized by increased inflammation, we also tested the anti-inflammatory steroidal agent prednisolone alone, or in combination with LV.NAGLU, to understand the importance of inflammation on behaviour. NAGLU enzyme was substantially increased in the brain of LV.NAGLU and LV.NAGLU-IGFII-treated mice, with little expression in wild-type bone marrow transplanted mice. LV.NAGLU treatment led to behavioural correction, normalization of heparan sulphate and sulphation patterning, reduced inflammatory cytokine expression and correction of astrocytosis, microgliosis and lysosomal compartment size throughout the brain. The addition of prednisolone improved inflammatory aspects further. Substantial correction of lysosomal storage in neurons and astrocytes was also achieved in LV.NAGLU-IGFII-treated mice, despite limited enzyme secretion from engrafted macrophages in the brain. Interestingly both wild-type bone marrow transplant and prednisolone treatment alone corrected behaviour, despite having little effect on brain neuropathology. This was attributed to a decrease in peripheral inflammatory cytokines. Here we show significant neurological disease correction is achieved using haematopoietic stem cell gene therapy, suggesting this therapy alone or in combination with anti-inflammatories may improve neurological function in patients.

Near-Complete Correction of Profound Metabolomic Impairments Corresponding to Functional Benefit in MPS IIIB Mice after IV rAAV9-hNAGLU Gene Delivery

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with complex CNS and somatic pathology due to a deficiency in α-N-acetylglucosaminidase (NAGLU). Using global metabolic profiling by mass spectrometry targeting 361 metabolites, this study detected significant decreases in 225 and increases in six metabolites in serum samples from 7-month-old MPS IIIB mice, compared to wild-type (WT) mice. The metabolic disturbances involve virtually all major pathways of amino acid, peptide (58/102), carbohydrate (18/28), lipid (111/139), nucleotide (12/24), energy (2/9), vitamin and cofactor (11/16), and xenobiotic (11/28) metabolism. Notably, the reduced metabolites included eight essential amino acids, vitamins (C, E, B2, and B6), and neurotransmitters (serotonin, glutamate, aspartate, tryptophan, and N-acetyltyrosine). The metabolic impairments appear to emerge early during disease progression before the age of 2months. Importantly, the restoration of NAGLU activity with an intravenous (i.v.) injection of rAAV9-hNAGLU vector led to near-complete correction of all serum metabolite abnormalities, with 201 (87%) metabolites normalized and 30 (13%) over-corrected. While the mechanisms are unclear, our data demonstrate that the lack of NAGLU activity triggers profound functional metabolic disturbances in MPS IIIB. These metabolic impairments respond well to a systemic rAAV9-hNAGLU gene delivery, supporting the surrogate biomarker potential of serum metabolomic profiles for MPS IIIB therapies.

A GLP-Compliant Toxicology and Biodistribution Study: Systemic Delivery of an rAAV9 Vector for the Treatment of Mucopolysaccharidosis IIIB.

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU gene delivery in WT C57BL/6 mice at 1 × 10(14) vg/kg and 2 × 10(14) vg/kg (n = 30/group, M:F = 1:1), and non-GLP testing in MPS IIIB mice at 2 × 10(14) vg/kg. Importantly, no adverse clinical signs or chronic toxicity were observed through the 6 month study duration. The rAAV9-mediated rNAGLU expression was rapid and persistent in virtually all tested CNS and somatic tissues. However, acute liver toxicity occurred in 33% (5/15) WT males in the 2 × 10(14) vg/kg cohort, which was dose-dependent, sex-associated, and genotype-specific, likely due to hepatic rNAGLU overexpression. Interestingly, a significant dose response was observed only in the brain and spinal cord, whereas in the liver at 24 weeks postinfection (pi), NAGLU activity was reduced to endogenous levels in the high dose cohort but remained at supranormal levels in the low dose group. The possibility of rAAV9 germline transmission appears to be minimal. The vector delivery resulted in transient T-cell responses and characteristic acute antibody responses to both AAV9 and rNAGLU in all rAAV9-treated animals, with no detectable impacts on tissue transgene expression. This study demonstrates a generally safe and effective profile, and may have identified the upper dosing limit of rAAV9-CMV-hNAGLU via systemic delivery for the treatment of MPS IIIB.

78. An IND-Enabling GLP-Toxicology and Biodistribution Study Assessing Systemic rAAV9-hNAGLU Gene Delivery for Treating MPS IIIB: Genotype- and Sex-Specific Dose-Limiting Acute Liver Toxicity in Male Wild Type C57BL/6 Mice

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU administration in wt C57BL/6 mice (6-8wk-old), at 1e14 vg/kg or 2e14 vg/kg (n=30/group, M:F=1:1). These were 2 and 4-fold higher, respectively, than our proposed clinical high dose. No adverse clinical signs were observed during the entire 6mo study duration. NAGLU activity above wt levels and vector genomes were detected in all tested CNS and somatic tissues in rAAV-treated mice through 6mo. However, acute mortality was observed in 5 male mice at 2e14 vg/kg dose on Days 6 and 8 pi. All 5 animals exhibited liver discoloration correlating with severe hepatic necrosis and vacuolation, without detectable leukocyte infiltration. At 6, 12 and 24wk pi, minimal single cell hepatocyte necrosis and/or vacuolation, and minimal cardiomyopathy were noted in a dose-dependent manner, with slight transient elevation of ALT or AST at 6wk pi. T-cell responses to AAV9 capsid and rNAGLU were detected at 6wk pi, but partially and completely diminished at 12wk pi, respectively. ELISA showed antibody responses to both AAV9 and rNAGLU in treated animals. In response to the acute mortality in the GLP-tox test, we performed a 6-wk non-GLP study mimicking the GLP-tox study conditions in male MPS IIIB and wt littermates, to further assess the approach and obtain data prior to early mortality. We treated the mice with the same batch of rAAV9-CMV-hNAGLU at the high dose, 2e14 vg/kg, and performed necropsy at 5 days and 6wk pi (n=4/group). No mortality or clinical signs were observed in either rAAV9-treated MPS IIIB or wt mice. At Day 5 pi necropsy, of 4 rAAV9-treated male wt mice, 1 exhibited liver discoloration, correlating with multifocal, periportal and midzonal hepatocyte necrosis, and 2 showed minimal single cell hepatocyte necrosis. Minimal liver damage and elevation of ALT/AST in wt mice persisted to 6wk pi. However, no abnormal changes attributed to vector treatment were detected in the liver in MPS IIIB mice. Therefore, we have identified genotype- and sex-specific dose-limiting toxicity of systemic rAAV9-hNAGLU delivery, supporting a safe profile for our proposed approach for treating MPS IIIB in patients.

172. Correction of Profound Metabolic Impairments in MPS IIIB Mice by a Systemic rAAV9-hNAGLU Gene Delivery

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease caused by deficiency in α-N-acetylglucosaminidase (NAGLU) leading to complex CNS and somatic pathology. This study was designed to characterize the metabolic impairments in MPS IIIB mice, and assess their responsiveness to effective gene therapy treatment. Using mass spectrometry targeting 361 metabolites, we detected significant decreases in 225 metabolites, and increases in 6, in serum from 7mo-old MPS IIIB mice, compared to wt mice. The metabolic disturbances in MPS IIIB mice emerge early, and involve virtually all major pathways of amino acid, peptide (58/102), carbohydrate (18/28), lipid (111/139), nucleotide (12/24), energy (2/9), vitamin and co-factors (11/16) and xenobiotics (11/28) metabolism. Notably, the reduced metabolites included 8 essential amino acids, Vitamins (C, E, B2, B6) and neurotransmitters (serotonin (5HT, glutamate, tryptophan, and N-acetyltyrosin). Importantly, the restoration of NAGLU activity with an IV injection of rAAV9-hNAGLU vector led to the efficient correction of these metabolic abnormalities, with 184 metabolites (79.7%) normalized, 6 (2.6%) partially corrected and 41 (17.7%) over-corrected. While the mechanisms are unclear, our data demonstrates that the lack of NAGLU activity triggers profound metabolic disturbances in MPS IIIB, leading to severely compromised functions in virtually all metabolic pathways. These metabolic impairments respond well to restoration of NAGLU activity using an effective gene therapy approach, supporting the biomarker potential of serum metabolomic profiles for MPS IIIB.

Amyloidosis, Synucleinopathy, and Prion Encephalopathy in a Neuropathic Lysosomal Storage Disease: The CNS-Biomarker Potential of Peripheral Blood

Mucopolysaccharidosis (MPS) IIIB is a devastating neuropathic lysosomal storage disease with complex pathology. This study identifies molecular signatures in peripheral blood that may be relevant to MPS IIIB pathogenesis using a mouse model. Genome-wide gene expression microarrays on pooled RNAs showed dysregulation of 2,802 transcripts in blood from MPS IIIB mice, reflecting pathological complexity of MPS IIIB, encompassing virtually all previously reported and as yet unexplored disease aspects. Importantly, many of the dysregulated genes are reported to be tissue-specific. Further analyses of multiple genes linked to major pathways of neurodegeneration demonstrated a strong brain-blood correlation in amyloidosis and synucleinopathy in MPS IIIB. We also detected prion protein (Prnp) deposition in the CNS and Prnp dysregulation in the blood in MPS IIIB mice, suggesting the involvement of Prnp aggregation in neuropathology. Systemic delivery of trans-BBB-neurotropic rAAV9-hNAGLU vector mediated not only efficient restoration of functional α-N-acetylglucosaminidase and clearance of lysosomal storage pathology in the central nervous system (CNS) and periphery, but also the correction of impaired neurodegenerative molecular pathways in the brain and blood. Our data suggest that molecular changes in blood may reflect pathological status in the CNS and provide a useful tool for identifying potential CNS-specific biomarkers for MPS IIIB and possibly other neurological diseases.

Biochemical evidence of the effects of SBC-103, a recombinant human alpha-N-acetylglucosaminidase in a mucopolysaccharidosis IIIB mouse model using an improved analytical method for substrate quantification

Biochemical evidence of the effects of SBC-103, a recombinant human alpha-N-acetylglucosaminidase in a mucopolysaccharidosis IIIB mouse model using an improved analytical method for substrate quantification

Peripheral Nervous System Neuropathology and Progressive Sensory Impairments in a Mouse Model of Mucopolysaccharidosis IIIB

The lysosomal storage pathology in Mucopolysaccharidosis (MPS) IIIB manifests in cells of virtually all organs. However, it is the profound role of the neurological pathology that leads to morbidity and mortality in this disease, and has been the major challenge to developing therapies. To date, MPS IIIB neuropathologic and therapeutic studies have focused predominantly on changes in the central nervous system (CNS), especially in the brain, and little is known about the disease pathology in the peripheral nervous system (PNS). This study demonstrates characteristic lysosomal storage pathology in dorsal root ganglia affecting neurons, satellite cells (glia) and Schwann cells. Lysosomal storage lesions were also observed in the myoenteric plexus and submucosal plexus, involving enteric neurons with enteric glial activation. Further, MPS IIIB mice developed progressive impairments in sensory functions, with significantly reduced response to pain stimulation that became detectable at 4–5 months of age as the disease progressed. These data demonstrate that MPS IIIB neuropathology manifests not only in the entire CNS but also the PNS, likely affecting both afferent and efferent neural signal transduction. This study also suggests that therapeutic development for MPS IIIB may benefit from targeting the entire nervous system.

Correction of Neurological Disease of Mucopolysaccharidosis IIIB in Adult Mice by rAAV9 Trans-Blood–Brain Barrier Gene Delivery

The greatest challenge in developing therapies for mucopolysaccharidosis (MPS) IIIB is to achieve efficient central nervous system (CNS) delivery across the blood-brain barrier (BBB). In this study, we used the novel ability of adeno-associated virus serotype 9 (AAV9) to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMV-hNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to the correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system (PNS) targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 months) neurological benefits in MPS IIIB mice, resulting in significant improvement in behavioral performance, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases.

Correction of neurological disease of Mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood-brain-barrier gene delivery

The greatest challenge in developing therapies for mucopolysaccharidosis (MPS) IIIB is to achieve efficient central nervous system (CNS) delivery across the blood–brain barrier (BBB). In this study, we used the novel ability of adeno-associated virus serotype 9 (AAV9) to cross the BBB from the vasculature to achieve long-term global CNS, and widespread somatic restoration of α-N-acetylglucosaminidase (NAGLU) activity. A single intravenous (IV) injection of rAAV9-CMV-hNAGLU, without extraneous treatment to disrupt the BBB, restored NAGLU activity to normal or above normal levels in adult MPS IIIB mice, leading to the correction of lysosomal storage pathology in the CNS and periphery, and correction of astrocytosis and neurodegeneration. The IV delivered rAAV9 vector also transduced abundant neurons in the myenteric and submucosal plexus, suggesting peripheral nervous system (PNS) targeting. While CNS entry did not depend on osmotic disruption of the BBB, it was significantly enhanced by pretreatment with an IV infusion of mannitol. Most important, we demonstrate that a single systemic rAAV9-NAGLU gene delivery provides long-term (>18 months) neurological benefits in MPS IIIB mice, resulting in significant improvement in behavioral performance, and extension of survival. These data suggest promising clinical potential using the trans-BBB neurotropic rAAV9 vector for treating MPS IIIB and other neurogenetic diseases.

Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.

Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response

BackgroundRecently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic.MethodsLymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system.ResultsOur results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFα, IFNα and Ifi30, and intraparenchymal lymphocyte infiltration.ConclusionsOur data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis.

Restoration of central nervous system α‐N‐acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno‐associated viral type 2 vector delivery

Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

Serum MIP‐1 α level: a biomarker for the follow‐up of lentiviral therapy in mucopolysaccharidosis IIIB mice

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by deficiency of alpha-N-acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement, with high mortality rates. Although some therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available. Moreover, assessing therapeutic efficacy is challenged by the lack of markers to for progression and severity. In this study, we examined the effect of brain-directed lentiviral (LV) gene therapy on serum levels of macrophage inflammatory protein 1 alpha (MIP-1alpha) and brain-derived neurotrophic factor (BDNF) proteins in the murine model of MPS IIIB to identify novel serum biomarkers. The cytokine MIP-1alpha was elevated in MPS IIIB mouse serum, and following gene therapy, it was reduced to normal levels. For neurotrophin BDNF, the difference in serum levels between MPS IIIB and normal mice was not statistically significant; after LV gene therapy, an increase in protein was found in treated mice, although the values were not statistically significant. Our studies suggest MIP-1alpha as the first serum biomarker that could be used to monitor disease progression and treatment for MPS IIIB disease.

Circadian rhythm and suprachiasmatic nucleus alterations in the mouse model of mucopolysaccharidosis IIIB

Mucopolysaccharidosis IIIB (MPSIIIB) is a lysosomal storage disease characterised by progressive central nervous system degeneration in patients, with death usually in the late teens. Serious behavioural problems have been reported in children at the early stages of the disease, such as hyperactivity and severe sleep disturbances, which suggest alterations in circadian rhythms. We investigated the circadian rhythm of locomotor activity of young and old MPSIIIB mice, under a 24-h light–dark (LD) cycle and under constant darkness (DD), and also examined neuropeptide expression in the suprachiasmatic nucleus (SCN), site of the principal biological pacemaker. We show that MPSIIIB mice have higher activity levels during the light (resting) phase of the LD cycle, together with weaker circadian rhythms, and a longer active phase due to a late peak of activity, in both LD and DD. In addition, young MPSIIIB mice showed shorter phase delays in response to a light pulse in DD. Increased lysosomal storage, neuroinflammation and changes in the expression of Arginine Vasopressin and Vasointestinal Polypeptide, two circadian neuropeptides, were observed in the SCN, which may be in part responsible for the changes in circadian behaviour observed in MPSIIIB mice. These findings suggest an alteration of the circadian system in MPSIIIB mice, and may inform better clinical management of circadian, sleep and behavioural disturbances in patients with MPSIII.

Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice

The presence of the blood-brain barrier (BBB) presents the most critical challenge in therapeutic development for mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological manifestation, because of alpha-N-acetylglucosaminidase (NaGlu) deficiency. Earlier, we showed a global central nervous system (CNS) transduction in mice by mannitol-facilitated entry of intravenous (IV)-delivered recombinant adeno-associated viral serotype 2 (rAAV2) vector. In this study, we optimized the approach and showed that the maximal transduction in the CNS occurred when the rAAV2 vector was IV injected at 8 min after mannitol administration, and was approximately 10-fold more efficient than IV delivery of the vector at 5 or 10 min after mannitol infusion. Using this optimal (8 min) regimen, a single IV infusion of rAAV2-CMV-hNaGlu vector is therapeutically beneficial for treating the CNS disease of MPS IIIB in adult mice, with significantly extended survival, improved behavioral performance, and reduction of brain lysosomal storage pathology. The therapeutic benefit correlated with maximal delivery to the CNS, but not peripheral tissues. This milestone data shows the first effective gene delivery across the BBB to treat CNS disease. The critical timing of vector delivery and mannitol infusion highlights the important contribution of this pretreatment to successful intervention, and the long history of safe use of mannitol in patients bodes well for its application in CNS gene therapy.

Innate and adaptive immune activation in the brain of MPS IIIB mouse model

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

Early Neurodegeneration Progresses Independently of Microglial Activation by Heparan Sulfate in the Brain of Mucopolysaccharidosis IIIB Mice

BackgroundIn mucopolysaccharidosis type IIIB, a lysosomal storage disease causing early onset mental retardation in children, the production of abnormal oligosaccharidic fragments of heparan sulfate is associated with severe neuropathology and chronic brain inflammation. We addressed causative links between the biochemical, pathological and inflammatory disorders in a mouse model of this disease.Methodology/Principal FindingsIn cell culture, heparan sulfate oligosaccharides activated microglial cells by signaling through the Toll-like receptor 4 and the adaptor protein MyD88. CD11b positive microglial cells and three-fold increased expression of mRNAs coding for the chemokine MIP1α were observed at 10 days in the brain cortex of MPSIIIB mice, but not in MPSIIIB mice deleted for the expression of Toll-like receptor 4 or the adaptor protein MyD88, indicating early priming of microglial cells by heparan sulfate oligosaccharides in the MPSIIIB mouse brain. Whereas the onset of brain inflammation was delayed for several months in doubly mutant versus MPSIIIB mice, the onset of disease markers expression was unchanged, indicating similar progression of the neurodegenerative process in the absence of microglial cell priming by heparan sulfate oligosaccharides. In contrast to younger mice, inflammation in aged MPSIIIB mice was not affected by TLR4/MyD88 deficiency.Conclusions/SignificanceThese results indicate priming of microglia by HS oligosaccharides through the TLR4/MyD88 pathway. Although intrinsic to the disease, this phenomenon is not a major determinant of the neurodegenerative process. Inflammation may still contribute to neurodegeneration in late stages of the disease, albeit independent of TLR4/MyD88. The results support the view that neurodegeneration is primarily cell autonomous in this pediatric disease.