Lentiviral Gene Therapy for Mucopolysaccharidosis II with Tagged Iduronate 2-Sulfatase Prevents Life-Threatening Pathology in Peripheral Tissues But Fails to Correct Cartilage.

Deficiency of iduronate 2-sulfatase (IDS) causes Mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder characterized by systemic accumulation of glycosaminoglycans (GAGs), leading to a devastating cognitive decline and life-threatening respiratory and cardiac complications. We previously found that hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) employing tagged IDS with insulin-like growth factor 2 (IGF2) or ApoE2, but not receptor-associated protein minimal peptide (RAP12x2), efficiently prevented brain pathology in a murine model of MPS II. In this study, we report on the effects of HSPC-LVGT on peripheral pathology and we analyzed IDS biodistribution. We found that HSPC-LVGT with all vectors completely corrected GAG accumulation and lysosomal pathology in liver, spleen, kidney, tracheal mucosa, and heart valves. Full correction of tunica media of the great heart vessels was achieved only with IDS.IGF2co gene therapy, while the other vectors provided near complete (IDS.ApoE2co) or no (IDSco and IDS.RAP12x2co) correction. In contrast, tracheal, epiphyseal, and articular cartilage remained largely uncorrected by all vectors tested. These efficacies were closely matched by IDS protein levels following HSPC-LVGT. Our results demonstrate the capability of HSPC-LVGT to correct pathology in tissues of high clinical relevance, including those of the heart and respiratory system, while challenges remain for the correction of cartilage pathology.

Tagged IDS causes efficient and engraftment-independent prevention of brain pathology during lentiviral gene therapy for Mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (OMIM 309900) is a lysosomal storage disorder caused by iduronate 2-sulfatase (IDS) deficiency and accumulation of glycosaminoglycans, leading to progressive neurodegeneration. As intravenously infused enzyme replacement therapy cannot cross the blood-brain barrier (BBB), it fails to treat brain pathology, highlighting the unmet medical need to develop alternative therapies. Here, we test modified versions of hematopoietic stem and progenitor cell (HSPC)-mediated lentiviral gene therapy (LVGT) using IDS tagging in combination with the ubiquitous MND promoter to optimize efficacy in brain and to investigate its mechanism of action. We find that IDS tagging with IGF2 or ApoE2, but not RAP12x2, improves correction of brain heparan sulfate and neuroinflammation at clinically relevant vector copy numbers. HSPC-derived cells engrafted in brain show efficiencies highest in perivascular areas, lower in choroid plexus and meninges, and lowest in parenchyma. Importantly, the efficacy of correction was independent of the number of brain-engrafted cells. These results indicate that tagged versions of IDS can outperform untagged IDS in HSPC-LVGT for the correction of brain pathology in MPS II, and they imply both cell-mediated and tag-mediated correction mechanisms, including passage across the BBB and increased uptake, highlighting their potential for clinical translation.

Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy.

OBJECTIVE: To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II. STUDY DESIGN: Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue. RESULTS: Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10 years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity in vitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers. CONCLUSIONS: Patients with the neuronopathic form and lack of IDS protein expression were most at risk to develop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity in vitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels.

Broad variation in phenotypes for common GAA genotypes in Pompe disease.

Patients with the common c.-32-13T > G/null GAA genotype have a broad variation in age at symptom onset, ranging from early childhood to late adulthood. Phenotypic variation for other common GAA genotypes remains largely unexplored. Here, we analyzed variation in age at symptom onset for the most common GAA genotypes using the updated and extended Pompe GAA variant database. Patients with the c.2647-7G > A/null genotype invariably presented symptoms at adulthood, while the c.-32-13T > G/null, c.546G > T/null, c.1076-22T > G/null, c.2238G > C/null, and c.2173C > T/null genotypes led to presentations from early childhood up to late adulthood. The c.1309C > T/null genotype was associated with onset at early to late childhood. Symptom onset shifted toward higher ages in homozygous patients. These findings indicate that a broad variation in symptom onset occurs for various common GAA genotypes, suggesting the presence of modifying factors. We identified three new compound heterozygous c.-32-13T > G/null patients who carried the genetic modifier c.510C > T and who showed symptom onset at childhood. While c.510C > T acted by lowering GAA enzyme activity, other putative genetic modifiers did not at the group level, suggesting that these act in trans on processes downstream of GAA enzyme activity.

Anesthesiological approach to the floppy child.

Children with symptoms of hypotonia (reduction of postural tone of lower limbs and trunk with or without changes in phasic tone) are frequently anesthetized for diagnostic and therapeutic interventions. This review outlines the underlying causes and classifications, and the anesthesiologic pre- and peri-operative management of hypotonic children. Hypotonia may have a large range of etiologies that are categorized into central and peripheral hypotonia. A multidisciplinary approach towards the (differential) diagnosis of the underlying cause of the symptoms in cooperation with a pediatrician and/or pediatric neurologist is emphasized. Anesthetic management involves the anticipation of an increased risk in difficult airway management because of macroglossia, reduced mouth opening, obesity and limited neck mobility, which increases with age. There are no specific restrictions towards the use of intravenous or inhalational anesthetics. Short acting opioids and hypnotics, avoiding neuromuscular blockade, and locoregional techniques are preferred. Most patients are sensitive to the cardiac and depressive effects of anesthetics and all dystrophic myopathies are considered at risk of malignant hyperthermia. Depolarizing neuromuscular blockers are contraindicated. The use of a peripheral nerve stimulator is recommended to detect the severity of muscle relaxation before extubating. Accurate control and management of IV fluids, electrolytes and temperature is mandatory. Adequate postoperative pain treatment is essential to limit stress and metabolic alteration. Preferably a locoregional technique is used to reduce the increased risk of respiratory depression. A multidisciplinary preoperative approach taking into account the differential diagnosis of the underlying disease of the floppy child is recommended.

Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience.

Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI.

Identification and characterization of disease-associated variants in monogenic disorders is an important aspect of diagnosis, genetic counseling, prediction of disease severity, and development of therapy. However, the effects of disease-associated variants on pre-mRNA splicing and mRNA degradation are difficult to predict and often missed. Here we present a generic assay for unbiased identification and quantification of arylsulfatase B (ARSB) mRNA for molecular diagnosis of patients with mucopolysaccharidosis VI (MPS VI). We found that healthy control individuals have inefficient ARSB splicing because of natural skipping of exon 5 and inclusion of two pseudoexons in introns 5 and 6. Analyses of 12 MPS VI patients with 10 different genotypes resulted in identification of a 151-bp intron inclusion caused by the c.1142+2T>C variant and detection of low ARSB expression from alleles with the c.629A>G variant. A special case showed skipping of exon 4 and low ARSB expression. Although no disease-associated DNA variant could be identified in this patient, the molecular diagnosis could be made based on RNA. These results highlight the relevance of RNA-based analyses to establish a molecular diagnosis of MPS VI. We speculate that inefficient natural splicing of ARSB may be a target for therapy based on promotion of canonical splicing.

A genetic modifier of symptom onset in Pompe disease.

BACKGROUND: Neonatal screening for Pompe disease is complicated by difficulties in predicting symptom onset in patients with the common c.-32-13T>G (IVS1) variant/null (i.e. fully deleterious) acid α-glucosidase (GAA) genotype. This splicing variant occurs in 90% of Caucasian late onset patients, and is associated with a broad range of symptom onset. METHODS: We analyzed a cohort of 143 compound heterozygous and 10 homozygous IVS1 patients, and we assessed ages at symptom onset, the presence of cis-acting single nucleotide variants (SNVs), and performed splicing analysis and enzyme activity assays. FINDINGS: In compound heterozygous IVS1 patients, the synonymous variant c.510C>T was uniquely present on the IVS1 allele in 9/33 (27%) patients with childhood onset, but was absent from 110 patients with onset in adulthood. GAA enzyme activity was lower in fibroblasts from patients who contained c.510C>T than it was in patients without c.510C>T. By reducing the extent of leaky wild-type splicing, c.510C>T modulated aberrant splicing caused by the IVS1 variant. The deleterious effect of c.510C>T was also found in muscle cells, the main target cells in Pompe disease. In homozygous IVS1 patients, the c.510C>T variant was absent in 4/4 (100%) asymptomatic individuals and present in 3/6 (50%) symptomatic patients. In cells from homozygous IVS1 patients, c.510C>T caused reduced leaky wild-type splicing. INTERPRETATION: c.510C>T is a genetic modifier in compound heterozygous and homozygous IVS1 patients. This finding is important for neonatal screening programs for Pompe disease. FUND: This work was funded by grants from Sophia Children's Hospital Foundation (SSWO, grant S17-32) and Metakids (2016-063).

Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arrays.

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.

Perioperative management of children with glycogen storage disease type II-Pompe disease.

BACKGROUND: Pompe disease is a rare metabolic disorder caused by a deficiency of the lysosomal enzyme acid α-glucosidase. Glycogen accumulation damages skeletal, cardiac, and smooth muscles, causing a progressive and debilitating muscle weakness and cardiomyopathy. As life expectancy has much improved since the introduction of enzyme replacement therapy an increasing number of patients are referred for surgical procedures. Due to the potential cardiopulmonary complications, these patients form a high-risk group for the anesthesiologist. AIMS: In this study, we investigated the incidence of perioperative complications in children with Pompe disease treated in our hospital since the introduction of enzyme replacement therapy. METHODS: Anesthetic and perioperative data of children with Pompe disease treated between 1999 and 2015 in the Erasmus MC-Sophia Children's Hospital, University Medical Centre, Rotterdam, The Netherlands, were collected, retrospectively. RESULTS: Of the 65 children with Pompe disease, 34 patients underwent in total 77, mostly low-risk, surgical procedures. Twenty-one children had the classic infantile form and 13 had a nonclassic presentation of Pompe disease. In 13 (16.8%) procedures, 1 or more perioperative complications occurred. Perioperative desaturation was the main complication (12.9%), followed by arrhythmia (3.8%) and heart failure requiring diuretic treatment (2.6%). One child died 2 days postoperatively, but this was considered unrelated to the procedure. CONCLUSION: Despite the potentially high anesthetic risk for children with Pompe disease under enzyme replacement therapy, the incidence of perioperative complications in our study was relatively low. Our data suggest that with proper precautionary measures and a critical choice of timing of the operation, general anesthesia in children with Pompe disease could be relatively safe nowadays.

Up to five years experience with 11 mucopolysaccharidosis type VI patients.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is a rare progressive metabolic disorder characterized by coarse facial features, hepatosplenomegaly, restrictive pulmonary function, cardiac abnormalities and stiff joints. The disease is caused by a deficiency of the lysosomal enzyme N-acetyl galactosamine 4-sulfatase which leads to glycosaminoglycan (GAG) storage in various tissues. It presents as a clinical spectrum with varying disease progressions and severities. While the phases I/II/III studies proved the effectiveness of enzyme-replacement therapy (ERT) with recombinant human arylsulfatase B, long-term data are still scarce. Over treatment periods ranging from 1.3 to 5.4 years, this prospective open-label follow-up study in 11 Dutch mucopolysaccharidosis type VI patients (age 2-18 years) showed that ERT had significant positive effects on cardiac-wall diameters (IVSd and LVMI), left and right shoulder flexions (p<0.001), liver size and spleen size (p<0.001), urinary GAG excretion (p<0.001), and the scales of quality of life (motor functioning and body functioning). ERT did not affect cardiac valve regurgitation or hearing function; HRQoL decreased slightly in two domains ('anxiety' and 'negative emotions'), and patients with the rapid and slow progressive forms of the disease differed with regard to baseline GAG excretion and GAG decrease during treatment. In conclusion, ERT had an effect on several clinical parameters. This effect was established in an open cohort of young mucopolysaccharidosis type VI patients.

Hearing loss in infantile Pompe's disease and determination of underlying pathology in the knockout mouse.

Hearing deficit occurs in several lysosomal storage disorders but has so far not been recognized as a symptom of Pompe's disease (glycogen storage disease type II). We discovered quite unexpectedly 30-90 dB hearing loss in four infants with Pompe's disease, who participated in a study on the safety and efficacy of enzyme replacement therapy. Three other patients with juvenile Pompe's disease did not have this symptom. The ABR (auditory brainstem response) thresholds but not the interpeak latency times were increased. This pointed to middle or inner ear pathology rather than to involvement of the central auditory nervous system. The possible occurrence of cochlear pathology was supported by the absence of oto-acoustic emissions. We investigated this hypothesis in a knockout mouse model of Pompe's disease and found glycogen storage in the inner and outer hair cells of the cochlea, the supporting cells, the stria vascularis, and the spiral ganglion cells. We conclude that cochlear pathology is the most likely cause of hearing loss in infantile Pompe's disease and possibly a characteristic feature of this clinical subtype.

The natural course of infantile Pompe's disease: 20 original cases compared with 133 cases from the literature.

OBJECTIVE: Infantile Pompe's disease is a lethal cardiac and muscular disorder. Current developments toward enzyme replacement therapy are promising. The aim of our study is to delineate the natural course of the disease to verify endpoints of clinical studies. METHODS: A total of 20 infantile patients diagnosed by the collaborative Dutch centers and 133 cases reported in literature were included in the study. Information on clinical history, physical examination, and diagnostic parameters was collected. RESULTS: The course of Pompe's disease is essentially the same in the Dutch and the general patient population. Symptoms start at a median age of 1.6 months in both groups. The median age of death is 7.7 and 6 months, respectively. Five percent of the Dutch patients and 8% of all reported patients survive beyond 1 year of age. Only 2 patients from literature became older than 18 months. A progressive cardiac hypertrophy is characteristic for infantile Pompe's disease. The diastolic thickness of the left ventricular posterior wall and cardiac weight at autopsy increase significantly with age. Motor development is severely delayed and major developmental milestones are generally not achieved. For the Dutch patient group, growth deviates significantly from normal despite start of nasogastric tube feeding. Levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, or creatine kinase-myocardial band isoenzyme are typically elevated, although aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase increase significantly with age. The patients have fully deleterious mutations. Acid alpha-glucosidase activity is severely deficient. CONCLUSIONS: Survival, decrease of the diastolic thickness of the left ventricular posterior wall, and achievement of major motor milestones are valid endpoints for therapeutic studies of infantile Pompe's disease. Mutation analysis and measurement of the alpha-glucosidase activity should be part of the enrollment program.

Morphological changes in muscle tissue of patients with infantile Pompe's disease receiving enzyme replacement therapy.

Pompe's disease (glycogen storage disease type II) is an autosomal recessive myopathy caused by lysosomal alpha-glucosidase deficiency. Enzyme replacement therapy (ERT) is currently under development for this disease. We evaluated the morphological changes in muscle tissue of four children with infantile Pompe's disease who received recombinant human alpha-glucosidase from rabbit milk for 72 weeks. The patients were 2.5-8 months of age at entry. Prior to treatment, all patients showed lysosomal glycogen storage in skeletal and smooth muscle cells, vascular endothelium, Schwann cells, and perineurium. The first response to treatment was noticed in vascular endothelium and in peripheral nerves after 12 weeks of treatment at an enzyme dose of 15-20 mg/kg. Increasing the dose to 40 mg/kg led, after 72 weeks of treatment, to a reduction of glycogen storage and substantial improvement of muscle architecture in the least affected patient. Not all patients responded equally well, possibly due to differences in degree of glycogen storage and concomitant muscle pathology at the start of treatment. We conclude that intravenous administration of recombinant human alpha-glucosidase from rabbit milk can improve muscle morphology in classic infantile Pompe's disease when treatment is started before irreversible damage has occurred.

Enzyme therapy for Pompe disease: from science to industrial enterprise.

UNLABELLED: Pompe disease or glycogen storage disease type II (OMIM 232300) is a metabolic myopathy with a broad clinical spectrum. Generalised muscle weakness combined with cardiomegaly presents within the first 3 months after birth, if the lysosomal alpha-glucosidase (AGLU) deficiency is complete. Residual enzyme activity prevents cardiac involvement and delays onset of muscle weakness. Enzyme therapy, by intravenous administration of acid AGLU, aims to supplement the missing enzyme activity. At the SHS symposium on Glycogen Storage Diseases Type I and II, in Fulda, two interim accounts were given of studies on the efficacy of enzyme therapy for Pompe disease; one with recombinant human acid AGLU produced in Chinese hamster ovary cells and the other with the same enzyme produced in the milk of transgenic rabbits. CONCLUSION: this review focuses on the latter study, discusses the scientific, technological and commercial aspects of the enterprise, and addresses the prospects and challenges of enzyme therapy for Pompe disease.