Long‐term safety and efficacy of velmanase alfa treatment in children under 6 years of age with alpha‐mannosidosis: A phase 2, open label, multicenter study

Alpha‐mannosidosis (AM) is a rare, autosomal recessive, lysosomal storage disorder caused by alpha‐mannosidase deficiency that leads to the accumulation of mannose‐rich oligosaccharides. AM symptoms and severity vary among individuals; consequently, AM is often not diagnosed until late childhood. Velmanase alfa (VA), a recombinant human lysosomal alpha‐mannosidase product, is the first enzyme replacement therapy indicated to treat non‐neurological symptoms of AM in Europe. Previous studies suggested that early VA treatment in children may produce greater clinical benefit over the disease course than starting treatment in adolescents or adults; however, long‐term studies in children are limited, and very few studies include children under 6 years of age. The present phase 2, multicenter, open‐label study evaluated the safety and efficacy of long‐term VA treatment in children under 6 years of age with AM. Five children (three males) received VA weekly for ≥24 months, and all children completed the study. Four children experienced adverse drug reactions (16 events) and two experienced infusion‐related reactions (12 events). Most (99.5%) adverse events were mild or moderate, and none caused study discontinuation. Four children developed antidrug antibodies (three were neutralizing). After VA treatment, all children improved in one or more efficacy assessments of serum oligosaccharide concentrations (decreases), hearing, immunological profile, and quality of life, suggesting a beneficial effect of early treatment. Although the small study size limits conclusions, these results suggest that long‐term VA treatment has an acceptable safety profile, is well tolerated, and may provide potential benefits to patients with AM under 6 years of age.


| INTRODUCTION
Alpha-mannosidosis (AM) is a rare, autosomal recessive, lysosomal storage disorder caused by alpha-mannosidase deficiency leading to the accumulation of mannose-rich oligosaccharides in many organ systems and tissues. 1,2 The prevalence of AM is estimated to be 1 in 500 000 live births worldwide, and the disease is not specific to any gender or ethnic group. 1,3,4 The clinical variability of AM signs and symptoms is broad, and the disorder presents on a continuum of severity over a patient's life span, becoming more debilitating over time. Symptoms include mental retardation, motor function disturbances, speech and hearing impairments, progressive coarsening of facial features, recurrent infections, impaired immune response, skeletal abnormalities, muscular pain and/or weakness, and ocular changes. 1,5 While patients' life expectancy can span several decades, many individuals with AM become increasingly dependent on caregivers, both physically and socially. 1 Diagnosis is based on detecting reduced alpha-mannosidase activity in leukocytes or fibroblasts and is confirmed by identifying two homozygous or heterozygous pathogenic variants in the MAN2B1 gene. 1,6 Currently, there is no cure for AM, and disease-modifying treatments are limited and primarily focused on symptom management. 7 Velmanase alfa (VA; Lamzede ® ; Chiesi Farmaceutici S.p.A. Pharmaceuticals, Parma, Italy), a recombinant human lysosomal alpha-mannosidase approved as a product for intravenous (IV) use, is the first enzyme replacement therapy (ERT) indicated for the treatment of nonneurological symptoms of AM. 8 Previous studies have demonstrated the safety and efficacy of VA in pediatric and adult patients and suggest that VA treatment may produce a greater clinical benefit when administered earlier in the disease course than later. 9,10 A recent case study of a 7-month-old infant treated with VA showed reduced oligosaccharide levels in the urine and serum after 2 months. 11 However, long-term clinical studies in multiple younger pediatric patients are limited, with many studies only including children 6 years of age and older. Further, due to the rarity of the disease, the safety and efficacy of VA in children under 6 years are largely unknown. Thus, the objective of the present study was to evaluate the long-term safety and efficacy of VA treatment in children with AM less than 6 years of age.

| Study design
In this phase 2, 24-month, multicenter, open-label study (NCT02998879), children under the age of 6 years with a confirmed AM diagnosis were administered 1 mg/kg of VA IV once a week for at least 24 months. After 24 months, one child was moved to the rhLAMAN07 study, where they continued to receive VA. This study collected data for an additional 16 months until VA was approved and available in their country of residence. Children were screened for eligibility (À1 Visit [V]), then underwent a baseline visit (V0), weekly dosing visits (V1-V109 [V1-V171 for the child who received 40 months of treatment]), and evaluation visits (V26a, V52a, V78a, V104a, and V166a) every 6 months. An "end-of-trial" visit was also completed to confirm no additional safety concerns. Children were provided with the option to continue VA treatment in a commercial setting or a clinical setting (if VA was commercially unavailable in their country of residence).

| Patients
At the time of the screening visit, patient eligibility was determined based on confirmation of AM diagnosis, defined as low alpha-mannosidase activity (<10% of normal activity) in leukocytes or fibroblasts, and a current age less than 6 years. Additionally, custodial/parental informed consent was provided. Children were excluded from the study if an AM diagnosis could not be confirmed; a chromosomal abnormality affecting psychomotor development other than AM was present; any medical conditions precluding study participation were present; or participated in another clinical trial within the previous 3 months. There were no specific enrollment criteria based on disease severity or age at first disease manifestation. The patients' compliance with the treatment regimen was calculated based on receiving VA during the protocol time period, and did not exclude interruptions.

| Endpoints
The study's primary objective was to evaluate the safety and tolerability of VA by routinely monitoring children for the emergence of adverse events (AEs), including infusion-related reactions (IRRs), in addition to changes in vital signs, clinical laboratory measures, and the development of anti-drug antibodies (ADAs) against VA, including neutralizing antibodies (nAbs). A secondary objective was to evaluate the efficacy of VA by assessing changes from baseline to month 24 in serum oligosaccharide concentrations, functional capacity, endurance/ physical functioning, hearing, immunological profiles, and quality of life (QoL). Lastly, the pharmacokinetic (PK) profile of VA after the first dose and at 6 months (defined as the steady state) was evaluated.
ADA and nAb status were measured at baseline, evaluation visits, and every 4 weeks for the first 3 months, then every 8 weeks. ADA IgG concentrations were determined using enzyme-linked immunosorbent assays (ELISAs). NAb ELISAs were performed with biotinylated-VA to bind anti-VA antibodies, captured on a streptavidin-coated ELISA plate ( Figure S1). VA activity was determined using a chromogenic substrate, measured for absorbance at 450 nm. Neutralizing activity of anti-VA antibodies was detected by a decrease in absorbance.
Serum oligosaccharide concentrations and immunological profiles were measured at baseline and each evaluation visit and served as a surrogate marker of efficacy. Oligosaccharides were qualitatively analyzed via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for assessment of different n-mannose oligosaccharides (GlcNAc[Man]n), including GlcNAc(Man)2, validated at the SGS Laboratory (Geneva, Switzerland). Serum samples were prepared by solid phase extraction. The established method used derivatization with 2-aminobenzamide followed by LC-MS/MS analysis to quantify internal standards and GlcNAc(Man)n levels in serum samples. The LC-MS/MS quantification was performed using isomaltotriose as a surrogate reference standard, and maltotetraose as an internal standard since the oligosaccharides of interest were unavailable to use as references. Immunological analyses were performed at TIGET San Raffaele Hospital (Milan, Italy).
Functional capacity was measured via the Peabody Developmental Motor Scale, Second Edition (PDMS-2), 12,13 and the Mullen Scales of Early Learning (MSEL), which are quantitative scales used to measure each patient's cognitive ability in addition to language, motor, and perceptual abilities. 14,15 PDMS-2 is indicated for children 0-5 years of age and MSEL is indicated for children 0-68 months of age; the tests were occasionally used out of range for some tests in some patients. Endurance/ physical functioning was measured with the 6-min walk test (6MWT) and 3-min stair climb test (3MSCT). 16,17 It should be noted that Bruininks-Oseretsky Test of Motor Proficiency Second Edition (BOT-2) scores and 2-min walk test (2MWT) [data not shown] were included in the protocol, but data collection for these tests was not available at similar time points for each child. Hearing impairment was measured by automated auditory brainstem response (A-ABR). 18 QoL was assessed by parents' responses to the Pediatric Evaluation of Disability Inventory (PEDI) questionnaire. 19 The PK profile was determined by collecting plasma samples and measuring VA concentrations at the first VA dose (V1) and 6-month evaluation visit (V26a; steady state). Plasma samples were collected pre-dose and at the following times after the end of infusion: 10 min ±2 min, 4 h ±10 min, 8 h ±10 min, 24 h ±30 min, and 46 h ±30 min.

| Statistical and data analyses
All statistical analyses and data processing were performed using Statistical Analysis System software (version 9.3). All analyses were descriptive in nature. Continuous variables presented were the number of nonmissing values, mean, standard deviation (SD), minimum, median, and maximum. For categorical variables, absolute and relative frequencies are reported. Percentages are based on the number of patients with data and are not calculated for missing categories.

| Patient demographics and characteristics
Six children diagnosed with AM under the age of 6 from five countries (Germany, Austria, France, Denmark, and Italy) were screened. Five children were enrolled and completed the study; one child was excluded at screening due to numerous ventricular extrasystoles. Enrollment was not based on disease severity. Individual baseline demographics and physical characteristics are presented in Table 1. The mean (SD) age was 4.5 (0.8) years, ranging from 3.7 to 5.9 years. Three (60%) children were male, and the four children with available data on race were identified as white. At baseline, mean (SD) weight was 18.9 (2.5) kg, head circumference was 51.8 (3.7) cm, height was 104.16 (5.4) cm, and body-mass index (BMI) was 17.4 (1.1) kg/m 2 (Table 1A). After ≥24 months of VA treatment, weight and height increased in all children (n = 5). At baseline and end of the study, most patient's growth percentiles were within the standard range for gender and age, except for patient 2's head circumference, consistent with a history of macrocephaly and hydrocephalus.  Table 1C lists the MAN2B1 variants for each patient, including two compound heterozygous children and three homozygous children.
The mean (SD) total dose of VA administered over the study treatment period was 2597.5 (1243.0) mg, and the median (range) was 2137.8 (1661.4-4780.4) mg. The mean (SD) duration of VA exposure was 120.6 (27.5) weeks, and the median (range) was 108.3 (105.4-169.6) weeks. Treatment compliance ranged from 79.6% to 97.2%, with a mean (SD) compliance rate of 91.3 (7.1). Four children had compliance rates ranging from 89.8% to 97.2% (compliance cut-off, <80%), while 1 child (patient 3) had a compliance rate of 79.6% due to personal and social reasons. Thirty-eight treatmentemergent AEs (TEAEs), unrelated to study treatment or infusion, interrupted treatment in 4 children. Study treatment was interrupted in patient 2 for $1 month due to COVID-19 restrictions at the treatment center from V155 to V160.

| Safety assessment
Safety assessments during VA treatment were routinely completed and documented throughout the study (see Section 2). Four children experienced 10 AEs prior to receiving VA treatment (Table 2), highlighting the severe burden of disease in children. All children (n = 5) experienced TEAEs (184 events; Table 2). Most (99.5%) TEAEs were mild (107 events) to moderate (76 events) in intensity, and 1 TEAE was severe (a concussion that was unrelated to study treatment). AEs were distributed across the course of treatment. The most frequent AEs (>50% of children) were vomiting, pyrexia, cough, otitis media, nasopharyngitis, rhinitis, and diarrhea. Overall, 16 adverse drug reaction (ADR) events were reported in four children (Table 2). Patient 2 experienced chills (three events), hyperthermia (two events), and cyanosis (two events); the hyperthermia and chills were serious adverse events, requiring hospitalization. Patient 3 experienced pyrexia (one event); patient 4 developed urticaria (five events) and required a reduction in VA dose; patient 5 experienced anal pruritus (three events).
All ADRs reported, except pyrexia and anal pruritus, were also considered IRRs. Patients 2 and 4 cumulatively experienced 12 IRRs (Table 2) and both tested positive for ADAs. During the study, both were treated with antiallergic prophylaxis-including corticosteroids and antihistamines-to mitigate subsequent IRRs (Table S1). All IRRs resolved without withdrawal from the study. Patients 1 and 3 transiently developed low ADA concentrations (≤0.77 U/mL); but only patient 3 had a positive nAb test at 1 evaluation and no IRRs were reported in either patient (Figures S1 and S2). The uncommon observation of anal pruritus that occurred in patient 5 was initially categorized as an IRR based on its temporality to the infusion. It was later excluded as an IRR because anal pruritus is usually not considered an immune reaction, and the patient was ADA-negative. However, there is a small unlikely possibility that this AE was an IRR.
At baseline, all children were negative for ADAs (n = 5). For all five patients, IRRs and ADA-/nAb-status are shown over time in Figure S1. Figure S2 shows ADA concentration relative to occurrence of IRRs over time. Three ADA-positive children (patients 2, 3, and 4) developed nAbs during VA treatment, and two children with ADAs (patients 2 and 4) experienced IRRs. Patient 2 experienced serious IRRs (chills and hyperthermia) at V54, for which overnight hospitalization was required, though the events resolved the same day. V54 occurred approximately 10 months after ADAs were first detected (V13); the patient remained ADA-and nAbpositive until the end of the study ( Figure S1). The V54 infusion was not considered to be completed because only half of the required 10 mL of rinse solution was administered after completion of the VA dose. In response to the IRRs at V54, the patient was acutely administered sodium chloride (15 mL/kg, IV) and dexchlorpheniramine maleate (5 mg, IV) for chills and paracetamol (300 mg, IV) for hyperthermia. The patient also received oxygen by inhalation at a flow of 1.5 mL/min for 5 min. After V54, premedication treatment included dexchlorpheniramine maleate (5 mg, IV) from V55 to V81 and paracetamol (400 mg, IV) from V57 to V81 (Table S1). Patient 2 did not experience serious IRRs again but did experience nonserious IRRs at V67 (chills, cyanosis, and hyperthermia) and V69 (chills and cyanosis). At the dose visit prior to the serious IRRs (V54) ADA concentration was 105 U/mL. ADA concentrations during the VA treatment period ranged from 4.63 to 174 U/ mL. The highest ADA concentration occurred at V45, 9 weeks before the first IRR; from its peak, the concentration trended downward for about a year before stabilizing at $5-25 U/mL ( Figure S2). There were no substantial dose changes in response to IRRs. Treatment was interrupted in patient 2 from V155 to V160 (Section 3.1); no IRRs were reported directly preceding or following the pause in treatment.
Patient 4 experienced IRRs (urticaria) at V32, V44, V59, V70, and V82 within 1-13 weeks of ADA detection ( Figure S1). ADA concentrations in patient 4 ranged from 0.51 to 6.03 U/mL over the VA treatment period, and nAbs were only detected at evaluation V104a. Between the first (V32) and last (V82) urticaria events, the maximum ADA concentration was 3.64 U/mL across 5 ADA positive tests during this timespan ( Figures S1 and S2). Patient 4 received cetirizine hydrochloride as premedication for the entire study (Table S1). At V44, patient 4 had a dose reduction, receiving 0.7 mg/kg VA instead of 1.0 mg/kg; the dosing returned to 1.0 mg/kg at subsequent visits.
These findings suggest that the safety profile of VA over 24 months was acceptable, and the drug was well tolerated in all children in the present study.

| Efficacy assessments
VA efficacy was also preliminarily assessed for each patient using pharmacodynamic, functional, and QoL measures. Serum oligosaccharides were used as a surrogate marker of efficacy and were measured at baseline and over the VA treatment period (Figure 1). After 24 months of VA treatment, GlcNAc(Man)2 serum oligosaccharide concentrations decreased from baseline by 32% to 83% in the five children ( Figure 1A). GlcNAc(Man)3 ( Figure 1B) and GlcNAc(Man) 4 ( Figure 1C) serum concentrations also decreased compared to baseline, although data were limited for some children ( Figure 1). GlcNAc(Man)5 and GlcNAc(Man)6 serum concentrations remained under the lower limit of quantification in all children at all evaluated visits. Although three children (patients 2, 3, and 4) developed nAbs ( Figure S1), their GlcNAc(Man)2 serum levels remained below baseline during treatment. Patient 4's serum oligosaccharide concentrations for GlcNAc(Man)2 increased from 4.8 to 6.8 μmol/L between 12 and 24 months ( Figure 1A) and this patient had high ADA levels and nAbs at 24 months. No other ADApositive children in the study showed an increase in oligosaccharide levels. Thus, serum oligosaccharide levels were reduced in all children with available data treated with VA over 24 months ( Figure 1A-C). Changes in functional capacity were measured over the VA treatment period via PDMS-2 and MSEL assessments. After VA treatment, PDMS-2 sum of standard score percent increased from baseline in 2 children (patients 1 and 5) and decreased from baseline in 2 children (patients 3 and 4; Figure 2A). Although PDMS-2 assessment was completed in patient 2, standard scores could not be calculated for this child due to assessmentrelated age validation limitations. PDMS-2 score age equivalents are provided in Figure 2B, and MSEL age equivalent scores compared to patient age for each child are shown in Figure 2C. Most patients showed an increase in age-equivalent and raw MSEL scores, even though the magnitude and slope varied between children.
Endurance/physical functioning was assessed over the VA treatment period for each child via 6MWT ( Figure 3A) and 3MSCT ( Figure 3B). The mean (SD) distance walked at baseline and after 24 months of treatment were 295.8 (68.4) and 317.0 (105.1) meters, respectively. Mean (SD) 3MSCT results at baseline and 24 months were 138.8 (30.1) and 114.0 (58.6) steps, respectively. One child was uncooperative (patient 5) during the assessment, and the investigators noted that interpretation of test results might be limited due to high variability in baseline endurance/physical functioning capabilities and the feasibility of testing at a young age.
Hearing impairment in children has been cited as an early sign of AM and may contribute to earlier diagnosis and subsequent treatment 20 ; hearing was assessed in four children at baseline and 12 months after VA treatment via A-ABR audiometry ( Figure 5). At baseline, all children (n = 4) demonstrated clinically significant hearing impairment. After 12 months of treatment with VA, 3 children showed improved hearing in at least 1 ear, as measured by the A-ABR test. Two patients were The immunological profile of each child was assessed over the VA treatment period; serum concentrations of immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) were measured at baseline, 6, 12, 18, and 24 months ( Figure 4A). Baseline mean (SD) serum concentrations (n = 5) of IgG, IgA, and IgM were 6.1 (0.6), 1.0 (0.2), and 0.6 (0.1) g/L, respectively. After 24 months, mean (SD) serum concentrations (n = 4) of IgG, IgA, and IgM were 9.6 (2.5), 1.5 (0.6), 0.6 (0.3) g/L, respectively. As shown in Figure 4B, serum IgG levels trended toward an increase in each child over the VA treatment period. Additionally, at ≥24 months, serum IgA concentrations trended toward an increase in four of five children and trended toward a decrease in one child (patient 1) compared to baseline; serum IgM concentrations trended toward a decrease in four children and trended toward an increase in one child (patient 2) compared to baseline.
The PK profile of VA at the first dose and steady state (6 months after beginning VA treatment) was assessed to determine whether the VA dose at first infusion was metabolized comparably at both time points. Overall, the PK parameters measured were similar following the first VA dose and at steady state ( Table 3).
QoL of the children was assessed by administering the PEDI questionnaire to their parents at baseline, 12 months, and 24 months (with an additional assessment at 40 months in patient 2), as shown in Table S2. After at least 24 months of VA treatment, raw and scaled scores improved in all children in all domains compared to baseline. Percent changes in self-care, mobility, and social function from baseline to the final assessment ranged from 8.4% to 36.4%, 1.9% to 33.0%, and 2.4% to 58.6%, respectively.

| DISCUSSION
The present study evaluated VA's long-term safety and efficacy in children with AM aged less than 6 years. The safety and tolerability of VA were considered acceptable in all children who participated in the study. Even though all children experienced AEs, most were mild to moderate, and none (IRRs or ADRs) resulted in discontinuation of the study. Efficacy assessment results suggested that long-term treatment with VA may improve serum oligosaccharide levels, hearing impairment, IgG and IgA serum levels, and QoL. Further, children in the study did not demonstrate a marked decline in functional capacity or endurance/physical functioning over the VA treatment period, though additional studies controlling for age-based development are required. The PK profile of VA between the first dose and steady state was comparable.

| Safety outcomes
This is the first clinical investigation of the long-term safety profile of VA in children with AM under 6 years of age. While previous studies suggest that early treatment may provide greater clinical benefit to patients with AM F I G U R E 3 Endurance/physical functioning over VA treatment period (A) 6MWT and (B) 3MSCT. A lack of motivation was noted for patient 5, and data were only collected at the 12-month evaluation visit in (A) and at the 18-and 24-month evaluation visits in (B). 3MSCT, 3-minute stair climb test; 6MWT, 6-minute walk test; VA, velmanase alfa than when treatment is started later, 9 studies focusing on the natural history of AM are limited. Recent reports utilize retrospective data to forecast AM severity and prognosis in young children. 21 Thus, the safety findings of this study in young children are both timely and relevant. A common side effect of long-term ERT treatment is the development of ADAs that can impact drug efficacy and precipitate unwanted side effects. 22 Notably, safety assessments in the present study of children treated with VA revealed that while children did develop ADAs and experience IRRs, these events were well managed and did not prevent patients from completing the study. Specifically, one child developed nAbs and experienced IRRs; another child experienced IRRs before and after developing nAbs but did not experience IRRs simultaneously or shortly after developing nAbs; one child developed nAbs without experiencing any IRRs. While the safety data suggest that VA may be a safe and tolerable ERT for children with F I G U R E 4 Immunological profile over VA treatment period (A) Change in serum immunological profile during VA treatment (B) Individual changes in serum IgG levels over VA treatment period. ID, identification; SD, standard deviation; VA, velmanase alfa AM under the age of 6 years, the relationship between ADA status and the occurrence of IRRs is unclear. Thus, additional studies are warranted to assess the safety of VA.

| Efficacy outcomes
Multiple efficacy assessments described in the present study support previous findings that VA may provide clinical benefits in patients with AM. 9 In the present study, VA potentially demonstrated efficacy in all children for at least one assessment that examined common clinical symptoms of AM, including, but not limited to, a decrease in serum oligosaccharides and improvements in hearing, serum immunoglobulin levels, and/or QoL.
While the small size of the present study limits conclusions about VA efficacy, the findings are promising and support the need for more robust studies. For example, all patients' serum oligosaccharide GlcNAc(Man2) concentrations decreased from baseline. Notably, the magnitude by which VA reduced serum oligosaccharide isomers concentrations varied by child and nAb status. F I G U R E 5 Change in A-ABR audiometry from baseline by patient. Over the VA treatment period, hearing impairment improved numerically in each child. Auditory assessment at baseline was not performed for patient 1 (data not shown). Assessments were performed at unscheduled visits at approximately 9, 21, and 24 months after beginning VA treatment; after 24 months of treatment, an improvement in V wave thresholds was observed in both ears. Descriptors and hearing loss definitions were obtained from the American Academy of Audiology, Centers for Disease Control, and UCSF Audiology Otolaryngology. 26 A-ABR, automated auditory brainstem response; BL, baseline; CS, clinically significant; dB nHL, decibel above normal adult hearing level; mo, month; VA, velmanase alfa GlcNAc(Man2) and GlcNAc(Man3) (Figure 1) serum concentrations in 1 child (patient 4) showed an upward trend toward baseline levels that coincided with positive nAbs status at 24 months; other clinical measures, such as 6MWT, 3MSCT, and IgG concentration, remained relatively stable at 24 months compared to the prior assessment with a negative nAbs status. In contrast, another child (patient 2) with persistently positive nAb status demonstrated a consistent reduction in serum oligosaccharides for up to 40 months of VA treatment. While some patients may be more sensitive to nAb effects that can develop after long-term VA use, the relationship between nAb status and treatment efficacy remains unclear and requires further investigation. The present study indicates that hearing impairment may improve after long-term VA treatment. Patients with AM typically have mixed hearing loss, comprised of conductive and sensorineural impairment, the latter of which is often permanent. 23 A previous case study of a pediatric patient with AM reported that conductive hearing and serum IgG levels improved during VA treatment. 24 Children in the present study were assessed for hearing loss. One child (patient 3) had sensorineural hearing loss in both ears at baseline and after VA treatment but demonstrated an improvement of 20 dB nHL in the V wave threshold in the right ear at 12 and 24 months. Patient 5 was classified as having severe deafness at baseline and demonstrated a relevant improvement to moderate hypoacusia in both ears after VA treatment. Future studies are warranted using pure tone audiometry (PTA) to further assess hearing problems, including the conductive and sensorineural components of hearing loss. Such studies may help elucidate the potential therapeutic effect of VA on different types of hearing loss in pediatric patients. However, since PTA is not practical in very young children, tympanometry has only been assessed in older patients with more advanced disease to date. While some children in the present study may not have achieved clinically relevant improvements in hearing while receiving VA treatment, they all demonstrated numerical changes (improvements) from baseline in V wave threshold in one or both ears, and none demonstrated a worsening of hearing while on VA treatment ( Figure 5). Thus, further studies on the benefit of VA on hearing loss in pediatric patients are warranted. Impairment or deficiency in immunological profiles is also a common finding in patients with AM. 5 All children in the present study had serum IgG levels within or near the normal international standard range for IgG 25 at baseline and showed increases in serum IgG levels during VA treatment. Additionally, other serum immunoglobulins measured changed similarly, suggesting that long-term VA treatment may benefit the immune response in children. While this study did not directly investigate immune response, it would be interesting for future studies to consider the role VA may play in preventing frequent infections commonly associated with AM. 5 Further, these findings suggest that long-term VA treatment in young children may beneficially impact immunoglobulin levels. Thus, more robust studies are needed.
Social and physical aspects of patients' QoL are severely impacted as AM progresses, and early treatment may provide a more positive prognosis for patients as they age. PEDI results from the present study suggest that QoL improved over the long-term VA treatment period. However, expectation bias in surveys is an established phenomenon that could confound these results. Moreover, the findings of this study provide baseline data that may be useful to healthcare providers when monitoring their patients' overall QoL and ability to engage in ageappropriate life skills as they mature. Future studies should aim to elucidate whether VA treatment may delay AM-associated symptom progression, potentially improving the prognosis of patients' social dependence on caregivers and decreasing their likelihood of being wheelchair-bound later in life.
The present study also assessed changes in the functional capacity and endurance/physical functioning of children treated with long-term VA. As these results were highly variable between children, age-independent and additional studies are needed to determine whether longterm VA treatment provides any clinically meaningful benefit in these areas. Specifically, more data are needed on whether changes in PDMS-2, MSEL, 6MWT, and 3MSCT are independent of their "normal development potential", such as from placebo-controlled or natural history studies of patients with AM. Only patient 2 had reduced endurance/physical functioning at 24 months compared to baseline (possibly related to the nAbs-positive status of this patient at the corresponding time point); however, at 24 months, patient 2 also had reduced oligosaccharide concentrations, higher MSEL scores in three of four categories, maintenance in PDMS-2 scores, and increased IgG concentration. Nevertheless, the baseline data collected from this patient population provide valuable insights into the natural history of AM in patients under 6 years old, which was largely unknown previously. In addition to further understanding the natural history of AM, follow-up studies regarding efficacy and response to treatment in this patient population are warranted.
This study also elucidated the PK profile of VA in children under 6 years of age. PK data revealed that systemic drug availability of the first dose of VA was comparable to the PK profile of VA at steady state in this young AM patient population.

| Limitations
Taken together, the present study provides critical insights into AM disease progression in the younger pediatric population, although conclusions are limited due to the small sample size and lack of a randomized control arm. This study included functional assessments, including the PDMS, 6MWT, and 3MSCT, which can be challenging to administer to children of a young age since these evaluations rely on participant cooperation. Additionally, these evaluations can be associated with high variability at baseline and may have inherent limitations to age-related validation. Inherently, alpha-mannosidosis is associated with heterogeneous disease manifestations that vary across patients and can make comparisons difficult among participants. While this study includes data collected up to 3 years, there is limited information available at some timepoints for certain children, including serum oligosaccharide evaluations. Additionally, 3 years of follow-up may not be sufficiently long enough to assess or reveal VA-associated improvements or stabilization of disease given the slow progression of AM. Further limitations may include utilization of serum oligosaccharide levels as surrogate marker of treatment efficacy.

| CONCLUSIONS
Notably, after 24 months of treatment, most children with available baseline data showed maintenance of or increased endurance/physical functioning in both the 6MWT and 3MSCT, though further investigation of the normal developmental potential as an explanation is warranted. Despite the development of ADAs, AEs were mild or moderate, including IRRs, which were readily managed, and children were able to continue long-term treatment. The improvements observed in the efficacy outcomes support the view that long-term VA treatment in children <6 years may be clinically beneficial. Finally, this study offers meaningful insights into the management of AM and provides promising results for the benefits of early and viable ERT treatment in young pediatric patients with AM that have not been previously described.

PLAIN LANGUAGE SUMMARY
A plain language summary of this study is available as supplemental material.

AUTHOR CONTRIBUTIONS
Ferdinando Ceravolo and Allan Lund provided substantial contributions to the conception and design of the work. All authors provided substantial contributions to the acquisition, analysis, and interpretation of data for this analysis. All authors contributed to drafting/revising the work and provided final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. consultant for Chiesi Farmaceutici S.p.A., Parma, Italy. Andrea Ballabeni is employed at Chiesi Farmaceutici S.p. A., Parma, Italy. Allan Lund has received hospital grants as a PI for this study from Chiesi Farmaceutici S.p.A. as well as consulting fees and/or honoraria/travel support from Alexion, BioMarin, Chiesi Farmaceutici S.p.A, Sanofi Genzyme, Shire DATA AVAILABILITY STATEMENT At this time, we will approve or deny data requests from external parties on a case-by-case basis. Chiesi Farmaceutici S.p.A. reserves the right to deny requests for any and all legally appropriate reasons. Data requests that risk sharing participant-level data or proprietary information will not be approved.

ETHICS STATEMENT
The study protocol, patient information, and patient informed consent forms were reviewed and approved by an Independent Ethics Committee (De Videnskabsetiske Komiteer Region Hovedstaden; Hillerød, Denmark) and a Regulatory Agency (Danish Medicines Agency; København S, Denmark) complying with the requirements of European Federal regulations and the International Conference on Harmonisation (ICH) before enrollment of patients. This study was conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki 1964, as revised in 2013), ICH good clinical practice guidelines, local guidelines, and applicable regulations when developing, obtaining, and documenting the patient informed consent.

INFORMED CONSENT STATEMENT
Informed consent was obtained from all individual participants included in the study.