Long-term efficacy and safety of vestronidase alfa enzyme replacement therapy in pediatric subjects < 5 years with mucopolysaccharidosis VII

Long-term efficacy and safety of vestronidase alfa enzyme replacement therapy in pediatric subjects < 5 years with mucopolysaccharidosis VII Heather A. Lau a, ⁎ ,1 , David Viskochil b , Pranoot Tanpaiboon c , Antonio Gonzalez-Meneses Lopez d , Esmeralda Martins e , Julie Taylor f , Betsy Malkus f , Lin Zhang f , Agnieszka Jurecka f,2 , Deborah Marsden f a NYU Grossman School of Medicine, Department of Neurology, New York, NY, USA b University of Utah, Department of Pediatrics, Salt Lake City, UT, USA c Rare Disease Institute, Children's National Health System, Washington, DC, USA d Hospital Universitario Virgen del Rocío and Universidad de Sevilla, Seville, Spain e Centro Hospitalar Universitário do Porto, Hospital de Santo António, Porto, Portugal f Ultragenyx Pharmaceutical Inc., Novato, CA, USA abstractarticle info Article history: Received 5 November 2021 Received in revised form 3 March 2022 Accepted 4 March 2022 Available online 9 March 2022 Mucopolysaccharidosis (MPS) VII is an ultra-rare, autosomal-recessive, metabolic disease caused by a deficiency of β-glucuronidase, a lysosomal enzyme that hydrolyzes glycosaminoglycans (GAGs), including dermatan sulfate (DS), chondroitin sulfate, and heparan sulfate (HS). β-glucuronidase deficiency leads to progressive accumulation of undegraded GAGs in lysosomes of affected tissues, which may cause hydrops fetalis, short stature, hepatosplenomegaly, and cognitive impairment. An open-label, multicenter, phase II study was conducted in 8 pediatric subjects <5 years of age with MPS VII. Subjects received the recombinant human β-glucuronidase vestronidase alfa 4 mg/kg by intravenous infusion every other week for 48 weeks (treatment period). Those who completed the 48-week treatment were offered to continue treatment with vestronidase alfa 4 mg/kg for up to 240 weeks or until withdrawal of consent, discontinuation, or study termination (continuation period). The level of GAG excreted in urine (uGAG) above normal has been shown to correlate with disease severity and clinical outcomes in MPS diseases. Therefore, the primary efficacy endpoint of this study was to determine the mean percentage change in uGAG DS excretion from baseline to week 48. Statistically significant reductions in uGAG DS from baseline were observed at each visit (p< 0.0001), with a least square mean (standard error) percentagechangeof−60% (6.6) at week 4 (first post-baseline assessment) and −61% (6.41) at week 48 (final assessment during treatment period). Secondary efficacy endpoints included change from baseline to week 48 in growth and hepatosplenomegaly. Positive trends were observed toward increased standing height Z-score (mean [standard deviation] at baseline, −2.630 [1.17], n=8;atweek48,−2.045 [0.27], n= 7) and growth velocity (mean [SD] Z-score at baseline, −2.59 [1.49], n=4;atweek48,−0.39 [2.10], n=4;p= 0.27). Hepatomegaly was resolved in 3 of 3 subjects assessed by ultrasound and in 5 of 6 subjects assessed by physical examination; splenomegaly was resolved in 1 of 3 subjects assessed by ultrasound and in 2 of 2 subjects assessed by physical examination. There were no new safety signals identified during this study. Mild-to-moderate infusion-associated reactions occurred in 4 (50%) subjects. In conclusion, long-term vestronidase alfa treatment demonstrated a rapid and sustained reduction in uGAGs, maintained growth, and improved hepatosplenomegaly in pediatric subjects with MPS VII <5 years of age. Trial registration: NCT02418455. © 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Keywords: Vestronidase alfa Mucopolysaccharidosis VII Urinary glycosaminoglycan Growth Hepatosplenomegaly Pediatric patients. Molecular Genetics and Metabolism 136 (2022) 28–37 Abbreviations: ADA, anti-drug antibody; BiPAP, bilevel positive airway pressure; CGI-C, Clinical Global Impression of Change; CPAP, continuous positive airway pressure; CS, chondroitin sulfate; DS, dermatan sulfate; ERT, enzyme replacement therapy; GAG, glycosaminoglycan; h, hour; HS, heparan sulfate; IAR, infusion-associated reaction; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LS, least squares; m, minute; MPS, mucopolysaccharidosis; MS-DS, mass spectrometry-serum disaccharide; NIHF, non-immune hydrops fetalis; NRE, non-reducing end; QOW, every other week; SAE, serious adverse event; SD, standard deviation; SE, standard error; SpO 2 , oxygen saturation; suppl O 2 , supplemental oxygen; TEAE, treatment-emergent adverse event; uGAG, urinary glycosaminoglycan; ULN, upper limit of normal.. ⁎Corresponding author at: Ultragenyx Pharmaceutical Inc., Novato, CA, USA. E-mail addresses: [email protected] (H.A. Lau), [email protected] (D. Viskochil), [email protected] (P. Tanpaiboon), [email protected] (A.G.-M. Lopez), [email protected] (E. Martins), [email protected] (J. Taylor), [email protected] (B. Malkus), [email protected] (L. Zhang), [email protected] (A. Jurecka), [email protected] (D. Marsden). 1 Present affiliations: Ultragenyx Pharmaceutical Inc., Novato, CA, USA; Yale University School of Medicine, New Haven, CT, USA. 2 Present affiliation: BridgeBio, Palo Alto, CA, USA. https://doi.org/10.1016/j.ymgme.2022.03.002 1096-7192/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Contents lists available at ScienceDirect Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme 1. Introduction Mucopolysaccharidoses (MPSs) are a heterogenous group of inherited metabolic diseases caused by the deficiency of any of the 11 lysosomal enzymes that catalyze the stepwise degradation of glycosaminoglycans (GAGs). Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is an ultra-rare, autosomal-recessive, multi-symptom, metabolic disease caused by mutations in the GUSB gene, which encodes β-glucuronidase, an acid hydrolase involved in the breakdown of specific GAGs, namely dermatan sulfate (DS), chondroitin sulfate (CS) [1,2], and heparan sulfate (HS) [3,4]. β-glucuronidase deficiency leads to progressive accumulation of these partially degraded GAGs in the lysosomes of affected tissues, causing cellular damage and organ dysfunction. Patients with MPS VII present with short stature, growth imbalance, hepatosplenomegaly, pulmonary disease, cardiovascular complications, skeletal abnormalities, and variable cognitive impairment [3]. In its most serious form, MPS VII can manifest as non-immune hydrops fetalis (NIHF), occurring in 48% of patients with MPS VII [5], and cause stillbirth or death within the first few weeks of life [3,4]. The highly variable clinical expressivity of MPS VII means that the use of traditional clinical study designs and standard endpoints may not be useful for evaluating the effectiveness of therapies for MPS VII. In contrast, biomarker endpoints indicating disease progression could enable assessment of response to therapy in patients showing a wide range of heterogeneous phenotypes. The level of GAG excreted in urine (uGAG) has been shown to correlate with disease severity and clinical outcomes in MPS diseases [1,6–9]. Hence, uGAG can be used as a relevant and important endpoint in assessing interventions [10]. Skeletal and cartilaginous growth and development are markedly affected in patients with MPS from birth, manifesting clinically as dysostosis multiplex. However, in many types of MPS, accelerated linear growth is typically observed at birth through the first 2–4 years of age; macrosomia and/or large for gestational age can be suggestive of MPS [11,12]. In the early stages of the disease, growth velocity slows and stunted growth becomes noticeable with age [11]. There is a scarcity of literature reporting detailed anthropometric data of patients with MPS VII. The first and only systematic analysis of growth impairment in MPS VII was reported in 2016 by Montaño et al. [13], in which short stature was one of the most common manifestations observed in patients; however, length/height was measured for only 12 patients. Vestronidase alfa, a recombinant human β-glucuronidase, is the first approved enzyme replacement therapy (ERT) for the treatment of pediatric and adult patients with MPS VII [3]. Vestronidase alfa was developed based on the successful outcome of ERT in other MPS disorders and preclinical evidence supporting the use of recombinant mouse βglucuronidase as ERT in MPS VII murine models [14–16]. In a pivotal clinical trial, the safety and efficacy of vestronidase alfa was investigated in subjects 8–25 years of age with MPS VII. A significant reduction in uGAG levels, as well as clinically meaningful improvements in functional outcomes, were observed, with no associated serious safety concerns [17]. Furthermore, Wang et al. [18] demonstrated the long-term safety and durability of clinical efficacy of vestronidase alfa treatment in these subjects in an open-label extension study. Previously, it was shown that an early intervention with ERT in the first few months to years of life amelioratesthe development of some disease complications and results in relatively normal growth and development in other MPS disorders [19–24]. Therefore, in the present study, we evaluated the efficacy, safety, and tolerability of vestronidase alfa in the treatment of MPS VII in pediatric subjects <5 years of age. 2. Materials and methods 2.1. Compliance and informed consent The study was approved by the relevant institutional review boards/ ethics committees and conducted in accordance with the Declaration of Helsinki, FDA regulations, ICH Good Clinical Practices guidelines, EU Clinical Trials Directive 2001/20/EC, and local ethical and regulatory requirements. Informed consent was obtained from the parent or caregiver of the subjects before the initiation of the study. The trial is registered with ClinicalTrials.gov (NCT02418455). 2.2. Study design This was an open-label, single-arm, multicenter, phase II study. Subjects received vestronidase alfa 4 mg/kg by intravenous infusion every other week (QOW) for 48 weeks (treatment period; Fig. 1). Major assessment visits (weeks 12, 24, 36, and 48) were scheduled at 12-week intervals (± 7 days). Those who completed the 48-week treatment were offered to continue treatment with vestronidase alfa 4 mg/kg for up to 240 weeks or until withdrawal of consent, discontinuation, or study termination (continuation period) with additional assessments conducted at 12-, 24-, or 48-week intervals. 2.3. Inclusion/exclusion criteria Study subjects met the following inclusion criteria: they had a confirmed diagnosis of MPS VII based on leukocyte or fibroblast glucuronidase enzyme assay or genetic testing; they were <5 years of age at the time of informed consent; and their parent/caregiver had provided a written informed consent. The study allowed the inclusion of subjects with prior exposure to vestronidase alfa based on the discretion of the investigator. Study subjects were excluded from the study based on the following criteria: they had previously undergone a successful bone marrow or stem cell transplant or if they had undergone unsuccessful bone marrow or stem cell transplant but chimerism with donor cells was detectable by bone marrow biopsy; they had used investigational drugs other than vestronidase alfa 30 days before screening for the present study; there was presence of a critical/acute condition that interfered with participation in the study; or there was presence of a concurrent disease/ condition that increased the risk of poor treatment compliance, discontinuation, interference with participation in the study, or adverse events. The risk of fluid overload was considered in assessing eligibility of infants with NIHF; however, the risk of death before 1 year of age was not considered sufficient to exclude such patients. 2.4. Intervention Vestronidase alfa was diluted with normal saline and administered at a dose of 4 mg/kg QOW by slow intravenous infusion over approximately 4 h, or longer for those with fluid overload. Subjects were treated with an appropriate dose of non-sedating antihistamine approximately 30–60 m before intravenous infusions. For subjects with a history of infusion-associated reactions (IARs) or other risk factors, such as allergies, a sedating antihistamine (e.g., diphenhydramine or chlorpheniramine) was allowed.Antipyretic drugs,such as ibuprofenor acetaminophen, were allowed at the discretion of the investigator. IARs were mitigated by a slow infusion rate; pre-treatment with H 2 blockers, additional antihistamines, or glucocorticoids was allowed. 2.5. Endpoints The primary efficacy endpoint was the mean percentage change in uGAG excretion from baseline to week 48. To this end, uGAG DS was measured using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) at the Greenwood Genetics Center and the Associated Regional and University Pathologists (ARUP) Laboratory. The decision to measure uGAG DS vs total uGAGs was based on previously established assessment of urinary DS during clinical studies of vestronidase alfa therapy in patients with MPS VII [17,18]. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 29 Secondary efficacy endpoints included changes in growth and hepatosplenomegaly from baseline at week 48. Growth was assessed using standing height/recumbent length (cm) and growth velocity. Length was measured for subjects up to 2 years of age, recumbent length was measured for subjects who were non-ambulatory and non-weight bearing, and height was measured in subjects over 2 years of age who could bear weight. Analyses of changes in height Z-score from baseline were based on Centers for Disease Control and Prevention standardized age- and sex-adjusted stature. Additional anthropometric measurements, including head circumference and weight, were also recorded. Hepatosplenomegaly was assessed by palpation of liver and spleen on examination and, when feasible, by ultrasound. Spleen size was determined by obtaining a coronal view that included the hilum and measuring the greatest longitudinal distance between the dome of the spleen and the tip (spleen length) as described by Rosenberg et al. [25]. Analyses of changes in liver and spleen size from baseline were based on age- and sex-adjusted standardized size as reported by Dhingra et al. [26]. Tertiary efficacy endpoints included the change from baseline in the following: serum GAG levels as assessed by non-reducing end (NRE) LC-MS/MS at the ARUP Laboratory, Clinical Global Impression of Change (CGI-C), functional development and motor outcome as assessed by Bayley-III scale and the Gross Motor Milestone Checklist, cardiac ventricular mass assessed by echocardiogram, and pulmonary function measured by pulse oximetry. The CGI-C scale is a well-established endpoint of response to treatment in which physicians consider the patient's history, psychosocial circumstances, symptoms, behavior, and quality of life [27]. An MPS VII disease-specific and age-appropriate version of the CGI-C was developed and used in this study. With this tool, physicians provided a global assessment of change using a 7-point scale ranging from −3 (severe worsening) to +3 (significant improvement), with 0 denoting no change. The CGI-C score was supported by reported changes relative to baseline or identification of a new finding based on 12 diseasespecific abnormalities considered to respond to treatment: short stature/slow growth rate, developmental delay, limited visual acuity, diminished walking ability/mobility, frequent infections, joint stiffness or contractures, limited endurance/fatigue/lack of energy/malaise, enlarged abdomen/thorax, shortness of breath/difficulty breathing on exertion/impaired respiratory function, pain/discomfort, respiratory support needed, and poor appetite. Bayley-III is a widely used, validated tool to assess cognitive development in infants/toddlers 1–42 months of age. The tool measures domains of language (receptive and expressive), motor (fine and gross), social/emotional, and adaptive functioning [28]. It consists of a series of developmental play tasks and requires 45–60 m to administer [28]. In this study, the total raw score was converted into a scaled score and a composite score with percentile rank to compare the performance with healthy controls. Unless the subject achieved the highest raw score on the test, the Bayley-III assessments continued throughout the study to monitor the progress of the subject. In addition, if valid and reliable administration of the instrument was not possible based on the clinical judgment of the investigator, the test was excluded at that visit. A Gross Motor Milestone Checklist was developed to assess gross motor function and development. The checklist allowed for assessment of the functional status of the subject and required limited language interaction and physical handling. Test items were grouped into 4 positions for which specific skills were assessed, including supine (10 skills), prone (10 skills), sitting (6 skills), and standing (16 skills). All skills listed on the checklist were expected to be achieved by 18 months of age. 2.6. Safety Safety outcomes included the incidence, frequency, and severity of treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs; defined as occurring during and up to 24 h following infusion). In addition, we studied the clinically significant changes from baseline to scheduled time points in vital signs and subject weight measurements, physical examination findings, clinical laboratory evaluations, concomitant drug use, immunoglobulin G antibody production in response to vestronidase alfa (as measured by anti-drug antibodies [ADAs]), and complement C3, C4, and CH50 levels indicative of immune response to drug-related IARs. Neutralizing antibodies were measured using a cell-based assay that evaluates the potential for antibodies to recombinant human ß-glucuronidase present in a subject's serum to impact the uptake of recombinant human ß-glucuronidase into ß-glucuronidase-deficient fibroblasts. The IARs were characterized into 4 distinct categories based on timing of onset relative to infusion and specific clinical manifestations: anaphylactoid, anaphylaxis, urticarial (with or without angioedema), and immune complex. Individual TEAEs were determined in parallel to assessing IARs characterization. ADA data were analyzed based on the number of subjects converting to a positive antibody assay value (defined as an increase in titer of ≥4-fold over baseline) and the timing of onset of a positive antibody response. 2.7. Statistical analysis Approximately 15 subjects with a confirmed diagnosis of MPS VII were planned, including approximately 5 subjects with NIHF. Given the low prevalence of MPS VII, the sample size was primarily based on enrollment of eligible subjects according to the inclusion and exclusion criteria and not on a formal power calculation. Data were analyzed using descriptive statistics. Continuous variables were described using mean, standard deviation (SD), median, minimums, and maximums; categorical variables were represented by counts and percentages of subjects in corresponding categories. Efficacy and safety analyses were based on the full analysis set. All statistical tests were 2-sided; a p-value <0.05 was considered significant. Mean percentage change in uGAG excretion (primary efficacy endpoint) and other selected efficacy endpoint changes from baseline were established using the generalized estimating equation (GEE) analysis method. The Z-score and percentiles calculated for anthropometric measures (secondary efficacy endpoints) were based on the Centers for Disease Control and Prevention/National Center for Health Statistics Fig. 1. Study design. Abbreviation: QOW, every other week. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 30 Clinical Growth Charts and were standardized and adjusted by age and sex. The change in the Z-score of the anthropometric measurements from baseline to week 48 were summarized by visits. The growth velocity Z-score was calculated based on Tanner's standard with standardized age- and sex-adjusted [29]. The change in growth velocity and its Z-score from pre- to post-baseline were summarized by visit. 3. Results 3.1. Disposition and baseline characteristics Overall, 8 subjects who were screened met all eligibility criteria and were enrolled at sites in the United States, Spain, and Portugal. Of these, 1 (12.5%) subject had received prior treatment with vestronidase alfa under an emergency investigational new drug (eIND) request at 5 months of age until study entry at age 18 months; the remaining 7 (87.5%) subjects were treatment-naïve at baseline. Of the 8 subjects, 3 had NIHF. These 8 subjects comprised the full analysis set. One (12.5%) subject discontinued following the week 42 visit for reasons not related to the study. The remaining 7 (87.5%) subjects completed the 48-week treatment period and entered the continuation period for up to an additional 88 weeks; following the regulatory approval of vestronidase alfa, these subjects transitioned to the commercial product. Mean (SD) age of the subjects was 3.25 (1.2) years at the time of parental/caregiver informed consent (range, 1.6–5.0 years), and there were 5 (62.5%) males and 4 non-Hispanic or Latino (50.0%) subjects (Table 1). All subjects had confirmed GUSB mutations (3 with a c526>T [p.L176F] homozygous mutation [2 were Hispanic or Latino, 1 had unknown ethnicity] and 2 with a c.1120C>T [p.R374C]; c.1136A>G [p.N379S] heterozygous mutation [both were Asian]) and insufficient β-glucuronidase enzyme activity. Three subjects were diagnosed with MPS VII as infants and had a medical history of NIHF. Derived baseline levels of uGAG were as high as 47-fold above the upper limit of normal (ULN; for normal reference ranges see Supplementary Tables 1 and 2). 3.2. Primary efficacy endpoint At baseline, mean (SD) uGAG DS was 2.09 (1.33) g GAG/g creatinine (range, 0.83–4.90 g GAG/g creatinine; n= 8). At week 48, mean (SD) uGAG DS was 0.70 (0.42) g GAG/g creatinine (range, 0.31–1.50 g GAG/ gcreatinine;n= 7). As assessed by the GEE analysis method, a statistically significantreduction in DS from baselinewas observed at each visit (p< 0.0001), with least squares (LS) mean(standard error; SE) percentage change of −60% (6.6) at week 4 (first post-baseline assessment) and −61% (6.41) at week 48 (final assessment during treatment period). Reductions in mean percentage change from baseline in uGAG DS were achieved during the continuation period, which were sustained up to final follow-up at week 132 (Fig. 2). At week 4 following vestronidase alfa treatment initiation, 87.5% of subjects were considered responders to treatment (defined as ≥50.0% reduction in uGAG DS). All subjects achieved ≥50.0% reduction in uGAG during the 48-week treatment period. The subjects' uGAG levels could also be determined by comparing against normative reference values [26]. This demonstrated progressive improvement over time following vestronidase alfa treatment. Mean uGAG DS was 25.9-fold above the ULN at baseline (n= 8), 9.7-fold above the ULN at week 48 (n=7),and1.9-foldabovetheULNatweek 132 (n=1). Table 1 Demographics and baseline characteristics. Characteristic Vestronidase alfa (N=8) Age at informed consent (years) Mean (SD) 3.25 (1.2) Median 3.14 Min, max 1.6, 5.0 Sex, n(%) Male 5 (62.5) Female 3 (37.5) Race, n(%) American Indian or Alaska Native 0 Asian 2 (25.0) Black or African American 1 (12.5) Native Hawaiian or other Pacific Islander 0 White 3 (37.5) Other 2 (25.0) Ethnicity, n(%) Hispanic or Latino 3 (37.5) Non-Hispanic or Latino 4 (50.0) Unknown 1 (12.5) Relevant medical history, n(%) Dysmorphism 6 (75.0) Macrocephaly 4 (50.0) Kyphosis (curvature of spine) 5 (62.5) Developmental delay 4 (50.0) Hypotonia 4 (50.0) Hepatosplenomegaly 3 (37.5) Body height below normal 4 (50.0) Non-immune hydrops fetalis 3 (37.5) Abbreviations: max, maximum; min, minimum; SD, standard deviation. Fig. 2. Change from baseline (%) in uGAG (LC-MS/MS-DS) through the continuation period. For the 1 patient treated prior to study enrollment, the percentage change was based on their initial uGAG level prior to treatment. Abbreviations: DS, dermatan sulfate; LC-MS, liquid chromatography-mass spectrometry; MS-DS, mass spectrometry-serum disaccharide; SE, standard error; uGAG, urinary glycosaminoglycan. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 31 3.3. Secondary efficacy endpoints 3.3.1. Growth We observed an increase in height (or recumbent length), head circumference, and weight of the subjects at the end of the 48-week treatment period, indicating a positive overall trend (Table 2). At baseline, all 8 subjects had impaired growth, with greater impairment in the subject previously treated with vestronidase alfa (Z-score, −5.35) compared with others. This previously treated subject had been born prematurely, had genetic and other medical factors impacting on their growth as well as having parents of below average height, and had been critically ill for 2 years prior to treatment. Mean (SD) standing height Z-score was −2.630 (1.17) at baseline (n= 8) and had improved by a mean (SD) of +0.196 (0.30) by week 48 (n= 7). Overall, a positive trend toward increased height Z-scores was observed throughout the treatment and continuation periods (Fig. 3). Growth velocity was analyzed before and during vestronidase alfa treatment in subjects with available pre-treatment historical data. Mean (SD) growth velocity was 5.06 (1.89) cm/year in subjects with historical data (n= 5; within 2 years of initiation of treatment). After treatment initiation, mean (SD) growth velocity increased to 6.20 (1.95) cm/year. Similar increases were observed relative to historical data within 1 year. As indicated by changes in slope, post-baseline growth velocities were increased in 3 subjects relative to pretreatment (Fig. 4). Pre-treatment growth velocity Z-scores based on historical data (within 2 years) and post-baseline growth velocity Z-scores were available for 4 subjects. Overall, despite a small sample size, a positive trend toward increased growth velocity was observed after vestronidase alfa treatment (mean [SD] Z-score at baseline, −2.59 [1.49]; −0.39 [2.10] at week 48; p=0.27). 3.3.2. Hepatosplenomegaly Ultrasonography of the liver was performed on 7 subjects. At baseline, mean (SD) linear liver measurement was 10.70 (1.14) cm (range, 9.20–12.70 cm). Significant changes in liver size from baseline were observed at week 12 and were maintained throughout the treatment period. At week 48, LS mean (SE) change from baseline was −0.98 (0.31) cm (p= 0.0016). Based on physical examination, hepatomegaly was observed at baseline in 6 subjects. Of these, 5 had not been treated previously with vestronidase alfa and had a normal liver size after 48 weeks of treatment. The remaining 1 subject had been treated previously with vestronidase alfa and did not undergo a physical examination at week 48. Overall, the degree of splenomegaly was not pronounced in the study population. Ultrasonography of the spleen was performed on all 8 subjects. At baseline, mean (SD) spleen size was 8.17 (1.11) cm (range, 7.00–10.30 cm). No significant change in mean spleen size from baseline was observed during the course of the study. At week 48, LS mean (SE) change from baseline was −0.15 (0.39) cm (p= 0.70). During physical examination, splenomegaly was observed at baseline in 2 subjects who had not been treated previously with vestronidase alfa; spleen assessments were found to be normal in both subjects after 48 weeks of treatment. 3.4. Tertiary endpoints 3.4.1. Serum GAG Abnormal serum GAG levels were assessed by NRE LC-MS/MS (for normal reference ranges see Supplementary Table 2). Mean (SD) baseline serum GAG level of the subject (n= 1) who had been treated previously with vestronidase alfa was 0.530 mg/L, whereas mean baseline serum GAG level of subjects who had not been treated previously with vestronidase alfa (n= 7) was 0.730 (0.153) mg/L. Beginning at week 12, serum GAG also showed substantial reduction and was maintained throughout the treatment period. At week 48, mean (SD) serum GAG levels decreased to 0.488 (0.066) mg/L in subjects not treated previously with vestronidase alfa, representing a −28.90% reduction from baseline (Fig. 5). 3.4.2. Clinical global impression of change Reductions in uGAG were accompanied by improvements in\\CGI-C. Overall, CGI-C scores showed improvement or no change at the last Table 2 Changes in anthropometric measurements from baseline to week 48. Vestronidase alfa a (N=8) Baseline Week 48 Change from baseline Standing height (cm) n777 Mean (SD) 89.34 (7.20) 95.91 (7.02) 6.57 (1.06) Min, max 78.2, 100.0 85.2, 106.5 5.0, 8.0 Median Z-score −2.360 −2.108 +0.121 Head circumference (cm) n777 Mean (SD) 51.57 (2.13) 52.36 (2.21) 0.79 (0.27) Min, max 48.5, 54.0 49.0, 55.0 0.5, 1.0 Weight (kg) n888 Mean (SD) 12.77 (4.03) 15.41 (3.37) 2.64 (1.81) Min, max 4.3, 17.4 10.6, 21.0 0.4, 6.3 Abbreviation: max, maximum; min, minimum; SD, standard deviation. a Height and head circumference data at week 48 were missing for the subject previously treated with vestronidase alfa. Fig. 3. Change in standing height Z-scores from baseline. Abbreviation: SE, standard error. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 32 available assessment except for one subject assessed with worsening (−1) in visual acuity (Fig. 6). Improvements in the CGI-C scores were noted in ≥1disease-specific abnormality in all subjects. Of the 8 subjects assessed, significant improvements (+3) were observed in joint stiffness (n= 1), mobility (n= 1), respiratory symptoms (n=1), fatigue/endurance (n= 1), and frequency of infections (n=1). 3.4.3. Functional development Bayley-III was used to assess 2 subjects; both showed consistent trends of improvement or stabilization in raw scores from baseline in all domains of the assessment. Bayley-III was not used in the majority of subjects based on the clinical judgment of the investigator or because the subject was above the upper age limit for the instrument. 3.4.4. Motor functioning The Gross Motor Milestone Checklist enabled the assessment of functional status without extensive language interaction or physical handling. At the initial assessment, 1 subject achieved all the motor milestones on the checklist and maintained functional status through the last assessment. Two subjects did not show any changes in motor milestones between initial assessment and at last available assessment. The remaining 5 subjects showed improvements in motor milestones following the initial assessment. At the end of the study, 4 subjects achieved all milestones on the Gross Motor Milestone Checklist. 3.4.5. Cardiac ventricular mass Most of the subjects did not show clinically significant abnormalities in any echocardiogram parameter. Two subjects experienced mitral valve regurgitation at both baseline and at all other post-baseline assessments. All eight subjects showed normal left ventricular mass index. Overall, there was no substantial change in cardiac ventricular mass. 3.4.6. Pulmonary function Overall, there were no apparent clinically meaningful changes in oxygen saturation (SpO 2 ) or in the requirement for respiratory support. The baseline SpO 2 of all subjects was ≥95.0%. Throughout the study, routine assessments of SpO 2 remained ≥94.0%. Two (25.0%) Fig. 4. Subject heights before and after ERT with vestronidase alfa treatment. a Informed consent. b Received treatment pre-study from 5 to 18 months of age. Abbreviation: ERT, enzyme replacement therapy. Fig. 5. Change from baseline (%) in serum GAG levels. Abbreviations: GAG, glycosaminoglycan; NRE-CS/DS, non-reducing end–chondroitin sulfate/dermatan sulfate; SE, standard error. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 33 subjects required respiratory support; of these, one subject required continuous mechanical ventilation throughout the study period, and the other required oxygen supplementation from baseline through the week 48 visit. Furthermore, 75.0% of the subjects reported use of systemic corticosteroids at some point during the study, and 62.5% received concomitant medication for obstructive airway disease throughout the study period. 3.5. Safety All 8 subjects experienced ≥1TEAE(Table 3), and 5 (62.5%) experienced drug-related TEAEs. The most common TEAE was pyrexia (n= 5, 62.5%). Other TEAEs reported by 50.0% of subjects each included gastroenteritis, otitis media, diarrhea, and vomiting. Three (37.5%) subjects experienced SAEs, including adenoidal hypertrophy, bradycardia, febrile convulsion, decreased oxygen saturation, spinal column stenosis, spinal instability (atlantoaxial instability), cervical cord compression, cervical spinal stenosis, device fastener issue (halo pin loosening), and otitis media acute. Most SAEs were associated with known complications of MPS VII. One (12.5%) subject had a treatment-related SAE (febrile convulsion). The subject had received a diphtheria, pertussis, and tetanus vaccination within 72 h of infusion with vestronidase alfa, and this was considered a possible contributing factor. The subject skipped 2 treatments and upon resumption received prophylactic acetaminophen and loratadine followed by a reduced rate of infusion with vestronidase alfa; no additional events of febrile convulsion occurred, and the subject continued on therapy. No treatment-related Grade 4 TEAE was observed. There were no deaths, and no subject discontinued treatment or withdrew from the study due to a TEAE. Four (50%) subjects experienced treatment-related IARs, which were either mild or moderate in severity. Most IARs caused temporary study drug interruption; all IARs were resolved with or without additional treatment. One subject experienced an anaphylactoid reaction; diphenhydramine was administered and the symptoms resolved on the same day, allowing treatment to be continued. At subsequent infusions (weeks 22 and 24), blood samples for complement testing were taken before and immediately after infusion to monitor changes in C3, C4, and CH50 levels. Complement levels were within normal range, and no further hypersensitivity reactions occurred. Overall, no IAR was considered serious, and no subject was discontinued. Complement tests in 3 subjects who experienced possible drug-related hypersensitivity reactions were mostly within normal range. Clinical laboratory tests, vital signs, physical examinations, andother observations related to safety were unremarkable and consistent with the age/condition of the study population. All subjects were ADA-negative at baseline. Antibodies specifically against vestronidase alfa were detected in all subjects at ≥1 point. The first post-baseline positive titers for each subject were detected beginning at approximately week 8 (n= 2), weeks 16–20 (n= 3), weeks 36–40 (n= 2), and week 132 (n= 1). Median peak titer was 1:6400 (range, 1:40–1:20,480). Neutralizing antibodies against vestronidase alfa were detected in 3 subjects. There was no apparent association between the development of ADAs or neutralizing antibodies and uGAG reduction. Of the 3 subjects who developed neutralizing antibodies, 2 experienced IARs. Fig. 6. Clinical Global Impression of Change scoresby domain at last available assessment. The figure includes only subjects with CGI findings observed throughout the study. Subjects with no report of an item are not included. Abbreviations: BiPAP, bilevel positive airway pressure; CGI-C, Clinical Global Impression of Change; CPAP, continuous positive airway pressure; suppl O 2 , supplemental oxygen H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 34 4. Discussion In this study, we investigated the efficacy, safety, and tolerability of vestronidase alfa in the treatment of MPS VII in pediatric subjects <5 years of age. Extensive clinical research has shown that uGAG correlates with disease severity and clinical outcomes in MPS diseases [1,6,8,9], and a recent retrospective study showed that uGAG could be used as a biomarker of disease progression and response to treatment in patients with a variety of MPS disorders [10]. In that study, a reduction in uGAG levels following treatment correlated with growth rate and other functional outcomes, including the 6-min walk test, pulmonary function, and CGI-C [10]. In thepresentstudy, we have shown that a uGAG reduction of at least 50% is associated with clinically meaningful changes. The baseline uGAG levels ranged from 10- to 47-fold above the ULN. Upon vestronidase alfa treatment, the reduction in uGAG excretion was observed as early as week 4 and sustained through week 48. Similar to other ERTs and previous studies on vestronidase alfa [17,18], uGAG levels reduced by at least 50% from pre-treatment baseline in all subjects by week 4, and these reductions were sustained throughout treatment. The strong reduction in uGAG is a clinically relevant outcome of the study because it is a direct indicator of successful clearance of the partially degraded GAGs in lysosomes, which otherwise cause cellular dysfunction resulting in tissue injury and multi-system organ damage. Clearance of GAG, reflected by decreases in both urine and serum GAG concentrations, restores normal cellular function and improves symptoms of MPS VII. The goals of therapy in pediatric patients <5 years of age are to stabilize current disease burden (skeletal deformities, growth delay, respiratory compromise) and to prevent the development of chronic disease, including cardiovascular diseases (valvular deformities, hypertrophic cardiomyopathy), respiratory dysfunction, pain, and disability. Early initiation and long-term treatment with vestronidase alfa are needed to assess disease stabilization and achieve maximum improvement in individual patients. Fluctuations in uGAG levels observed in individual patients were temporally associated with missed doses. Other potential variabilities could not be verified due to difficulties in obtaining the first morning void in pediatric patients. The variation in DS and CS excretion observed in some patients could also be due to changes in the growth rate or illnesses occurring during the study period. Rapid metabolic turnover of connective tissue during postnatal development is thought to be the main cause of increased DS and CS excretion [30]. It has also been shown that uGAGs increase when pediatric patients experience a growth spurt [30]. Given the rarity of MPS VII, the young age of the study population, the variable expressivity of the disorder, and the duration of the observation period, it was difficult to determine treatment-induced changes in anthropometric variables in this study. At the time of enrollment, all subjects had a short stature/slow growth rate, which is characteristic of MPS VII. In the study conducted by Montaño et al. [13], the heights of male subjects 2–13 years of age ranged between the 5th and 90th centiles; after 13 years of age, a rapid decline in growth velocity was observed in untreated subjects. In the present study, the baseline height of 4 of 5 male subjects was below the 5th centile, despite the young age, thereby implying a more serious disease in this cohort compared with the study population assessed by Montaño et al. [13]. During the present trial period, most of the male subjects maintained growth near the 5th centile. Two male subjects appeared to show a slower growth velocity and “fell off”the normative growth curve; both subjects had NIHF as infants. Of note, 5 subjects had kyphosis and 1 had kyphoscoliosis, which may affect height and length. Montaño et al. [13] demonstrated that growth impairment was more pronounced in female subjects, and a clear decrease in attained height was observed after 24 months of age, with values below the 25th centile. In the present study, the baseline height of the 3 female subjects ranged from ≤5th centile up to and including the 10th centile, despite their young age, thereby implying a more serious disease in this younger cohort. Followingvestronidase alfa treatment, the 3 female subjects showed sustained growth after 24 months of age; however, only limited historical data before 24 months of age were available. Further longer-term studies are required for a complete growth assessment in pediatric patients with MPS VII. MPS VII is associated with chronic hepatosplenomegaly, where spleen enlargement, if present, is usually mild without significant effects on liver function or platelet count [31–33]. The incidence and severity of hepatosplenomegaly in the present study population was not pronounced. In subjects with hepatomegaly, liver size decreased after vestronidase alfa treatment; physical examination findings were mostly normal by the last available assessment. Within the present study, 3 subjects had a prior history of NIHF, a common presentation in patients with MPS VII and appears to be a transient state that is not universally fatal but significantly impacts the patient's stability in the first year of life [13]. Historically, many underlying variants have been identified in patients with MPS VII and NIHF, with considerable variability in both clinical and biochemical manifestations [13,34,35]. Therefore, it is important to consider the efficacy of vestronidase alfa in this subpopulation. In subjects with a history of NIHF in the current study, treatment with vestronidase alfa resulted in sustained, significant reductions from baseline in uGAG levels (when the subjects remained compliant with treatment). One subject had NIHF at the time of treatment with vestronidase alfa and prior to study enrollment. Vestronidase alfa therapy led to stabilization of the respiratory status, which was sustained throughout the study. All 3 subjects experienced resolution of hepatomegaly as detected by ultrasound Table 3 Overall summary of TEAEs. TEAEs Vestronidase alfa (N=8) n(%) ≥1 TEAE 8 (100) SAEs 3 (37.5) Treatment-related TEAEs 5 (62.5) Treatment-related SAEs 1 (12.5) Grade 3 or 4 TEAEs 2 (25.0) TEAEs leading to treatment discontinuation 0 TEAEs leading to study discontinuation 0 TEAEs leading to death 0 Infections and infestations 8 (100) Gastroenteritis 4 (50.0) Otitis media 4 (50.0) Upper respiratory tract infection 3 (37.5) Ear infection 2 (25.0) Otitis media acute 2 (25.0) Pharyngitis 2 (25.0) Sinusitis 2 (25.0) General disorders 6 (75.0) Pyrexia 5 (62.5) Investigations 6 (75.0) Hepatic enzyme increased 2 (25.0) Oxygen saturation decreased 2 (25.0) Musculoskeletal and connective tissue disorder 6 (75.0) Arthralgia 2 (25.0) Pain in extremity 2 (25.0) Respiratory, thoracic, and mediastinal disorder 6 (75.0) Cough 3 (37.5) Rhinorrhea 3 (37.5) Adenoidal hypertrophy 2 (25.0) Nasal congestion 2 (25.0) Gastrointestinal disorders 4 (50.0) Diarrhea 4 (50.0) Vomiting 4 (50.0) Nausea 2 (25.0) Injury, poisoning, and procedural complications 4 (50.0) Fall 2 (25.0) Skin abrasion 2 (25.0) Ear and labyrinth disorder 3 (37.5) Ear pain 2 (25.0) Abbreviations: SAE, serious adverse event; TEAE, treatment-emergent adverse event. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 35 and/or physical examination. Together, these results indicate efficacy of vestronidase alfa across all patients with MPS VII. Additional efficacy variables were investigated to assess the improvement in clinical features of MPS VII. First, disease-specific CGI-C improvements were observed in ≥1disease-specific abnormality in all subjects. Improvements in Bayley-III functional assessments were also noted in 2 siblings; the majority of subjects achieved additional motor milestones throughout the study. Second, we investigated cardiac function of subjects because cardiac diseases are commonly associated with MPS VII [3]. Notably, 2 subjects experienced mitral valve regurgitation; theremaining 6 subjects showed normal leftventricularmass index and cardiac function. Third, many patients with MPS experience progressive pulmonary problems because of airway obstruction. The subjects of the present study were very young, and some had cognitive impairment. Compliance with verbal commands for standard pulmonary function tests would be challenging, thus oxygen saturation was used as a surrogate of pulmonary function. Throughout the study, routine assessments for SpO 2 remained ≥94.0%, with no apparent clinically meaningful changes or increased requirements for respiratory support. Overall, the safety profile of vestronidase alfa observed in the pediatric population of the present study was similar to that of older children and adults [17,18], and no new safety signals were observed. The most common TEAEs reported may not be easily attributable to vestronidase alfa initiation given the events recorded in this study are those commonly observed in healthy children. All IARs were mild to moderate in severity. One subject experienced a moderate (Grade 2) anaphylactoid reaction. The study drug was interrupted and the subject was treated with diphenhydramine, resulting in resolution of symptoms on the same day. Complement testingwas within the normal range, the patient was able to continue vestronidase alfa treatment, and hypersensitivity reactions did not recur with subsequent infusions. This was not an unexpected occurrence because anaphylactic and hypersensitive reactions have been observed previously following vestronidase alfa administration [17,18]. Therefore, it remains important to have the appropriate medical support on hand to manage and mitigate potential anaphylactoid events during vestronidase alfa administration. Vestronidase alfa is a recombinant protein; therefore, there is potential for immunogenicity and drug neutralization. In the present study, all subjects developed low-to-moderate titers of anti-vestronidase alfa antibodies; 2 tested positive for neutralizing antibodies. There was no apparent association between the development of ADAs/neutralizing antibodies and reduction in uGAG excretion or hypersensitivity reactions, including IARs, with no need to implement immunotolerance therapies. Overall, the immunogenicity of vestronidase alfa observed in the present study was similar to those observed in previous studies on vestronidase alfa conducted in older children and adults [17,18]. This study had a few limitations. First, the disease manifestation and progression of MPS VII is highly variable; therefore, the present sample size was small and heterogenous. Second, developmental delays in subjects may have affected comprehension required to complete some of the assessments or led to surrogate assessments (pulse oximetry rather than pulmonary function tests). Unfortunately, there is no specifictool to evaluate cognitive function for MPS VII, and fine and gross motor delays due to dysostosis multiplex may impose physical limitations while cognitive abilities to problem-solve and communicate remain intact. In addition, the Bayley-III assessment can be scaled to 3 years of age, limiting its use in certain subjects. Third, measurement of liver and spleen size by ultrasonography and physical examination provided only linear measurements, which may lead to underestimation of the true volume of these organs. Finally, given the young age of the study population, spirometry could not be performed. 5. Conclusions In conclusion, the results of the present study indicate that longterm vestronidase alfa treatment in children <5 years of age with MPS VII is associated with substantial, rapid, and sustained reductions in uGAG levels, corresponding to reduced hepatomegaly. No new safety signals compared with older MPS VII populations were observed. In addition, stabilization or incremental improvement in height and growth velocity was observed following vestronidase alfa ERT. These results support the long-term use of vestronidase alfa as a safe and effective treatment for younger pediatric patients with MPS VII. Funding This work was supported by Ultragenyx Pharmaceutical Inc. Declaration of Competing Interest Heather A. Lau, David Viskochil, Pranoot Tanpaiboon, Antonio Gonzalez-Meneses Lopez, and Esmeralda Martins served as principal investigators for this study. Heather A. Lau is now a full-time employee of Ultragenyx Pharmaceutical Inc.; served on advisory boards for Adult Polyglucosan Body Disease Research Foundation, Amicus Therapeutics, BioMarin Pharmaceutical Inc., Chiesi, Enzyvant, National Tay-Sachs & Allied Diseases Association, SanofiGenzyme, Takeda, and Taysha Therapeutics; received honoraria from Actelion, Amicus Therapeutics, BioMarin Pharmaceutical Inc., Chiesi, Enzyvant, Fabry Support & Information Group, Muscular Dystrophy Association, National Gaucher Foundation, SanofiGenzyme, Takeda, and Ultragenyx Pharmaceutical Inc.; served as a consultant for Actelion, Amicus Therapeutics, ASPA Therapeutics, BioMarin Pharmaceutical Inc., Chiesi, Pfizer, Prevail Therapeutics, SanofiGenzyme, Takeda, Taysha Therapeutics, and Ultragenyx Pharmaceutical Inc.; received grants from National Tay-Sachs & Allied Diseases Association and SanofiGenzyme; and received research support from Amicus Therapeutics, ASPA Therapeutics, BioMarin Pharmaceutical Inc., Intrabio, Mallinckrodt Pharmaceuticals, Orphazyme, Ovid Therapeutics, Pfizer, Protalix BioTherapeutics, Sangamo Therapeutics, Sanofi Genzyme, Takeda, and Ultragenyx Pharmaceutical Inc. David Viskochil received research support from Levo Therapeutics, Soleno Therapeutics, SpringWorks Therapeutics, Takeda, and Ultragenyx Pharmaceutical Inc.; served on advisory boards for SanofiGenzyme and Takeda; and received honoraria from AstraZeneca and SanofiGenzyme. Pranoot Tanpaiboon is an employee of Quest Diagnostics. Antonio Gonzalez-Meneses Lopez has served on advisory boards for Sanofiand Ultragenyx Pharmaceutical Inc., and received payments for travel expenses from Sanofi, Shire, and Ultragenyx Pharmaceutical Inc. Esmeralda Martins received research support from Ultragenyx Pharmaceutical Inc. Julie Taylor, Betsy Malkus, Lin Zhang, and Deborah Marsden are employees and shareholders of Ultragenyx Pharmaceutical Inc. Agnieszka Jurecka is an employee of BridgeBio (formerly an employee of Ultragenyx Pharmaceutical Inc.). Acknowledgments The authors acknowledge the contributions of Tricia Cimms, MPH; Ting Chang, PhD; Erin Schwartz; Regina Pierre; Kathleen Crosby, MS, CGC; and Debra S. Regier, MD, PhD. Medical writing assistance was provided by Kimberly Denis-Mize, PhD, and Jack Pike, PhD, CMPP, of Envision Pharma Group. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ymgme.2022.03.002. References [1] J. Muenzer, J.E. Wraith, M. Beck, R. Giugliani, P. Harmatz, C.M. Eng, A. Vellodi, R. Martin, U. Ramaswami, M. Gucsavas-Calikoglu, S. Vijayaraghavan, S. Wendt, A.C. Puga, B. Ulbrich, M. Shinawi, M. Cleary, D. Piper, A.M. Conway, A. Kimura, A phase II/III clinical study of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II (Hunter syndrome), Genet. Med. 8 (2006) 465–473. H.A. Lau, D. Viskochil, P. Tanpaiboon et al. Molecular Genetics and Metabolism 136 (2022) 28–37 36