Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome.

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients' CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

Otorhinolaryngological manifestations in 61 patients with mucopolysaccharidosis.

OBJECTIVES: The mucopolysaccharidoses (MPS) are inherited lysosomal storage disorders with multisystemic and highly variable clinical manifestation. ENT symptoms are common and early signs of MPS. The most common ENT diagnoses are chronic/recurrent rhinosinusitis, acute otitis media, otitis media with effusion, hearing loss and airway obstruction. METHODS: A single-centre retrospective chart review of 61 patients (36 M/25F) with different MPS subtypes (MPS I (n = 15), MPS II (n = 10), MPS III (n = 17), MPS IV (n = 15) and MPS VI (n = 4)) was conducted. The age of ENT presentation and frequency of ENT symptoms, surgeries and their distribution among MPS subtypes was studied. The relationship between ENT presentation, first ENT surgery and the age of diagnosis was also evaluated. RESULTS: Median age at the first ENT manifestation was 2.8 years, median age at MPS diagnosis 4.1 years. The great majority of patients (90%) manifested at least one ENT diagnosis; often before the diagnosis of MPS (75%). Chronic/recurrent rhinosinusitis was the most prevalent ENT diagnosis (77%), followed by upper airway obstruction (65%) and hearing loss (53%). Chronic/recurrent rhinosinusitis was the first ENT symptom to appear (median age 2.2 years), followed by otitis media with effusion (3.7 years) and hearing loss (4.5 years). At least one ENT surgery was performed in 57% of patients; in 69% before MPS diagnosis was established. Median age of the first ENT surgery was 4.1 years. ENT symptoms and surgical procedures were earliest present in MPS II. CONCLUSIONS: Our study documents high and early occurrence of various otolaryngologic symptoms in MPS and thus highlights the role of ENT specialist in prompt diagnosis of these rare diseases and their long-term management.

Aortopathies in mouse models of Pompe, Fabry and Mucopolysaccharidosis IIIB lysosomal storage diseases.

INTRODUCTION: Lysosomal storage diseases (LSDs) are rare inherited metabolic diseases characterized by an abnormal accumulation of various toxic materials in the cells as a result of enzyme deficiencies leading to tissue and organ damage. Among clinical manifestations, cardiac diseases are particularly important in Pompe glycogen storage diseases (PD), in glycosphingolipidosis Fabry disease (FD), and mucopolysaccharidoses (MPS). Here, we evaluated the occurrence of aortopathy in knock out (KO) mouse models of three different LSDs, including PD, FD, and MPS IIIB. METHODS: We measured the aortic diameters in 15 KO male mice, 5 for each LSD: 5 GLA-/- mice for FD, 5 NAGLU-/- mice for MPS IIIB, 5 GAA-/- mice for PD, and 15 wild type (WT) mice: 5 for each strain. In order to compare the aortic parameters between KO and WT mice deriving from the same colonies, different diameters were echocardiographically measured: aortic annulus, aortic sinus, sino-tubular junction, ascending aorta, aortic arch and descending aorta. Storage material content and aortic defects of the KO mice were also analyzed by histology, when available. RESULTS: Compared to their correspondent WT mice: GAA-/- mice showed greater diameters of ascending aorta (1.61mm vs. 1.11mm, p-value = 0.01) and descending aorta (1.17mm vs 1.02mm, p-value 0.04); GLA-/- mice showed greater diameters of aortic annulus (1.35mm vs. 1.22mm, p-value = 0.01), sinus of Valsalva (1.6mm vs. 1.38mm, p-value<0.01), ascending aorta (1.57mm vs. 1.34mm, p-value<0.01), aortic arch (1.36mm vs. 1.22mm, p-value = 0.03) and descending aorta (1.29mm vs. 1.11mm, p-value<0.01); NAGLU-/- mice showed greater diameters of sinus of Valsalva (1.46mm vs. 1.31mm, p-value = 0.05), ascending aorta (1.42mm vs. 1.29mm, p-value<0.01), aortic arch (1.34mm vs. 1.28mm, p-value<0.01) and descending aorta (1.18mm vs. 1.1mm, p-value 0.01). CONCLUSIONS: We evaluated for the first time the aortic diameters in 3 LSD mouse models and identified different aortopathy patterns, in concordance with recent human findings. Our results are relevant in view of using KO mouse models for efficiently testing the efficacy of new therapies on distinct cardiovascular aspects of LSDs.

Avoiding diagnostic delay for mucopolysaccharidosis IIIB: do not overlook common clues such as wheezing and otitis media.

Mucopolysaccharidosis IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disorder. In comparison to Hurler syndrome (MPS I) and Hunter syndrome (MPS II), characteristic facial and physical features tend to be milder and progression of neurological symptoms may initially be slower. Obvious neurological and behavioural symptoms may not appear until age 2-6 years, but once they begin, progression is relentless, leading to death by the early 20s. Although there is currently no known cure for MPS IIIB, enzyme replacement clinical trials are showing hope for delay in the progression of symptoms. Early diagnosis is therefore necessary before neurological symptoms have progressed. In our case, MPS IIIB was diagnosed at an early age because recurrent wheezing and otitis media in conjunction with hepatomegaly were recognised as more than trivial findings. A thorough examination and a definitive proactive decision to perform a liver biopsy resulted in early diagnosis of a rare 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.

The Murine Model of Mucopolysaccharidosis IIIB Develops Cardiopathies over Time Leading to Heart Failure.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal disease due to the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The disease is characterized by mild somatic features and severe neurological disorders. Very little is known on the cardiac dysfunctions in MPS IIIB. In this study, we used the murine model of MPS IIIB (NAGLU knockout mice, NAGLU(-/-)) in order to investigate the cardiac involvement in the disease. Echocardiographic analysis showed a marked increase in left ventricular (LV) mass, reduced cardiac function and valvular defects in NAGLU(-/-) mice as compared to wild-type (WT) littermates. The NAGLU(-/-) mice exhibited a significant increase in aortic and mitral annulus dimension with a progressive elongation and thickening of anterior mitral valve leaflet. A severe mitral regurgitation with reduction in mitral inflow E-wave-to-A-wave ratio was observed in 32-week-old NAGLU(-/-) mice. Compared to WT mice, NAGLU(-/-) mice exhibited a significantly lower survival with increased mortality observed in particular after 25 weeks of age. Histopathological analysis revealed a significant increase of myocardial fiber vacuolization, accumulation of HS in the myocardial vacuoles, recruitment of inflammatory cells and collagen deposition within the myocardium, and an increase of LV fibrosis in NAGLU(-/-) mice compared to WT mice. Biochemical analysis of heart samples from affected mice showed increased expression levels of cardiac failure hallmarks such as calcium/calmodulin-dependent protein kinase II, connexin43, α-smooth muscle actin, α-actinin, atrial and brain natriuretic peptides, and myosin heavy polypeptide 7. Furthermore, heart samples from NAGLU(-/-) mice showed enhanced expression of the lysosome-associated membrane protein-2 (LAMP2), and the autophagic markers Beclin1 and LC3 isoform II (LC3-II). Overall, our findings demonstrate that NAGLU(-/-) mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.

Repeated administrations of human umbilical cord blood cells improve disease outcomes in a mouse model of Sanfilippo syndrome type III B.

Sanfilippo syndrome type III B (MPS III B) is an inherited disorder characterized by a deficiency of α-N-acetylglucosaminidase (Naglu) enzyme leading to accumulation of heparan sulfate in lysosomes and severe neurological deficits. We have previously shown that a single administration of human umbilical cord mononuclear cells (hUCB MNCs) into Naglu knockout mice decreased behavioral abnormalities and tissue pathology. In this study, we tested whether repeated doses of hUCB MNCs would be more beneficial than a single dose of cells. Naglu mice at 3 months of age were randomly assigned to either a Media-only group or one of three hUCB MNC treatment groups--single low dose (3 × 10(6) cells), single high dose (1.8 × 10(7) cells), or multiple doses (3 × 10(6) cells monthly for 6 months) delivered intravenously; cyclosporine was injected intraperitoneally to immune suppress the mice for the duration of the study. An additional control group of wild-type mice was also used. We measured anxiety in an open field test and cognition in an active avoidance test prior to treatment and then at monthly intervals for 6 months. hUCB MNCs restored normal anxiety-like behavior in these mice (p < 0.001). The repeated cell administrations also restored hippocampal cytoarchitecture, protected the dendritic tree, decreased GM3 ganglioside accumulation, and decreased microglial activation, particularly in the hippocampus and cortex. These data suggest that the neuroprotective effect of hUCB MNCs can be enhanced by repeated cell administrations.

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.

High prevalence of femoral head necrosis in Mucopolysaccharidosis type III (Sanfilippo disease): a national, observational, cross-sectional study.

BACKGROUND: Sanfilippo disease, or Mucopolysaccharidosis type III (MPS III), is a lysosomal storage disorder and a member of the mucopolysaccharidoses (MPSs). MPS III is clinically characterized by progressive neurodegeneration. Skeletal disease is not felt to be an important clinical component in MPS III patients, unlike in the other MPSs. We conducted radiographic studies in a relatively large group of MPS III patients and detected a high prevalence of osteonecrosis of the femoral head (ONFH). METHODS: Thirty-three patients were included in the study. All the patients underwent an X-ray of the pelvis (anteroposterior view). All the X-rays were evaluated by a single, blinded radiologist using a modified Ficat classification system for ONFH (the stages ranged from 0 to IV, with increasing stages signifying more severe abnormalities). Clinical symptoms possibly related to hip disease were recorded. The patients were divided into different phenotypes based on mutational analysis and their plasma heparan sulfate (HS) levels. RESULTS: In 21 of the 33 patients, the disease severity could be predicted by genotype. In 11 of the 12 remaining patients, the phenotype could be assessed via the plasma HS levels. Eight patients (24%) exhibited signs of ONFH (Ficat stage≥I), and 6 (75%) of them had bilateral changes. None of the patients with attenuated MPS III (n=14) had ONFH. In 6 of the patients with a severe phenotype, hip dysplasia was detected as an additional finding. The 7 patients with Ficat stages ≥ II reported hip pain. CONCLUSIONS: Femoral head disease, which resembles ONFH, is common in patients with the severe MPS III phenotype. An evaluation of hip disease should be included in follow-up visits with MPS III patients.

A retrospective audit of anesthetic techniques and complications in children with mucopolysaccharidoses.

OBJECTIVES AND AIMS: To document the incidence of difficult airway management and difficult intubation in the era of replacement therapy for Australian children with mucopolysaccharidosis (MPS). BACKGROUND: Medical treatment for MPS has developed significantly since 1980's with a large number of patients now being offered either bone marrow transplant or enzyme replacement. The impact of these therapies on the incidence of difficult airway management has not been adequately documented. Similarly, anesthesia techniques and airway devices have been developed, which are thought to have greatly increased the safety of managing these patients under anesthesia but their role in children with MPS has not been systematically described. METHODS: A retrospective chart review of 17 patients with MPS who had received anesthetics at the Royal Children's Hospital during the time frame January 1998-January 2011. The primary outcome was the incidence of difficult or failed intubation. Secondary outcomes were the relationship between the incidence of difficult intubation and treatment with enzyme replacement therapy (ERT) or bone marrow transplantation. RESULTS: Seventeen patients received 141 anesthetics for 214 procedures. Difficult face mask ventilation occurred in 20 anesthetics (14.2%). Difficult intubation occurred in 40 anesthetics (25%). Failed intubation occurred in two cases (1.6%).The incidence of difficult intubation was 12% in MPS I, 35% MPS II, 86.7% in MPS VI, and 0% in MPS III and IV. CONCLUSIONS: Hematopoietic stem cell transplantation prior to 2 years of age reduces the incidence of difficult mask ventilation and difficult intubation in children with MPS I. ERT was initiated late in the clinical course of MPS II and VI and induced improvements in upper airway patency but did not reduce the incidence of difficult airway management.

Hemiatrophy of the spinal cord in a patient with mucopolysaccharidosis type IIIB.

The authors describe a girl with mucopolysaccharidosis type IIIB (Sanfilippo disease). She presented with speech delay, macrocephaly, and left lower limb hypoatrophy. Her brain and spinal cord imaging revealed diffuse cystic brain lesions and hemiatrophy of her spinal cord on the left (thoracic levels 11/12). She had marked reduction of her α-N-acetylglucosaminidase activity assay consistent with the diagnosis of mucopolysaccharidosis type IIIB. Mutational analysis showed 2 mutations on exon 6 of the α-N-acetylglucosaminidase gene, both of which were identified in her parents. At 10 years of age the girl had minor learning difficulties and mild behavioral problems. Her spinal cord hemiatrophy, in association with mucopolysaccharidosis type III, has not previously been described in the literature.

[Postoperative complications in a man with San Filippo syndrome anesthetized for multiple tooth extraction]. / Conducta perioperatoria en un paciente con síndrome de San Filippo sometido a exodoncia múltiple.

San Filippo syndrome is a hereditary lysosomal disorder resulting in the accumulation of mucopolysaccharides (mucopolysaccharidosis type II). A deficit in the enzyme required to break down heparan sulfate leads to its deposition in the connective tissue of many organs, particularly the brain, liver, heart, and spleen. The first symptoms-including mental deterioration, dimorphism, and behavioral changes such as hyperkinesis and aggressivity-present in childhood. Because this rare disorder has many anesthetic implications, we report the case of a 20-year-old man with San Filippo syndrome who underwent multiple tooth extraction under general combined anesthesia and a block of the second and third branches of the trigeminal nerve. This anesthetic combination provided satisfactory surgical conditions and recovery from anesthesia was rapid. Following surgery the patient developed a respiratory infection that led to severe respiratory failure and death.

Precocious puberty in Sanfilippo IIIA disease: diagnosis and follow-up of two new cases.

To date the association between mucopolysaccharidosis (MPS) IIIA and precocious puberty has been found in only three polish patients. We observed two children affected by MPS IIIA with central precocious puberty (CPP) both treated with GnRH agonists. The occurrence of CPP in both patients with MPS IIIA suggests that it is necessary to look for an association between the two conditions. The follow-up of our two patients leads us to believe also that GnRH agonist treatment can have a beneficial effect on final height and probably on the improvement of behavioural problems.

Mucopolysaccharidosis IIIB, cerebral vasculopathy and recurrent subdural hematoma.

Mucopolysaccharidoses are characterized by involvement of multiple organs, including blood vessels. We present a case of mucopolysaccharidosis IIIB admitted with recurrent subdural hematoma; further evaluation and dural biopsy revealed an association with cerebral vasculopathy.

[Case of Sanfilippo syndrome type B and Wilson disease born to unrelated parents].

A 5-year-old boy visited a hospital because of macrocephalus, mental retardation and hepatic dysfunction, and was suspected to have Wilson's disease since his father had this disease. The serum level of ceruloplasmin was low, but urinary copper excretion was not increased markedly. He was treated with D-penicillamine. He was then reffered to our hospital because of his facial features suggesting mucopolysaccharidosis. Based on mucopolysacchariduria and the deficiency of N-acetylglucosaminidase, the diagnosis of Sanfilippo syndrome type B was made. Molecular analyses identified him as a compound heterozygote for both the ATP7B (A844V/2659delG) and alpha-N-acetylglucosaminidase (V241M/R482W) genes, responsible for Wilson's disease and Sanfilippo syndrome type B, respectively. Although born to non-consanguineous parents, he had two rare autosomal recessive diseases. In this case, liver dysfunction was attributed to Wilson's disease, and mental retardation to Sanfilippo syndrome.

Secondary skeletal involvement in Sanfilippo syndrome.

BACKGROUND: Sanfilippo syndrome, or mucopolysaccharidosis (MPS) type III, is a rare lysosomal storage disease, resulting from errors in the catabolism of heparan sulphate. AIM: To evaluate bone turnover and bone mineral density (BMD) in MPS type III patients. DESIGN: Clinical and observational study. METHODS: We evaluated serum markers of bone formation or resorption, and measured BMD using dual-energy X-ray absorptiometry (DEXA), in three patients with MPS type III. RESULTS: Serum vitamin D were low, and BMDs greatly reduced at lumbar and femoral sites, indicating the possibility of osteoporosis and osteomalacia. DISCUSSION: These skeletal effects probably result from nutritional deficiencies and inability to walk, rather than from the genetic defect itself. Secondary skeletal involvement in patients with MPS type III may represent a considerable cause of morbidity, and requires interventions to reduce the risk of pathological fractures.

Anaestethic problems in Sanfilippo syndrome. A rare case of adult patient.

The authors report the case of a female patient (41 years old) affected by mucopolysaccharidosis type III or Sanfilippo syndrome submitted to a gynecologic surgical procedure and describe the main anesthesiologic problems. A sub-arachnoid anesthesia with hyperbaric Bupivacain 0.5% was used. This technique proved to be safe and convenient without peri- and postoperative complications.