Untreated Mucopolysaccharidosis Research Articles

A phase III clinical trial of GC1111 as an enzyme replacement therapy in previously untreated mucopolysaccharidosis type II (Hunter syndrome) patients: A double-blind, randomized, active-controlled (part 1) and open-labeled, historical placebo-controlled (part 2) study

A phase III clinical trial of GC1111 as an enzyme replacement therapy in previously untreated mucopolysaccharidosis type II (Hunter syndrome) patients: A double-blind, randomized, active-controlled (part 1) and open-labeled, historical placebo-controlled (part 2) study

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme β-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human β-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4mg/kg) or a high dose (20mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

Effects of lithium administration on vertebral bone disease in mucopolysaccharidosis I dogs.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14days to 6months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.

Long-term outcomes of patients with mucopolysaccharidosis VI treated with galsulfase enzyme replacement therapy since infancy

ObjectiveLong-term outcomes of patients with mucopolysaccharidosis (MPS) VI treated with galsulfase enzyme replacement therapy (ERT) since infancy were evaluated. MethodsThe study was a multicenter, prospective evaluation using data from infants with MPS VI generated during a phase 4 study (ASB-008; Clinicaltrials.govNCT00299000) and clinical data collected ≥5 years after completion of the study. ResultsParents of three subjects from ASB-008 (subjects 1, 2, and 4) provided written informed consent to participate in the follow-up study. One subject was excluded as consent was not provided. Subjects 1, 2, and 4 were aged 0.7, 0.3, and 1.1 years, respectively, at initiation of galsulfase and 10.5, 7.9, and 10.5 years, respectively, at follow-up. All subjects had classical MPS VI based on pre-treatment urinary glycosaminoglycans and the early onset of clinical manifestations. At follow-up, subject 4 had normal stature for age; subjects 1 and 2 had short stature, but height remained around the 90th percentile of growth curves for untreated classical MPS VI. Six-minute walk distance was normal for age/height in subjects 1 (550 m) and 4 (506 m), and reduced for subject 2 (340 m). Subject 2 preserved normal respiratory function, while percent predicted forced vital capacity and forced expiratory volume in 1 s decreased over time in the other subjects. Skeletal dysplasia was already apparent in all subjects at baseline and continued to progress. Cardiac valve disease showed mild progression in subject 1, mild improvement in subject 4, and remained trivial in subject 2. All subjects had considerably reduced pinch and grip strength at follow-up, but functional dexterity was relatively normal for age and there was limited impact on activities of daily living. Bruininks-Oseretsky Test of Motor Proficiency (BOT-2) results showed that subjects 2 and 4 had numerous fine and gross motor competencies. Corneal clouding progressed in all subjects, while progression of hearing impairment was variable. Liver size normalized from baseline in subjects 1 and 4, and remained normal in subject 2. ConclusionVery early and continuous ERT appears to slow down the clinical course of MPS VI, as shown by preservation of endurance, functional dexterity, and several fine and gross motor competencies after 7.7–9.8 years of treatment, and less growth impairment or progression of cardiac disease than could be expected based on the patients' classical phenotype. ERT does not seem to prevent progression of skeletal or eye disease in the long term.

Ex Vivo Gene Therapy Treats Bone Complications of Mucopolysaccharidosis Type II Mouse Models through Bone Remodeling Reactivation

Mucopolysaccharidosis type II is a disease caused by organ accumulation of glycosaminoglycans due to iduronate 2-sulfatase deficiency. This study investigated the pathophysiology of the bone complications associated with mucopolysaccharidosis II and the effect of lentivirus-mediated gene therapy of hematopoietic stem cells on bone lesions of mucopolysaccharidosis type II mouse models in comparison with enzyme replacement therapy. Bone volume, density, strength, and trabecular number were significantly higher in the untreated mucopolysaccharidosis type II mice than in wild-type mice. Accumulation of glycosaminoglycans caused reduced bone metabolism. Specifically, persistent high serum iduronate 2-sulfatase levels and release of glycosaminoglycans from osteoblasts and osteoclasts in mucopolysaccharidosis type II mice that had undergone gene therapy reactivated bone lineage remodeling, subsequently reducing bone mineral density, strength, and trabecular number to a similar degree as that observed in wild-type mice. Bone formation, resorption parameters, and mineral density in the diaphysis edge did not appear to have been affected by the irradiation administered as a pre-treatment for gene therapy. Hence, the therapeutic effect of gene therapy on the bone complications of mucopolysaccharidosis type II mice possibly outweighed that of enzyme replacement therapy in many aspects.

Prevalence of intestinal disease as terminal event in mucopolysaccharidosis type III – A study of 136 deceased patients

Since we previously observed that in patients with mucopolysaccharidosis (MPS) the storage of undegraded glycosaminoglycans (GAG) occurs from birth, in the present study we aimed to compare normal, untreated MPS I mice (knockout for alpha-l-iduronidase-IDUA), and MPS I mice treated with enzyme replacement therapy (ERT, Laronidase, 1.2 mg/kg every 2 weeks) started from birth (ERT-neo) or from 2 months of age (ERT-ad). All mice were sacrificed at 6 months. Both treatments were equally effective in normalizing GAG levels in the viscera but had no detectable effect on the joint. Heart function was also improved with both treatments. On the other hand, mice treated from birth presented better outcomes in the difficult-to-treat aortas and heart valves. Surprisingly, both groups had improvements in behavior tests, and normalization of GAG levels in the brain and IDUA injection resulted in detectable levels of enzyme in the brain tissue 1 h after administration. ERT-ad mice developed significantly more anti-IDUA-IgG antibodies, and mice that didn't develop antibodies had better performances in behavior tests, indicating that development of antibodies may reduce enzyme bioavailability. Our results suggest that ERT started from birth leads to better outcomes in the aorta and heart valves, as well as a reduction in antibody levels. Some poor vascularized organs, such as the joints, had partial or no benefit and ancillary therapies might be needed for patients. The results presented here support the idea that ERT started from birth leads to better treatment outcomes and should be considered whenever possible, a observation that gains relevance as newborn screening programs are being considered for MPS and other treatable lysosomal storage disorders.

Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured.Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively.Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.

Aortic Root Dilatation in Mucopolysaccharidosis I-VII.

The prevalence of aortic root dilatation (ARD) in mucopolysaccharidosis (MPS) is not well documented. We investigated aortic root measurements in 34 MPS patients at the Children’s Hospital of Orange County (CHOC). The diagnosis, treatment status, age, gender, height, weight and aortic root parameters (aortic valve annulus (AVA), sinuses of Valsalva (SoV), and sinotubular junction (STJ)) were extracted by retrospective chart review and echocardiographic measurements. Descriptive statistics, ANOVA, and paired post-hoc t-tests were used to summarize the aortic dimensions. Exact binomial 95% confidence intervals (CIs) were constructed for ARD, defined as a z-score greater than 2 at the SoV. The patient age ranged from 3.4–25.9 years (mean 13.3 ± 6.1), the height from 0.87–1.62 meters (mean 1.24 ± 0.21), and the weight from 14.1–84.5 kg (mean 34.4 ± 18.0). The prevalence of dilation at the AVA was 41% (14/34; 95% CI: 25%–59%); at the SoV was 35% (12/34; 95% CI: 20%–54%); and at the STJ was 30% (9/30; 95% CI: 15%–49%). The highest prevalence of ARD was in MPS IVa (87.5%). There was no significant difference between mean z-scores of MPS patients who received treatment with hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) vs. untreated MPS patients at the AVA (z = 1.9 ± 2.5 vs. z = 1.5 ± 2.4; p = 0.62), SoV (z = 1.2 ± 1.6 vs. z = 1.3 ± 2.2; p = 0.79), or STJ (z = 1.0 ± 1.8 vs. z = 1.2 ± 1.6; p = 0.83). The prevalence of ARD was 35% in our cohort of MPS I–VII patients. Thus, we recommend screening for ARD on a routine basis in this patient population.

Deleterious effects of interruption followed by reintroduction of enzyme replacement therapy on a lysosomal storage disorder

Temporary interruption of enzyme replacement therapy (ERT) in patients with different lysosomal storage disorders may happen for different reasons (adverse reactions, issues with reimbursement, logistic difficulties, and so forth), and the impact of the interruption is still uncertain. In the present work, we studied the effects of the interruption of intravenous ERT (Laronidase, Genzyme) followed by its reintroduction in mice with the prototypical lysosomal storage disorder mucopolysaccharidosis type I, comparing to mice receiving continuous treatment, untreated mucopolysaccharidosis type I mice, and normal mice. In the animals which treatment was temporarily interrupted, we observed clear benefits of treatment in several organs (liver, lung, heart, kidney, and testis) after reintroduction, but a worsening in the thickness of the aortic wall was detected. Furthermore, these mice had just partial improvements in behavioral tests, suggesting some deterioration in the brain function. Despite worsening is some disease aspects, urinary glycosaminoglycans levels did not increase during interruption, which indicates that this biomarker commonly used to monitor treatment in patients should not be used alone to assess treatment efficacy. The deterioration observed was not caused by the development of serum antienzyme antibodies. All together our results suggest that temporary ERT interruption leads to deterioration of function in some organs and should be avoided whenever possible.

Enzyme replacement therapy for mucopolysaccharidosis VI: Growth and pubertal development in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase.

BACKGROUND AND METHODS: Growth failure is characteristic of untreated mucopolysaccharidosis type VI (MPS VI: Maroteaux-Lamy syndrome). Growth was studied in fifty-six MPS VI patients (5 to 29 years old) prior to and for up to 240 weeks of weekly infusions of recombinant human arylsulfatase B (rhASB) at 1 mg/kg during Phase 1/2, Phase 2, Phase 3 or Phase 3 Extension clinical trials. Height, weight, and Tanner stage data were collected. Pooled data were analyzed to determine mean height increase by treatment week, growth impacts of pubertal status, baseline urinary GAG, and age at treatment initiation. Growth rate for approximately 2 years prior to and following treatment initiation was analyzed using longitudinal modeling. RESULTS: Mean height increased by 2.9 cm after 48 weeks and 4.3 cm after 96 weeks on enzyme replacement therapy (ERT). Growth on ERT was not correlated with baseline urinary GAG. Patients under 16 years of age showed greatest increases in height on treatment. Model results based on pooled data showed significant improvement in growth rate during 96 weeks of ERT when compared to the equivalent pretreatment time period. Delayed pubertal onset or progression was noted in 10 patients entering the clinical trials; all of whom showed progression of at least one Tanner stage during 2 years on ERT, and 6 of whom (60%) completed puberty. CONCLUSION: Analysis of mean height by treatment week and longitudinal modeling demonstrate significant increase in height and growth rate in MPS VI patients receiving long-term ERT. This impact was greatest in patients aged below 16 years. Height increase may result from bone growth and/or reduction in joint contractures. Bone growth and resolution of delayed puberty may be related to improvements in general health, bone cell health, nutrition, endocrine gland function and reduced inflammation.

The effect of neonatal gene therapy on skeletal manifestations in mucopolysaccharidosis VII dogs after a decade

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disease due to deficient activity of β-glucuronidase (GUSB), and results in glycosaminoglycan accumulation. Skeletal manifestations include bone dysplasia, degenerative joint disease, and growth retardation. One gene therapy approach for MPS VII involves neonatal intravenous injection of a gamma retroviral vector expressing GUSB, which results in stable expression in liver and secretion of enzyme into blood at levels predicted to be similar or higher to enzyme replacement therapy. The goal of this study was to evaluate the long-term effect of neonatal gene therapy on skeletal manifestations in MPS VII dogs. Treated MPS VII dogs could walk throughout their lives, while untreated MPS VII dogs could not stand beyond 6months and were dead by 2years. Luxation of the coxofemoral joint and the patella, dysplasia of the acetabulum and supracondylar ridge, deep erosions of the distal femur, and synovial hyperplasia were reduced, and the quality of articular bone was improved in treated dogs at 6 to 11years of age compared with untreated MPS VII dogs at 2years or less. However, treated dogs continued to have osteophyte formation, cartilage abnormalities, and an abnormal gait. Enzyme activity was found near synovial blood vessels, and there was 2% as much GUSB activity in synovial fluid as in serum. We conclude that neonatal gene therapy reduces skeletal abnormalities in MPS VII dogs, but clinically-relevant abnormalities remain. Enzyme replacement therapy will probably have similar limitations long-term.

Enzyme replacement therapy started at birth improves outcome in difficult-to-treat organs in mucopolysaccharidosis I mice

Since we previously observed that in patients with mucopolysaccharidosis (MPS) the storage of undegraded glycosaminoglycans (GAG) occurs from birth, in the present study we aimed to compare normal, untreated MPS I mice (knockout for alpha-l-iduronidase-IDUA), and MPS I mice treated with enzyme replacement therapy (ERT, Laronidase, 1.2mg/kg every 2weeks) started from birth (ERT-neo) or from 2months of age (ERT-ad). All mice were sacrificed at 6months. Both treatments were equally effective in normalizing GAG levels in the viscera but had no detectable effect on the joint. Heart function was also improved with both treatments. On the other hand, mice treated from birth presented better outcomes in the difficult-to-treat aortas and heart valves. Surprisingly, both groups had improvements in behavior tests, and normalization of GAG levels in the brain and IDUA injection resulted in detectable levels of enzyme in the brain tissue 1h after administration. ERT-ad mice developed significantly more anti-IDUA-IgG antibodies, and mice that didn't develop antibodies had better performances in behavior tests, indicating that development of antibodies may reduce enzyme bioavailability. Our results suggest that ERT started from birth leads to better outcomes in the aorta and heart valves, as well as a reduction in antibody levels. Some poor vascularized organs, such as the joints, had partial or no benefit and ancillary therapies might be needed for patients. The results presented here support the idea that ERT started from birth leads to better treatment outcomes and should be considered whenever possible, a observation that gains relevance as newborn screening programs are being considered for MPS and other treatable lysosomal storage disorders.

Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

Clear cells in the atrioventricular valves of infants with severe human mucopolysaccharidosis (Hurler syndrome) are activated valvular interstitial cells

Severe mucopolysaccharidosis type I (Hurler syndrome) is an autosomal recessive lysosomal storage disease of childhood that results in accumulation of glycosaminoglycans within cardiac valves and consequent valve dysfunction. Valve thickening in mucopolysaccharidosis type I (Hurler syndrome) is due, in part, to the presence of glycosaminoglycan-laden cells (the so-called "clear" or "Hurler" cells) within the valve that remain largely unstudied with respect to identity, origin, and function. We hypothesized that the "clear" or "Hurler" cells within the atrioventricular valves from individuals with untreated mucopolysaccharidosis type I are activated valvular interstitial cells. We performed routine and immunohistochemical staining on atrioventricular valves from two infants with untreated severe mucopolysaccharidosis type I (Hurler syndrome) and compared them to atrioventricular valve tissue from two age-matched and gender-matched normal infants. Despite the marked differences in their histological appearances, mucopolysaccharidosis type I valve cells have an immunohistochemical fingerprint identical to that of normal infant valvular interstitial cells. Both mucopolysaccharidosis type I valvular interstitial cells and normal infant valvular interstitial cells have the phenotype of activated myofibroblasts, as evidenced by positive staining for vimentin, smooth muscle actin, and metalloproteinase-9. However, the number of mucopolysaccharidosis type I valvular interstitial cells is significantly increased when compared to that of normal cells (P<.0031). Both mucopolysaccharidosis type I (Hurler syndrome) cells and normal valvular interstitial cells express CD34(+), a hematopoietic and capillary endothelial progenitor cell marker, suggesting a common response to activation. We conclude that "clear" or "Hurler" cells are valvular interstitial cells with the immunohistochemical phenotype of activated myofibroblasts and may be engaged, albeit ineffectively, in valve repair.

Enzyme replacement therapy for mucopolysaccharidosis VI: evaluation of long‐term pulmonary function in patients treated with recombinant human N‐acetylgalactosamine 4‐sulfatase

Pulmonary function is impaired in untreated mucopolysaccharidosis type VI (MPS VI). Pulmonary function was studied in patients during long-term enzyme replacement therapy (ERT) with recombinant human arylsulfatase B (rhASB; rhN-acetylgalactosamine 4-sulfatase). Pulmonary function tests prior to and for up to 240 weeks of weekly infusions of rhASB at 1 mg/kg were completed in 56 patients during Phase 1/2, Phase 2, Phase 3 and Phase 3 Extension trials of rhASB and the Survey Study. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and, in a subset of patients, maximum voluntary ventilation (MVV), were analyzed as absolute volume in liters. FEV1 and FVC showed little change from baseline during the first 24 weeks of ERT, but after 96 weeks, these parameters increased over baseline by 11% and 17%, respectively. This positive trend compared with baseline continued beyond 96 weeks of treatment. Improvements from baseline in pulmonary function occurred along with gains in height in the younger group (5.5% change) and in the older patient group (2.4% change) at 96 weeks. Changes in MVV occurred earlier within 24 weeks of treatment to approximately 15% over baseline. Model results based on data from all trials showed significant improvements in the rate of change in pulmonary function during 96 weeks on ERT, whereas little or no improvement was observed for the same time period prior to ERT. Thus, analysis of mean percent change data and longitudinal modeling both indicate that long-term ERT resulted in improvement in pulmonary function in MPS VI patients.

Inhibition of Glycosaminoglycan Synthesis Using Rhodamine B in a Mouse Model of Mucopolysaccharidosis Type IIIA

Reduction of an enzyme activity required for the lysosomal degradation of glycosaminoglycan (gag) chains will result in a mucopolysaccharidosis (MPS) disorder. Substrate deprivation therapy (SDT), a potential therapy option for MPS with residual enzyme activity, aims to reduce the synthesis of gag chains, the natural substrate for the deficient enzyme. Reduced substrate levels would balance the reduced level of enzyme in patient cells, resulting in normalized gag turnover. Rhodamine B, a nonspecific inhibitor, reduced gag synthesis in a range of normal and MPS cells and also decreased lysosomal storage of gag in MPS VI (72%) and MPS IIIA (60%) cells. Body weight gain of male MPS IIIA mice treated with 1 mg/kg rhodamine B was reduced compared with untreated MPS IIIA mice and was indistinguishable from that of normal mice. Liver size, total gag content, and lysosomal gag was reduced in treated MPS IIIA animals as was urinary gag excretion. Lysosomal gag content in the brain was also reduced by treatment. The alteration in MPS IIIA clinical pathology by rhodamine B, combined with the observation that treatment had no effect on the health of normal animals, demonstrates the potential for SDT in general as a therapy for MPS disorders.

856. Neonatal Liver-Directed Gene Therapy Markedly Reduces Bone Disease in MPS I

Mucopolysaccharidosis (MPS) is due to deficiencies in enzymes that degrade glycosaminoglycans (GAGs). MPS I is due to deficient α-L-iduronidase activity (IDUA) and results in the accumulation of heparin sulfate (HS) and dermatan sulfate (DS). MPS VII is due to deficient β-glucuronidase activity and results in the accumulation of chondroitin sulfate (CS) in addition to HS and DS. Both disorders result in dysostosis multiplex in patients, which is characterized by short and thick bones. MPS can be treated with liver-directed gene therapy in which hepatocytes continuously secrete enzyme with mannose 6-phosphate, which can be taken up from blood. Although hepatic gene therapy reduced many manifestations of MPS VII, bone disease has not been completely corrected. The purpose of this study was to evaluate the effect of neonatal retroviral vector (RV)-mediated gene therapy on bone disease in MPS I mice. In contrast to untreated MPS VII mice in which long bones have only 81% to 87% of normal length, untreated MPS I mice had normal bone lengths at 6 weeks and 8 months after birth. Normal lengths were likely due to the fact that untreated MPS I mice had much less lysosomal storage material than did MPS VII mice in cartilage cells of the growth plate, which plays a pivotal role in bone elongation. This is likely due to the fact that CS is the major GAG in chondrocytes, and CS only accumulates in MPS VII. For untreated MPS I mice, bone mineral density (BMD) was normal at 6 weeks, but markedly increased at 0.069 gm/cm2 at 8 months [normal is 0.054 (p < 0.0001)]. The long bones of untreated MPS I mice had an increased diameter and were sclerotic, and there was substantial storage in osteocytes. The canine IDUA cDNA was cloned into a retroviral vector (RV) with a strong liver promoter, and the RV was injected into newborn MPS I mice at a high [1 × 109 transducing units (TU)/kg] or a low (1 × 108 TU/kg) dose. This resulted in transduction of liver cells and stable serum IDUA activity in most animals for 8 months at 1241 ± 147 and 110 ± 31 units (U)/ml for the high and low dose animals, respectively. At 8 months, mice that received a high dose of RV had normal long bone diameters without sclerosis, normal BMD [0.054 gm/cm2 (p < 0.0001 vs. values in untreated MPS I)], and marked reduction in lysosomal storage in the osteocytes. Mice that received a low dose of RV had some increase in diameter and sclerosis of the long bones, although they were not as severe as untreated MPS I mice. Similarly, the BMD (0.060 gm/cm2) was partially increased. We conclude that bone disease is much less severe in untreated MPS I mice than in untreated MPS VII mice. This is likely due to the fact that CS does not accumulate in MPS I, and CS is high in cartilage and accumulates in MPS VII. However, MPS I mice still had marked thickening and sclerosis of the bone, which were prevented with a high dose of RV at birth. However, the low dose of RV was less effective. These data help to define the serum activity necessary to prevent bone disease in MPS I.

696. Comparative Study of Lentiviral Vector-Mediated Gene Therapy in Neonatal and Young-Adult MPS I Mice by a Single Intravenous Injection

Mucopolysaccharidosis (MPS) I is due to deficient activity of α-L-iduronidase (IDUA) and is the most common MPS syndrome. Hepatic gene therapy might permanently correct the clinical manifestations of this disorder by programming liver cells to secrete IDUA with mannose 6-phosphate (M6P) that could be taken up by cells throughout the rest of the body via the M6P receptor. The canine IDUA cDNA was cloned into a retroviral vector (RV) with a strong liver promoter, and the RV was injected into newborn MPS I mice at a high [1×109 transducing units (TU)/kg] or a low (1×108 TU/kg) dose. This resulted in transduction of liver cells and stable serum IDUA activity in most animals for 8 months at 1241+/-147 and 110+/-31 units (U)/ml, and DNA copy number of 0.17 and 0.014 copies of RV/cell, for the high (N=25) and low dose (N=18) animals, respectively. Three of 18 mice that received the low dose of RV lost expression over time, which appeared to be due to a cytotoxic T lymphocyte response. Although serum IDUA activity was much higher than in normal mice (1 U/ml) or untreated MPS I mice (<0.01 U/ml), only 3% of the IDUA in serum of RV-treated MPS I mice contained M6P. In contrast, 70% of the IDUA in normal mice contained M6P. This suggests that the canine IDUA may be a poor substrate for modification with M6P in murine cells. Nevertheless, for animals that received the high dose of RV, IDUA activity in most organs was at least as high as in normal mice, while IDUA activity in brain was 10% of normal. MPS I mice that received the high dose of RV had normal echocardiograms, electroretinograms (ERG), and auditory brainstem responses (ABR) at 8 months after birth. In the heart, untreated MPS I mice had aortic insufficiency, aortic dilatation [ascending aorta diameter was 3.1 mm for MPS I vs 1.8 mm in high dose RV (p<0.001)], and cardiomegaly [left ventricular internal diameter at diastole of 4.3 mm in MPS I vs. 3.6 mm in high dose RV (p=0.02)]. In visual testing, MPS I mice had a markedly reduced response to light on electroretinograms [the a wave was-124 microvolts in MPS I vs.-329 in high dose RV (p<0.0001) and the b wave was 452 microvolts in MPS I vs. 682 in high dose RV (p=0.002)]. In hearing testing, untreated MPS I mice had a markedly increased threshold for hearing at frequencies of sound of 5 mHz [84 decibels in MPS I vs. 42 for high dose RV (p<0.0001)], 10 mHz [76 decibels for MPS I vs. 24 for high dose RV (p<0.0001)], and 20 mHz [77 decibels in MPS I vs. 55 in high dose RV (p=0.01)]. MPS I mice that received a low dose of RV had no improvements in echocardiograms, and only partial improvements in ERG and ABR. Pathological improvements correlated with clinical improvements. We conclude that neonatal gene therapy with a high dose of RV expressing IDUA corrects the cardiac, visual, and hearing abnormalities of MPS I, while a low dose of RV was less effective. These data help to define a target goal for serum IDUA activity for correcting these major manifestations of disease in MPS I.

857. Intravenous Retroviral Gene Therapy in Neonatal Mucopolysaccharidosis VI Cats

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by deficient activity of N-acetylgalactosamine 4-sulfatase (4S) resulting in an inability to degrade the glycosaminoglycan (GAG) dermatan sulfate, which accumulates in the lysosomes. Clinical features include skeletal abnormalities, growth retardation, facial dysmorphia, and corneal clouding. Two MPS VI cats were injected intravenously at 4 days of age with a Moloney murine leukemia virus-based retroviral vector containing the human alpha-1-antitrypsin promoter, the feline 4S (f4S) cDNA (kindly provided by John J. Hopwood), and the woodchuck hepatitis virus posttranscriptional regulatory element. Both animals received 7.2X10E9 transducing units/kg of body weight. Stable f4S activity has been detected in serum throughout the study and the most recent values from samples taken at 6 months were 23 and 9 times normal. The f4S activities in liver biopsies taken at 5 months were 82 and 38 times normal and the concentration of GAG in the biopsies was reduced to normal levels, 1.2 and 1.8 ug/mg protein, respectively. In comparison, the GAG concentration in liver biopsies from the untreated MPS VI littermates was 34.9 and 20.6 ug/mg. The body weights of the treated cats are 130% and 122% that of their two untreated MPS VI sex-matched control littermates (average of 1.89 kg) and are not different from normal unrelated age- and sex-matched controls. There are profound differences in the appearance of the treated and untreated cats. The MPS VI controls have short ears and tails and their faces are broad and flat. They have joint stiffness and major locomotor difficulties. The treated cats have only mild facial dysmorphia, a much freer range of pelvic motion, near normal gaits, and are more physically active than their MPS VI littermates. The cat with the higher f4S activity is nearly indistinguishable from a phenotypically normal cat except for the eyes, as there was no significant improvement in corneal clouding. MPS VI is particularly suited to liver-directed therapy because unlike other MPS disorders, CNS involvement is minimal. Because MPS VI is a progressive disease, the full extent of the clinical benefit will only become evident over time. We will present a 10-month update, including radiographs comparing the skeletons of normal, treated, and untreated MPS VI cats.

Monitoring dose response of enzyme replacement therapy in feline mucopolysaccharidosis type VI by tandem mass spectrometry.

Mucopolysaccharidosis type VI is an inherited disorder of glycosaminoglycan metabolism characterized by organomegaly, corneal clouding, and skeletal dysplasia. Recent developments in the use of tandem mass spectrometry to measure sulfated mono- and disaccharides have enabled us to perform noninvasive, biochemical monitoring during therapy regimes in mucopolysaccharidosis type VI cats in addition to established methods of disease evaluation. In this study, mucopolysaccharidosis type VI animals were given high-dose (20 mg/kg) enzyme replacement therapy for the first month after birth followed by low doses (1 mg/kg) for a further 2 mo and were compared with animals maintained on 1 mg/kg enzyme replacement therapy for 3 mo. A sulfated monosaccharide (N-acetylhexosamine) and a sulfated disaccharide (N-acetylhexosamine-uronic acid) were elevated in MPS VI cat urine and blood. These markers showed a clear discrimination between the treatment groups during the first 4 wk of therapy: values in the high-dose group were close to normal whereas those in the low-dose group were only slightly lower than the untreated mucopolysaccharidosis type VI cats. However, within 2 mo of cessation of the high-dose therapy there was minimal difference in the oligosaccharide levels, with both groups lying between the untreated and unaffected cats. At the completion of the trial, subjective minor improvement was noted in overall physical disease features and also in lysosomal vacuolation in tissues from animals on the initial high-dose enzyme replacement therapy compared to the low-dose therapy animals. Initial high-dose therapy reduced storage load in the animals but had no lasting clinical benefit over continuous low-dose therapy.