Treatment methods for late-onset Pompe disease in pediatric patients

Abstract

Provided herein are methods for treating Pompe disease in pediatric patients by administering to the subject a population of recombinant human acid α-glucosidase molecules, or a pharmaceutical composition or formulation thereof, and a pharmacological chaperone.

Description

[Technical Field]

[0001] The present disclosure relates to methods for treating late-onset Pompe disease (LOPD) in pediatric patients by administering recombinant human α-glucosidase. [Background technology]

[0002] Pompe disease (also known as glycogen storage disease type II (GSD II) or acid maltase deficiency) is an inherited lysosomal storage disorder caused by a deficiency in acid alpha-glucosidase (GAA) activity. People with Pompe disease lack or have low levels of GAA, the enzyme that breaks down glycogen into glucose, the main energy source for muscles. This enzyme deficiency causes excess glycogen to accumulate in lysosomes, intracellular organelles that normally contain enzymes that break down glycogen and other cellular debris or waste products.

[0003] The accumulation of glycogen in certain tissues, especially muscles, of subjects with Pompe disease impairs the ability of cells to function normally. In Pompe disease, glycogen is not metabolized properly and gradually accumulates in the lysosomes of skeletal muscle cells, especially in the infantile-onset form of the disease, and cardiac muscle cells. The accumulation of glycogen damages muscle and nerve cells, as well as cells in other affected tissues.

[0004] Current non-palliative treatments for Pompe disease involve enzyme replacement therapy (ERT) using recombinant alglucosidase alfa preparations sold under the trademarks LUMIZYME®, MYOZYME®, or NEXVIAZYME®. This conventional enzyme replacement therapy attempts to treat Pompe disease by administering rhGAA to replenish the missing GAA in lysosomes, thereby restoring the cell's ability to break down lysosomal glycogen. LUMIZYME®, MYOZYME®, and NEXVIAZYME® are U.S. Food and Drug Administration-approved conventional rhGAA products produced or sold as biologics by Sanofi Genzyme and are listed by reference in the Physician's Desk Reference (2014), which is hereby incorporated by reference. Summary of the Invention [Problem to be solved by the invention]

[0005] However, currently available ERTs, at best, provide limited improvement in muscle function, muscle strength, and respiratory function measures for a limited period of time, after which these parameters slowly deteriorate (Toscano and Schoser, (2013) J Neurol 260, 951-959; Wyatt et al., (2012) Health Technol Assess 16(39)). There is a need for Pompe disease treatments that provide long-term improvement.

[0006] Recently, novel rhGAA formulations have been developed that improve the efficacy of restoring cellular glycogenolytic capacity by increasing the binding efficiency of rhGAA to lysosomal receptors. Furthermore, the inventors have also found that combining rhGAA with pharmacological chaperones increases efficacy. However, these treatments have only been established for adult patient populations. There is a need for a treatment for late-onset Pompe disease in the pediatric (under 18 years of age) population. [Means for solving the problem]

[0007] Various aspects of the present disclosure relate to treating Pompe disease in pediatric patients by administering recombinant human α-glucosidase (rhGAA) in combination with a pharmacological chaperone. [Brief explanation of the drawings]

[0008] [Figure 1A] Non-phosphorylated high-mannose N-glycans, mono-M6P N-glycans, and bis-M6P N-glycans are shown, where each box represents N-acetylglucosamine (GlcNAc), each circle represents mannose, and each P represents phosphate. [Figure 1B] The chemical structure of the M6P group is shown. [Figure 2A] Productive targeting of rhGAA to target tissues (eg, muscle tissue in subjects with Pompe disease) by M6P-bearing N-glycans is described. [Figure 2B] This explains non-productive drug clearance to non-target tissues (eg, liver and spleen) or due to binding of non-M6P N-glycans to non-target tissues. [Figure 3] FIG. 1 is a schematic diagram of an exemplary production, capture, and purification process for recombinant lysosomal proteins. [Figure 4] 1 shows a DNA construct for transformation of CHO cells with DNA encoding rhGAA. [Figure 5]1 is a graph showing the results of CIMPR affinity chromatography of ATB200 rhGAA with (Embodiment 2) and without (Embodiment 1) capture on an anion exchange (AEX) column. [Figure 6A] Figures 6A to 6H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS / MS analytical techniques. Figure 6A shows the site occupancy of seven potential N-glycosylation sites for ATB200. [Figure 6B] Two analyses of the N-glycosylation profile of the first potential N-glycosylation site for ATB200 are shown. [Figure 6C] Two analyses of the N-glycosylation profile of the second potential N-glycosylation site for ATB200 are shown. [Figure 6D] Two analyses of the N-glycosylation profile of the third potential N-glycosylation site for ATB200 are shown. [Figure 6E] Two analyses of the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200 are shown. [Figure 6F] Two analyses of the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200 are shown. [Figure 6G] Two analyses of the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200 are shown. [Figure 6H] The relative percentages of mono- and bis-phosphorylated species for the first, second, third, fourth, fifth, and sixth potential N-glycosylation sites are summarized. [Figure 7] 1 is a graph showing the Polywax dissolution profiles of LUMIZYME® (thin line, eluting on the left) and ATB200 (thick line, eluting on the right). [Figure 8]1 is a table showing a summary of the N-glycan structure of LUMIZYME® compared to three different formulations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1, and ATB200-2. [Figure 9A] 1 is a graph showing the results of CIMPR affinity chromatography of LUMIZYME®. [Figure 9B] 1 is a graph showing the results of CIMPR affinity chromatography of MYOZYME®. [Figure 10A] 1 is a graph comparing ATB200 rhGAA (left line) with LUMIZYME® (right line) for CIMPR binding affinity. [Figure 10B] 1 is a table comparing bis-M6P content between LUMIZYME® and ATB200 rhGAA. [Figure 11A] 1 is a graph comparing ATB200 rhGAA activity (left trace) with LUMIZYME® rhGAA activity (right trace) inside normal fibroblasts at various GAA concentrations. [Figure 11B] 1 is a table comparing ATB200 rhGAA activity (left trace) with LUMIZYME® rhGAA activity (right trace) within fibroblasts from subjects with Pompe disease at various GAA concentrations. [Figure 11C] 1 is a table comparing Kuptake in fibroblasts from normal and Pompe disease subjects. [Figure 12] 1 illustrates the stability of ATB200 in acidic or neutral pH buffers as determined in a thermal stability assay using SYPRO orange, following an increase in dye fluorescence upon protein denaturation. [Figure 13]Figure 1 shows tissue glycogen content in WT mice or Gaa KO mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221, as determined using amyloglucosidase digestion. Bars represent the mean ± SEM of 7 mice per group. *p<0.05 compared to alglucosidase alfa in one-way ANOVA analysis using Dunnett's multiple comparison method. [Figure 14] LAMP1-positive vesicles are shown in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and are representative of seven mice from each group. Magnification = 200x (inset = 1,000x). [Figure 15A] LC3-positive aggregates in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and were representative of seven mice from each group. Magnification = 400x. [Figure 15B] Western blot analysis of LC3 II protein. A total of 30 mg of protein was loaded in each lane. [Figure 16] Figure 1 shows dysferlin expression in muscle fibers from Gaa KO or WT mice treated with vehicle, alglucosidase alfa, or ATB200 / AT2221. Images were taken from the vastus lateralis muscle and were representative of seven mice from each group. Magnification = 200x. [Figure 17] Co-immunofluorescence staining of LAMP1 (green) (see, e.g., "B") and LC3 (red) (see, e.g., "A") in a single fiber isolated from the white gastrocnemius muscle of Gaa KO mice treated with vehicle, alglucosidase alfa, or ATB200 is shown. "C" illustrates the clearance of autophagic debris and the absence of enlarged lysosomes. A minimum of 30 fibers were examined from each animal. [Figure 18]1 illustrates the stabilization of ATB200 by 17 μM AT2221 and 170 μM AT2221, respectively, compared to ATB200 alone. [Figure 19A] Figures 19A-19H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA, including the N-glycosylation profile for the seventh potential N-glycosylation site, using LC-MS / MS analysis of protease-digested ATB200. Figures 19A-19H provide average data for 10 lots of ATB200 produced at different scales. Figure 19A shows the average site occupancy of the seven potential N-glycosylation sites for ATB200. The N-glycosylation sites are provided according to SEQ ID NO: 1. CV = coefficient of variation. [Figure 19B] Figures 19B to 19H show site-specific N-glycosylation analysis of all seven potential N-glycosylation sites for ATB200, where the site numbers are provided according to SEQ ID NO: 5. The bars represent the maximum and minimum percentages of N-glycan species identified as specific N-glycan groups for the 10 lots of ATB200 analyzed. Figure 19B shows the N-glycosylation profile of the first potential N-glycosylation site for ATB200. [Figure 19C] 1 shows the N-glycosylation profile of the second potential N-glycosylation site for ATB200. [Figure 19D] 1 shows the N-glycosylation profile of the third potential N-glycosylation site for ATB200. [Figure 19E] 1 shows the N-glycosylation profile of the fourth potential N-glycosylation site for ATB200. [Figure 19F] 1 shows the N-glycosylation profile of the fifth potential N-glycosylation site for ATB200. [Figure 19G] 1 shows the N-glycosylation profile of the sixth potential N-glycosylation site for ATB200. [Figure 19H] 1 shows the N-glycosylation profile of the seventh potential N-glycosylation site for ATB200. [Figure 20A] Figures 20A-20B further characterize and summarize the N-glycosylation profile of ATB200, as also shown in Figures 19A-19H. Figure 20A shows 2-anthranilic acid (2-AA) glycan mapping and LC / MS-MS analysis of ATB200, summarizing the N-glycan species identified in ATB200 as a percentage of total fluorescence. The 2-AA glycan mapping and LC-MS / MS analysis data are also graphically represented in Table 7. [Figure 20B] The average site occupancy and average N-glycan profile, including total phosphorylation, monophosphorylation, bisphosphorylation, and sialylation, for all seven potential N-glycosylation sites of ATB200 are summarized. ND = not detected. [Figure 21] A schematic diagram of the ATB200-03 study design is shown. [Figure 22] Baseline 6-minute walk distance (6MWD) and seated forced vital capacity (FVC) characteristics of 122 subjects enrolled in the ATB200-03 study are shown. AT-GAA group: ATB200 / AT2221-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 23A] 6MWD and FVC data are plotted for the entire population (n=122), showing baseline, change from baseline at week 52 ("CFBL"), difference, and P-value. AT-GAA group: ATB200 / miglustat-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 23B] 6MWD and FVC data showing change from baseline over time are shown graphically for the total population (n=122). Sipaglucosidase alfa / miglustat group: ATB200 / miglustat-treated subjects; alglucosidase alfa / placebo: alglucosidase alfa / placebo-treated subjects. [Figure 24]6MWD and FVC data are illustrated for the ERT-experienced population (n=95), showing CFBL at baseline, week 52, difference, and P-values. AT-GAA group: ATB200 / miglustat-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 25] Figure 1 illustrates the relative change versus baseline in 6MWD and FVC at weeks 12, 26, 38, and 52 for the ERT-experienced population (n=95). [Figure 26A] 6MWD and FVC data are illustrated for the ERT-naive population (n=27), showing CFBL, difference, and P-values ​​at baseline, week 52. AT-GAA group: ATB200 / miglustat-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 26B] 6MWD and FVC data are plotted for the ERT-naive population (n=27), showing change from baseline over time. Sipaglucosidase alfa / miglustat group: ATB200 / miglustat-treated subjects; alglucosidase alfa / placebo: alglucosidase alfa / placebo-treated subjects. [Figure 27] Baseline characteristics for key secondary endpoints and biomarkers for the overall and ERT-experienced populations are depicted. AT-GAA group: ATB200 / miglustat-treated subjects; Alglucosidase alfa group: Alglucosidase alfa / placebo-treated subjects. [Figure 28] Relative change from baseline in lower extremity manual muscle testing (MMT) at weeks 12, 26, 38, and 52 is depicted for the overall population (left) and the ERT-experienced population (right). [Figure 29]Relative change from baseline in Walk-Stair-Gowers-Chair (GSGC) is depicted for the total population (left) and the ERT-experienced population (right) at weeks 12, 26, 38, and 52. Sipaglucosidase alfa / miglustat group: ATB200 / miglustat-treated subjects; alglucosidase alfa / placebo: alglucosidase alfa / placebo-treated subjects. [Figure 30] Patient-Reported Outcomes Measurement Information System (PROMIS) results for relative change from baseline in physical function at weeks 12, 26, 38, and 52 are shown for the overall population (left) and the ERT-experienced population (right). [Figure 31] PROMIS is illustrated for the relative change from baseline in fatigue at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right). [Figure 32] Relative change versus baseline in creatine kinase (CK) biomarkers at weeks 12, 26, 38, and 52 for the overall population (left) and the ERT-experienced population (right) is depicted. [Figure 33] Relative change versus baseline in urinary hexose tetrasaccharide (Hex4) biomarkers at weeks 12, 26, 38, and 52 for the total population (left) and the ERT-experienced population (right) is depicted. [Figure 34] Primary, secondary, and biomarker endpoint heat maps are shown for the overall population (left) and the ERT-experienced population (right). AT-GAA group: ATB200 / miglustat-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. [Figure 35] Safety data from the ATB200-03 study are summarized below. AT-GAA group: ATB200 / miglustat-treated subjects; alglucosidase alfa group: alglucosidase alfa / placebo-treated subjects. TEAE: treatment-emergent adverse event; IAR: infusion-related reaction. [Figure 36]Summarize the results of the ATB200-03 study. [Figure 37] Explain the test objectives and statistical methods of the ATB200-03 test. [Figure 38] The primary and secondary endpoints of the ATB200-03 study are described below. [Figure 39] Summarize patient allocation for the ATB200-03 study. [Figure 40] Summarize the baseline demographic characteristics of the ATB200-03 study. [Figure 41A-41B] Subgroup analyses of change from baseline in 6MWD and FVC by baseline status are shown for the total population (n=122) in the ATB200-03 study (Figure 41A) and for ERT-experienced patients (n=95) in the ATB200-03 study (Figure 41B). [Figure 42] Treatment-emergent adverse events (TEAEs) occurring in ≥10% of patients in any arm of the ATB200-03 study are listed below. [Figure 43] The study design for the Phase I / II ATB200-02 study is shown. An asterisk indicates that prior ERT was with 20 mg / kg alglucosidase alfa Q2W. Q2W, every 2 weeks [Figure 44] An overview of the endpoints and cohorts reported for the ATB200-02 study is provided below. [Figure 45] Baseline characteristics and patient disposition for the ATB200-02 study are shown. An asterisk indicates that one ERT-naive patient had received one dose of alglucosidase alfa more than 6 months prior to study enrollment. M means meters; M:F means male:female ratio; N / A means not applicable; SD means standard deviation. [Figures 46A-46B] FIG. 46 shows the mean change from baseline (CFBL) in 6-minute walk distance (6MWD) over time for ERT-experienced (FIG. 46A) and ERT-naive (FIG. 46B) subjects in the ATB200-02 study. [Figure 47A-47B]FIG. 47 shows mean change from baseline (CFBL) in percent versus predicted sitting forced vital capacity (FVC) over time for ERT-experienced ( FIG. 47A ) and ERT-naive ( FIG. 47B ) subjects in the ATB200-02 study. [Figure 48A-48B] FIG. 48 shows the mean change from baseline (CFBL) in manual muscle testing (MMT) lower extremity scores over time for ERT-experienced ( FIG. 48A ) and ERT-naive ( FIG. 48B ) subjects in the ATB200-02 study. [Figure 49A-49B] 49A and 49B show mean percentage change from baseline (CFBL) in urinary hexose tetrasaccharide (Hex4) levels (FIG. 49A) and plasma creatine kinase (CK) levels (FIG. 49B) for ERT-experienced and ERT-naive subjects in the ATB200-02 study. [Figure 50] A summary of treatment-emergent adverse events (TEAEs) in the ATB200-02 study is shown. Asterisks indicate diffuse large B-cell lymphoma. IAR means infusion-related reaction; TEAE means treatment-emergent adverse event with onset date on or after the first dose of study drug. [Figure 51] Figure 1 shows a comparison of the long-term effects of sipaglucosidase alfa / miglustat and avalglucosidase alfa on change from baseline in 6MWD and percentage-to-predicted FVC (sitting) in subjects with prior ERT. [Figure 52] Figure 1 shows a comparison of the long-term effects of sipaglucosidase alfa / miglustat and avalglucosidase alfa on change from baseline in 6MWD and percentage-to-predicted FVC (sitting) in ERT-naive subjects. [Figure 53A-53B] Figure 53B shows the 6-minute walk test (6MWT) percentage versus predicted value during treatment with alglucosidase alfa in subjects with a history of ERT. Figure 53B shows the data from Figure 53A replotted from 2 years onwards only. [Figure 54] Figure 1 shows FVC percentage versus predicted value during treatment with alglucosidase alfa in subjects with a history of ERT. [Figure 55]The following shows an overview of the endpoints and cohort for Cohort 2 of the ATB200-02 study (non-ambulatory patients with a history of ERT). [Figure 56] Baseline characteristics and patient disposition for Cohort 2 (non-ambulatory patients with prior ERT) of the ATB200-02 study are shown. An asterisk indicates that the baseline assessment is the last non-missing result at or before the administration of the first dose of study medication (20 mg / kg sipa glucosidase alfa + 260 mg miglustat co-administered dose). M:F means male:female ratio; SD means standard deviation. [Figure 57] Figure 1 shows mean change from baseline (CFBL) in percent-predicted sitting forced vital capacity (FVC) over time in Cohort 2 (non-ambulatory patients with a history of ERT). [Figure 58] This figure shows an overview of treatment-emergent adverse events (TEAEs) for Cohort 2 (non-ambulatory patients with a history of ERT) in the ATB200-02 study. An asterisk indicates urticaria, which was considered an IAR. IAR refers to an infusion-related reaction; TEAE refers to a treatment-emergent adverse event with an onset date on or after the first dose of study drug. [Figure 59] Baseline characteristics of seven clinical trials identified by systematic literature review (SLR) are presented. [Figure 60] For each of the identified studies, longitudinal efficacy results for the trials of 6MWD (m) as change from baseline and FVC (% predicted) are presented. [Figure 61] The network of 6MWD (m) and locus FVC (% predicted) is shown. [Figure 62] Forest plots of relative effect estimates and 95% credible intervals for 6MWD in the base-case scenario (primary analysis) are shown. [Figure 63] Forest plot of relative effect estimates and 95% credible intervals for FVC in the base-case scenario (primary analysis) is shown. [Figure 64]Forest plots of relative effect estimates and 95% credible intervals for 6MWD by ERT duration are shown. [Figure 65] Forest plots of relative effect estimates and 95% credible intervals for FVC by ERT duration are shown. [Figure 66] Forest plots of relative effect estimates and 95% credible intervals for 6MWD in the base-case scenario (sensitivity analysis) are shown. [Figure 67] Forest plot of relative effect estimates and 95% credible intervals for FVC in the base case scenario (sensitivity analysis) is shown. [Figure 68] Figure 1 shows total GAA protein with signature peptide T09 versus time after start of sipa glucosidase alfa infusion. [Figure 69] Predicted sipa glucosidase alfa exposure (AUC0-24h and Cmax) for adults (N=100) and adolescents (N=8) is shown. [Figure 70] 10 demonstrates the miglustat concentration-time profiles observed in adolescents and adults following administration of sipa glucosidase alfa and miglustat. [Figure 71] Predicted miglustat exposure (AUC0-24 and Cmax) for adults (N=100) and adolescents (N=8) is shown. [Figure 72] Shown are mean values ​​± SD (error bars) representing miglustat concentrations (ng / mL) over a 24 hour course for capsules or prototypes 1, 2, 3, or 4 in rabbits. [Figure 73] Shown are log-transformed means ± SD (error bars) representing miglustat concentrations (ng / mL) over a 24-hour course for capsules or prototypes 1, 2, 3, or 4 in rabbits. [Figure 74] PK summary plots of mean ± SD (error bars) and log-transformed mean ± SD (error bars) miglustat concentrations over a 72-hour time course following administration of either prototype 3 or 4 at a 260 mg dose to humans. [Figure 75]PK summary plots of mean ± SD (error bars) and log-transformed mean ± SD (error bars) miglustat concentrations over a 72-hour time course following administration of either prototype 3 or 4 at a dose of 1040 mg to humans. [Figure 76] 1 shows dose-proportional PK summary plots for mean ± SD (error bars) miglustat concentrations in humans following administration of prototype 3 or 4 at either a 260 mg or 1040 mg dose over a 72 hour time course. DETAILED DESCRIPTION OF THE INVENTION

[0009] Provided herein are methods for treating late-onset Pompe disease (LOPD), comprising administering recombinant human α-glucosidase (rhGAA) and a pharmacological chaperone to an individual under the age of 18. The methods may provide benefits such as improved cardiac function (e.g., LVMi or ejection fraction), exercise function, muscle strength, and / or pulmonary function. The methods provided herein also have a favorable safety profile. The benefits of the methods provided herein are improvements over enzyme replacement therapy (ERT), the current treatment option for Pompe disease.

[0010] Current ERT options for treating Pompe disease result in limited improvement followed by slow deterioration. In 2012, a systematic review of all studies conducted on subjects with late-onset Pompe disease (LOPD) was conducted (Toscano and Schoser, (2013) J Neurol 260, 951-959). This review included data on 368 LOPD subjects from published studies, including 27 juvenile subjects (age range: 2-17 years) and 251 adult subjects who had been treated with alglucosidase alfa for at least the previous 2 years. Results indicated that more than 30% of subjects did not achieve initial improvement upon treatment with alglucosidase alfa and continued to experience deterioration in muscle and respiratory function despite treatment. In the group of subjects who initially responded to alglucosidase alfa treatment, several further long-term studies revealed that improvement usually lasted only about 2 years. Afterwards, subjects generally reached a plateau before gradually beginning to deteriorate.

[0011] In 2012, the United Kingdom Health Technology Assessment programme (Wyatt et al., (2012) Health Technology Assessment) was launched as part of the UK National Institute for Health Research. 16 (39) issued recommendations derived from a review of longitudinal data on 81 patients with Pompe disease (including infantile-onset and late-onset (children and adults)) who received the currently approved standard of care ERT, alglucosidase alfa. Key markers of Pompe disease progression (forced vital capacity, ventilator dependence, mobility, 6-minute walk test, muscle strength, and body mass index) were assessed and modeled by time on treatment with alglucosidase alfa. Results of this evaluation indicated that improvements in FVC, 6-minute walk distance, and muscle strength in patients with LOPD occurred during the first 2 years after initiation of ERT with alglucosidase alfa, with deterioration occurring beyond this time frame as treatment continued. Additionally, a 3-year study of 38 subjects with LOPD receiving alglucosidase alfa found that subjects demonstrated improvement in motor function during the first year of treatment, which generally remained stable during the second year and began to deteriorate during the third year (Regnery et al. al., (2012) Journal of Inherited Metabolic Disease 35:837-845).

[0012] Furthermore, a report providing 10-year follow-up of the Phase 3 LUMIZYME® (Genzyme Corporation) study found that after some improvement in motor and pulmonary function during the first few years of treatment, subjects slowly began to deteriorate while continuing treatment (van der Ploeg et al., (2017) European journal of neurology 24.6:768-e31). In this study, there was an average deterioration of approximately 10% of baseline 6-minute walk distance percent predicted between the third and sixth years of therapy, with approximately 80% of subjects experiencing deterioration.

[0013] The most significant tolerability issue with alglucosidase alfa is the occurrence of infusion-related reactions (IARs), which in some cases can include life-threatening anaphylaxis or other severe allergic reactions (MYOZYME® Summary of Product Characteristics, December 2018). Management of these events includes dose reduction, reduction in infusion rate and prolonged infusion duration, and interruption or discontinuation of administration. Premedication (before infusion) with antihistamines and steroids is also commonly used to prevent and reduce the occurrence and severity of IARs and hypersensitivity reactions associated with alglucosidase alfa infusions. Despite these measures, IARs can still occur in Pompe disease patients, some of whom are unable to tolerate regular infusions of currently approved ERTs.

[0014] In 2017, the European Pompe Consortium, a network of experts in the field of Pompe disease from 11 European countries, conducted a systematic review of the literature (van der Ploeg et al., (2017) European Journal of Neurology 24.6:768-e31). The Consortium evaluated the evidence for the effectiveness of ERT at the group level based on data from one clinical trial and 43 observational studies, covering a total of 586 individual adult subjects. The current consensus of the European Pompe Consortium is that the occurrence of severe IAR or progressive clinical worsening of disease symptoms, as well as the development of high neutralizing antibody (Ab) titers, effectively renders existing ERT treatment ineffective and should lead to the discontinuation of ERT therapy. The European Pompe Consortium consensus also includes consideration of restarting ERT treatment if disease progression and clinical deterioration recurs after stopping ERT.

[0015] Cellular uptake of rhGAA molecules is facilitated by a specialized carbohydrate, mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on target cells, such as muscle cells. Upon binding, rhGAA molecules are internalized by the target cells and subsequently transported to intracellular lysosomes. However, many conventional rhGAA formulations lack a high total content of mono-M6P- and bis-M6P-bearing N-glycans (i.e., N-glycans bearing one or two M6P residues, respectively), limiting their CIMPR-mediated cellular uptake and lysosomal delivery, ultimately resulting in the ineffectiveness of conventional enzyme replacement therapy. For example, conventional rhGAA formulations at doses of 20 mg / kg or greater, while indeed improving some aspects of Pompe disease, are not sufficiently potent at reversing disease progression, particularly in terms of (i) treating underlying cellular dysfunction, (ii) restoring muscle structure, or (iii) reducing accumulated glycogen in many target tissues, such as skeletal muscle. Furthermore, higher doses can impose additional burdens on the subject and the medical professionals treating them, such as the longer infusion times required for intravenous administration of rhGAA.

[0016] Enzymatic modification of the glycosylation of GAA or rhGAA in vitro using phosphotransferases and uncoating enzymes, as described by Canfield et al. in U.S. Pat. No. 6,534,300, can create M6P groups. However, enzymatic glycosylation cannot be adequately controlled, potentially producing rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may contain only high-mannose oligosaccharides, all of which may be enzymatically phosphorylated in vitro by phosphotransferases or uncoating enzymes. The glycosylation pattern produced by enzymatic treatment of GAA in vitro is problematic because additional terminal mannose residues, especially non-phosphorylated terminal mannose residues, adversely affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified preparations are administered in vivo, their mannose groups increase the non-productive clearance of GAA and increase the uptake of enzymatically modified GAA by immune cells, resulting in less GAA reaching target tissues, such as skeletal muscle cells, thereby reducing the therapeutic efficacy of rhGAA. For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, leading to rapid clearance of enzymatically modified rhGAA and less rhGAA being targeted to target tissues. Furthermore, the glycosylation pattern of enzymatically modified GAA, which has high-mannose N-glycans with terminal non-phosphorylated mannose residues, is similar to that found in glycoproteins produced by yeast and mold, increasing the risk of immune or allergic reactions, such as life-threatening severe allergic (anaphylactic) or hypersensitivity reactions, to enzymatically modified rhGAA.

[0017] Compared with conventional recombinant rhGAA preparations and in vitro phosphorylated rhGAA, the rhGAA used in the binary therapy described herein has an optimized N-glycan profile for enhanced biodistribution and lysosomal uptake, thereby minimizing non-productive clearance of rhGAA after administration. These formulations provide patients with stable or worsening Pompe disease with an effective therapy that reverses disease progression at the cellular level—including by clearing lysosomal glycogen more efficiently than current standard treatments. Patients treated with binary therapy containing rhGAA and a pharmacological chaperone (e.g., miglustat) exhibit significant health improvements, including improvements in cardiac function, muscle strength, exercise function, and / or pulmonary function, and / or reversal of disease progression.

[0018] Furthermore, comparisons of three randomized clinical trials (LOTS: alglucosidase alfa vs. placebo; COMET: avalglucosidase alfa vs. alglucosidase alfa; and PROPEL: sipaglucosidase alfa / miglustat vs. alglucosidase alfa) share key primary or secondary endpoints: 6MWD and %-predicted FVC. Using patient-level data from the PROPEL randomized clinical trial (RCT) plus aggregated published data from other RCTs, phase I / II, and open-label extension studies, we performed multilevel network meta-regression, adjusting for various baseline covariates, including prior ERT duration. A base-case scenario included covariates similar to the PROPEL population (naive and ERT-experienced), and analyzed the change in 6MWD and FVC from baseline at 52 weeks. Sipaglucosidase alfa / miglustat compared favorably with alglucosidase alfa and avalglucosidase alfa for 6MWD and FVC, with a relative effect of 16.3 m (95% CI: 9.6-24.3) and 29.5 m (7.4-52.6), respectively, for 6MWD, and 3.1% (2.4-3.8) and 2.8% (1.0-4.6) for FVC. In naive subjects, the relative effect of sipaglucosidase alfa / miglustat remained significant for both endpoints, except for FVC, which was 0.4% (-0.8-1.7) for sipaglucosidase alfa / miglustat compared with avalglucosidase alfa. These findings suggest that sipaglucosidase alfa / miglustat may clinically differentiate patients with LOPD with respect to key motor and respiratory endpoints, particularly in the ERT-experienced population.

[0019] definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them. The articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical referent of the article. The term "or" means, and is used synonymously with, the term "and / or" unless the context clearly dictates otherwise. In this application, use of the singular includes the plural unless specifically stated otherwise. Furthermore, use of the term "including" and other forms, such as "includes" and "included," is not limiting. Any ranges described herein will be understood to include the endpoints and all values ​​between the endpoints. In this specification, unless the context otherwise requires by express language or necessary implication, the word "comprises" or variations such as "comprising" are used in an inclusive sense, i.e., to specify the presence of the indicated features but not to exclude the presence or addition of further features in various embodiments of the present disclosure.

[0020] The term "GAA" refers to the human acid α-glucosidase (GAA) enzyme, which catalyzes the hydrolysis of α-1,4- and α-1,6-glycosidic bonds in lysosomal glycogen, as well as insertion, relational, or substitution variants of the GAA amino acid sequence and fragments of the long GAA sequence that exhibit enzymatic activity. Human acid α-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) gene ID 2548), which has been mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). An exemplary amino acid sequence of GAA is NP 000143.2 (incorporated by reference). The present disclosure also encompasses DNA sequences encoding the amino acid sequence of NP 000143.2. Currently, over 500 mutations in the human GAA gene have been identified, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect the maturation and processing of this enzyme include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE (SEQ ID NO: 7) at amino acid residues 516-521 is required for the activity of the acid α-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the human acid α-glucosidase enzyme, while the italicized abbreviation "GAA" is intended to refer to the human gene encoding the human acid α-glucosidase enzyme. The italicized abbreviation "Gaa" is intended to refer to a non-human gene encoding a non-human acid alpha-glucosidase enzyme, including, but not limited to, a rat or mouse gene; the abbreviation "Gaa" is intended to refer to a non-human acid alpha-glucosidase enzyme.

[0021] The term "rhGAA" is intended to refer to recombinant human acid α-glucosidase enzyme and is used to distinguish endogenous GAA from synthetic or recombinantly produced GAA (e.g., GAA produced from CHO cells or other host cells transformed with DNA encoding GAA). The term "rhGAA" encompasses populations of individual rhGAA molecules. Properties of rhGAA molecule populations are provided herein. The term "conventional rhGAA formulation" is intended to refer to formulations containing alglucosidase alfa, such as LUMIZYME®, MYOZYME®, or avalglucosidase alfa, e.g., NEXVIAZYME®.

[0022] The terms "genetically modified" or "recombinant" refer to cells, such as CHO cells, that express a particular gene product, such as rhGAA, after introduction of a nucleic acid comprising a coding sequence that encodes the gene product along with regulatory elements that control expression of the coding sequence. Introduction of the nucleic acid may be accomplished by any method known in the art, including gene targeting and homologous recombination. As used herein, the terms also include cells that have been engineered, for example, by gene activation techniques, to express or overexpress an endogenous gene or a gene product that is not normally expressed by such cells.

[0023] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid alpha-glucosidase identified as [199-arginine, 223-histidine] prepro-alpha-glucosidase (human); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa is approved for sale in the United States by Genzyme under the formulations LUMIZYME®, MYOZYME®, and NEXVIAZYME®.

[0024] As used herein, the term "ATB200" is intended to refer to the recombinant human acid α-glucosidase described in PCT / 2015 / 053252, U.S. Pat. No. 10,208,299, and U.S. Pat. No. 10,961,522 (the disclosures of which are incorporated herein by reference). ATB200 is also referred to as "sipa glucosidase alfa" or "sipa glucosidase alfa-atga." In some embodiments, "ATB200" refers to rhGAA, which is enriched in N-glycans carrying mono-M6P and bis-M6P, and which is produced and purified from the GA-ATB200 cell line using the methods described herein. Sipa glucosidase alfa is currently sold under the name POMBILITI™ in the United States and POMBILITI® in the European Union. POMBILITI is currently indicated in combination with OPFOLDA for the treatment of adult patients with late-onset Pompe disease. The U.S. Prescribing Information and European Product Summary for POMBILITI are hereby incorporated by reference in their entireties.

[0025] As used herein, the term "glycan" refers to an oligosaccharide covalently attached to an amino acid residue on a protein or polypeptide. As used herein, the term "N-glycan" or "N-linked glycan" refers to a polysaccharide chain attached to an asparagine residue on a protein or polypeptide via a covalent bond with the nitrogen atom of the asparagine residue. In some embodiments, the N-glycan unit attached to rhGAA is determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an instrument such as a Thermo Scientific™ Orbitrap Velos Pro™ mass spectrometer, a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ mass spectrometer, or a Waters Xevo® G2-XS QTof mass spectrometer.

[0026] As used herein, forced vital capacity, or "FVC," is the volume of air that can be forcibly exhaled from a subject's lungs after the subject takes the deepest possible breath.

[0027] As used herein, the "6-minute walk test" (6MWT) is a test that measures the distance an individual can walk on a hard, flat surface in a total of six minutes. The test is conducted by having the individual walk as far as possible in six minutes.

[0028] As used herein, the "10 Meter Walk Test" (10MWT) is a test that measures the time it takes an individual wearing walking shoes to walk 10 meters on a flat surface.

[0029] As used herein, the compound miglustat, also known as N-butyl-1-deoxynojirimycin or NB-DNJ or (2R,3R,4R,5S)-1-butyl-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: [ka] It is a compound having the formula:

[0030] Or, [ka] is shown as:

[0031] One formulation of miglustat is marketed under the trademark ZAVESCA® as monotherapy for Gaucher disease type 1. In some embodiments, miglustat is referred to as AT2221.

[0032] Another formulation of miglustat is currently marketed under the trade name OPFOLDA™ in the United States and OPFOLDA® in the European Union. OPFOLDA is currently indicated in combination with POMBILITI for the treatment of adult patients with late-onset Pompe disease. The U.S. Prescribing Information and European Product Summary for OPFOLDA are hereby incorporated by reference in their entirety.

[0033] As discussed below, pharmaceutically acceptable salts of miglustat may also be used in the present disclosure. When a salt of miglustat is used, the dosage of the salt will be adjusted so that the dose of miglustat the patient receives is equivalent to the amount they would have received if miglustat free base had been used.

[0034] As used herein, the compound duvoglustat, also known as 1-deoxynojirimycin or DNJ or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, is a compound having the following chemical formula: [ka]

[0035] As used herein, the term "pharmacological chaperone," or sometimes simply the term "chaperone," is intended to refer to a molecule that specifically binds to acid α-glucosidase and has one or more of the following effects: Enhances the formation of stable molecular conformations of proteins; ·Enhancing the proper transport of proteins from the endoplasmic reticulum to another cellular site, preferably the native cellular site, in order to prevent endoplasmic reticulum-associated degradation of proteins; · Prevents aggregation of conformationally unstable or misfolded proteins; · at least partially restore and / or enhance wild-type function, stability, and / or activity of the protein; Improving the phenotype or function of cells harboring acid alpha-glucosidase; and / or Stabilizing acid alpha-glucosidase in vitro and / or in vivo (e.g., in the patient's bloodstream).

[0036] Thus, a pharmacological chaperone for acid α-glucosidase is a molecule that binds to acid α-glucosidase, resulting in proper folding, trafficking, non-aggregation, and / or activity of acid α-glucosidase. In at least one embodiment, the pharmacological chaperone is miglustat. Another non-limiting example of a pharmacological chaperone for acid α-glucosidase is duvoglustat.

[0037] As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition or other editions.

[0038] The term "pharmaceutically acceptable salt," as used herein, is intended to mean a salt that is generally water- or oil-soluble or dispersible and effective for its intended purpose, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable risk-to-benefit ratio. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. For a list of suitable salts, see, for example, S. M. Berge et al., J. Pharm. Sci., 1977, 66, pp. 1-19 (incorporated herein by reference). The term "pharmaceutically acceptable acid addition salt," as used herein, is intended to mean a salt formed with an inorganic acid that retains the biological effectiveness and properties of the free base and which is not biologically or otherwise undesirable. The term "pharmaceutically acceptable base addition salts," as used herein, is intended to mean salts formed with inorganic bases which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable.

[0039] As used herein, the term "buffer" refers to a solution containing a weak acid and its conjugate base or a weak base and its conjugate acid that helps prevent changes in pH.

[0040] As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to refer to the amount of acid α-glucosidase and / or the amount of miglustat and / or the amount of the two-component therapy sufficient to produce a therapeutic response in a subject.

[0041] Therapeutic response may also include molecular responses, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagic regions. Therapeutic response may be determined by comparing the physiological and molecular responses of muscle biopsies before and after treatment with the rhGAA described herein. For example, the amount of glycogen present in a biopsy sample can be used as a marker in determining therapeutic response. Another example includes biomarkers such as LAMP-1, LC3, and dysferlin, which can be used as indicators of lysosomal storage deficiency. For example, muscle biopsies taken before and after treatment with the rhGAA described herein may be stained with antibodies that recognize one or more of these biomarkers. Therapeutic response may also include a decrease in fatigue or an improvement in other patient-reported outcomes (e.g., activities of daily living, well-being, etc.).

[0042] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of a non-native purified enzyme into an individual deficient in such enzyme. The administered protein can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual who would otherwise require or benefit from the administration of the purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The introduced enzyme can be a purified recombinant enzyme made in vitro, or a protein purified from isolated tissue or body fluid, such as the placenta or milk of an animal, or from a plant.

[0043] As used herein, the term "binary therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In some embodiments, the results of a binary therapy are enhanced compared to the effects of each therapy when administered individually. Enhancement may include any improvement in the effects of the various therapies that may result in a favorable outcome compared to the results achieved by those therapies when administered alone. Enhanced effects or results may include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when administered alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy when administered alone; or a sub-additive effect, where the enhanced effect is less than the additive effect of each therapy when administered alone, but is still better than the effect of each therapy when administered alone. An enhanced effect may be measured by any means known in the art for measuring therapeutic efficacy or outcome.

[0044] "Pompe disease" refers to an autosomal recessive LSD characterized by impaired lysosomal glycogen metabolism due to a deficiency in acid alpha-glucosidase (GAA) activity. This enzymatic deficiency leads to lysosomal glycogen accumulation, resulting in progressive skeletal muscle weakness, cardiac decline, respiratory failure, and / or CNS dysfunction in end-stage disease. Genetic mutations in the GAA gene either result in decreased expression or give rise to mutant forms of the enzyme with altered stability and / or biological activity, ultimately leading to disease (for review see Hirschhorn R, 1995, "Glycogen Storage Disease Type II: Acid a-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease", Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile, and adult) correlate with the level of residual α-glucosidase activity (Reuser AJ et al., 1995, "Glycogenosis Type II (Acid Maltase Deficiency)," Muscle & Nerve Supplement 3, S61-S69). Infantile Pompe disease (Type I or Type A) is the most common and most severe form and is characterized by growth failure within the first two years of life, generalized hypotonia, cardiomegaly, and cardiopulmonary failure. Juvenile Pompe disease (Type II or Type B) is of intermediate severity and is characterized by a predominance of muscle symptoms and the absence of cardiomegaly. Individuals with juvenile Pompe disease usually die of respiratory failure before the age of 20.Adult Pompe disease (type III or type C) often presents as a slowly progressive myopathy in the teens or as late as the sixth decade (Felicia KJ et al., 1995, "Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature," Medicine 74, 131-135). In Pompe disease, α-glucosidase has been shown to undergo extensive post-translational modification by glycosylation, phosphorylation, and proteolytic processing. Optimal glycogen catalysis requires the conversion of a 110 kilodalton (kDa) precursor to the 76 and 70 kDa mature forms by lysosomal proteolysis. The formulations and administration regimens disclosed herein may be used, for example, to treat type I, type II, or type III Pompe disease.

[0045] Pompe disease is now considered a continuum of phenotypes, with the most clinically severe, rapidly progressive phenotype being classic IOPD and the less severe, slowly progressive phenotype being LOPD. The first symptoms of Pompe disease can appear at any time during life. The most severe form of IOPD appears at 3 months of age. Muscle weakness, cardiac dysfunction, and respiratory dysfunction characterize the progression of IOPD, and the average life expectancy is approximately 2 years. Atypical infantile, juvenile-onset, and nonclassic IOPD are some of the terms used to describe pediatric Pompe disease patients who do not have the significant, progressive hypertrophic cardiomyopathy seen in classic IOPD, and thus whose phenotype more closely resembles LOPD.

[0046] Late-onset Pompe disease can manifest in childhood or adulthood without clinically evident cardiac involvement (Leslie and Bailey, 2017). Late-onset Pompe disease, often referred to as juvenile-onset Pompe disease, occurs in the pediatric subpopulation of the LOPD category. LOPD progresses more slowly than classic IOPD, and most patients present with progressive limb-girdle muscle weakness and respiratory failure due to muscle involvement in the proximal lower and upper limbs, paraspinal muscles, and diaphragm. Clinical symptoms include difficulty walking and stair climbing, and progressive limitations in motor activities of daily living, which progress to the need for ambulatory assistance and subsequent wheelchair dependency (Reuser, et al., 2001). Clinical manifestations of the disease are compounded by respiratory involvement, initially manifesting as sleep-disordered breathing and orthopnea (shortness of breath while supine). The progressive nature of Pompe disease typically leads to the use of invasive mechanical ventilation.

[0047] As used herein, " significant " refers to statistical significance.This term refers to the statistical evidence that there is a difference between two treatment groups.It can be defined as the probability of making the decision to reject the null hypothesis when the null hypothesis is actually true.This decision is often made using the p-value<0.05 derived from suitable statistical analysis of comparison.See for example, Example 9.

[0048] A "subject" or "patient" is preferably a human, although other mammals and non-human animals with disorders involving glycogen storage can also be treated. The subject can be a fetus, newborn, child, juvenile, or adolescent with Pompe disease or another glycogen storage or accumulation disorder. One example of an individual to be treated is an individual (fetus, newborn, child, juvenile, or adolescent human) with GSD-II (e.g., infantile GSD-II or juvenile GSD-II). The individual may have residual GAA activity or no measurable activity. For example, an individual with GSD-II may have GAA activity less than about 1% of normal GAA activity (infantile GSD-II) or about 1-10% of normal GAA activity (juvenile GSD-II). In some embodiments, the subject or patient is under 18 years of age. In some embodiments, the subject or patient is an "ERT-experienced" or "ERT-switched" patient, which refers to a Pompe disease patient who has previously received enzyme replacement therapy. In some embodiments, an "ERT-experienced" or "ERT-switched" patient is a Pompe disease patient who has received or is currently receiving alglucosidase alfa for 24 months or more. In some embodiments, an "ERT-experienced" or "ERT-switched" patient is a Pompe disease patient who is currently receiving an approved ERT (e.g., MYOZYME®, LUMIZYME®, or NEXVIAZYME®) and is declining. In some embodiments, the subject has a confirmed diagnosis of late-onset Pompe disease (acid alpha-glucosidase (GAA) deficiency) and has previously received enzyme replacement therapy (ERT). In some embodiments, the subject has late-onset Pompe disease (lysosomal acid alpha-glucosidase [GAA] deficiency) and has experienced disease progression during the course of enzyme replacement therapy (ERT). In some embodiments, the subject or patient is an "ERT-naive" patient, which refers to a Pompe disease patient who has not previously received enzyme replacement therapy. In certain embodiments, the subject or patient is ambulatory (e.g., an ambulatory ERT-switched patient or an ambulatory ERT-naive patient). In certain embodiments, the subject or patient is non-ambulatory (e.g., a non-ambulatory ERT-switched patient).Ambulatory or non-ambulatory status may be determined by a 6-minute walk test (6MWT). In some embodiments, an ambulatory patient is a Pompe disease patient who can walk at least 200 meters in the 6MWT. In some embodiments, a non-ambulatory patient is a Pompe disease patient who cannot walk unassisted or is wheelchair-bound. In some embodiments, the subject is using an effective contraceptive method. In some embodiments, the subject and / or the subject's partner is using a highly effective contraceptive method, such as a contraceptive method that, when used consistently and correctly, results in a low failure rate (e.g., less than 1% per year). Examples of highly effective contraceptive methods include, but are not limited to, total abstinence; combined hormonal contraceptives (estrogen-containing and progestogen-containing) associated with ovulation suppression; oral, intravaginal, transdermal progestogen-only hormonal contraception associated with ovulation suppression: oral, injectable, implantable intrauterine device; intrauterine hormone-releasing system; bilateral tubal occlusion; and vasectomy. In some embodiments, the subject is postmenopausal. In some embodiments, the subject is not of childbearing potential. In some embodiments, the subject is on permanent contraception. In some embodiments, the subject is not pregnant. In some embodiments, the subject is not lactating.

[0049] In some embodiments, the patient has a diagnosis of late-onset Pompe disease based on documented evidence of at least one of the following: (1) a GAA enzyme deficiency; and / or (2) genotyping of the gene encoding human acid alpha-glucosidase (GAA). In some embodiments, the patient has previously received enzyme replacement therapy (ERT). In some embodiments, the patient is currently on and declining from an approved ERT (e.g., MYOZYME®, LUMIZYME®, or NEXVIAZYME®). In some embodiments, if of reproductive potential, both the male and female patient agree to use highly effective methods of contraception throughout the entire treatment period and for up to 90 days after the final dose. In some embodiments, patients taking a beta-2 receptor agonist or a non-selective beta-blocker (e.g., propranolol, nadolol, carvedilol) are maintaining a stable dose, as determined by the treating physician, as appropriate.

[0050] The terms "treat" and "treatment," as used herein, refer to the improvement of one or more symptoms associated with a disease, the delay in the onset of one or more symptoms of a disease, and / or the reduction in the severity or frequency of one or more symptoms of a disease. For example, treatment can refer to an improvement in cardiac condition (e.g., an increase in end-diastolic and / or end-systolic volume, or a reduction or improvement in the progressive cardiomyopathy typically seen in GSD-II) or an improvement in pulmonary function (e.g., an increase in crying vital capacity compared to baseline volume and / or a normalization of oxygen desaturation during crying); an improvement in neurodevelopment and / or motor skills (e.g., an increase in AIMS score); a reduction in glycogen levels in tissues of an individual affected by the disease; or any combination of these effects. In one embodiment, treatment includes an improvement in cardiac condition, particularly a reduction in GSD-II-associated cardiomyopathy.

[0051] The terms "improve," "increase," and "decrease," as used herein, refer to a value relative to a baseline measurement or a corresponding value from a control treatment, e.g., a measurement in the same individual before initiation of a treatment described herein, a measurement in a control individual(s) not receiving a treatment described herein, or a measurement after a control treatment. A control individual is an individual suffering from the same form of GSD-II (either infantile, juvenile, or adult-onset) as the individual under treatment and of approximately the same age as the individual under treatment (to ensure that the stage of disease between the treated individual and one or more control individuals is comparable). In some embodiments, the control treatment comprises administering alglucosidase alfa and a pharmacological chaperone placebo (see Example 9).

[0052] As used herein, the phrases "stabilizing motor function," "stabilizing pulmonary function," and similar terms refer to reducing or halting deterioration of motor and pulmonary function and / or restoring motor and / or pulmonary function. Because untreated Pompe disease patients are expected to exhibit significant declines in motor and pulmonary function over time, an increase in the rate of deterioration of motor and / or pulmonary function and / or an increase in motor and / or pulmonary function demonstrates the benefit of a therapy as described herein. Moreover, because Pompe disease patients with a history of ERT often continue to experience deterioration in motor and / or pulmonary function over time, stabilizing motor and / or pulmonary function using a therapy as described herein may include reducing and / or halting deterioration of motor and / or pulmonary function compared to such patients previously treated with ERT (e.g., MYOZYME®, LUMIZYME®, or NEXVIAZYME®).

[0053] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for the quantity being measured given the nature or precision of the measurement. For example, the degree of error may be indicated by the number of significant digits provided for that measurement, as understood in the art, and includes, but is not limited to, a variation of ±1 with the most accurate significant digit reported for the measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Numerical quantities given herein are approximate unless otherwise specified, meaning that the term "about" or "approximately" can be inferred when not specified.

[0054] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be construed as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0055] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0056] Recombinant human acid α-glucosidase (rhGAA) In some embodiments, the recombinant human acid alpha-glucosidase (rhGAA) is an enzyme having an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the rhGAA is encoded by a nucleotide sequence as set forth in SEQ ID NO: 2.

[0057] [Table 1]

[0058] [Table 2]

[0059] [Table 3]

[0060] [Table 4]

[0061] In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 1, as described in U.S. Patent No. 8,592,362, and has GenBank accession number AHE24104.1 (GI:568760974). In some embodiments, rhGAA has the GAA amino acid sequence as encoded by SEQ ID NO: 2, an mRNA sequence with GenBank accession number Y00839.1. In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 4, and has National Center for Biotechnology Information (NCBI) accession number NP_000143.2 or UniProtKB accession number P10253.

[0062] In some embodiments, rhGAA is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA as set forth in SEQ ID NO:1 or SEQ ID NO:4, and as this rhGAA undergoes intracellular processing, some amino acids, e.g., the first 56 amino acids, are removed. Accordingly, rhGAA secreted from host cells may have a shorter amino acid sequence than rhGAA initially expressed intracellularly. In some embodiments, this shorter protein has the amino acid sequence set forth in SEQ ID NO:5, which differs from SEQ ID NO:1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, resulting in a protein with 896 amino acids. In some embodiments, this shorter protein has the amino acid sequence set forth in SEQ ID NO:6, which differs from SEQ ID NO:4 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, resulting in a protein with 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions relative to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the rhGAA product includes a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0063] In some embodiments, rhGAA comprises an amino acid sequence at least 90%, 95%, 98%, or 99% identical to SEQ ID NO:4 or SEQ ID NO:6. Various alignment algorithms and / or programs may be used to calculate identity between two sequences, including FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), which may be used, for example, with default settings. For example, polypeptides having at least 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein, and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Unless otherwise indicated, similarity scores will be based on the use of BLOSUM62. When BLASTP is used, percent similarity is based on the BLASTP positive score, and percent sequence identity is based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues in the high-scoring sequence pair that are identical; and BLASTP "positive value" indicates the number and percentage of residues for which the alignment score is positive and which are similar to each other. Amino acid sequences having these degrees of identity or similarity to the amino acid sequences disclosed herein, or any intermediate degrees of identity or similarity, are contemplated and encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides can be inferred using the genetic code and obtained by conventional means, particularly by back-translating the amino acid sequence using the genetic code.

[0064] In some embodiments, rhGAA undergoes post-translational and / or chemical modifications to one or more amino acid residues in the protein. For example, methionine and tryptophan residues can undergo oxidation. As another example, the N-terminal glutamine of SEQ ID NO: 6 can be further modified to form pyroglutamic acid. As another example, an asparagine residue can undergo deamidation to aspartic acid. As yet another example, an aspartic acid residue can undergo isomerization to isoaspartic acid. As yet another example, an unpaired cysteine ​​residue in the protein can form a disulfide bond with free glutathione and / or cysteine. Accordingly, in some embodiments, the enzyme is initially expressed as having an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or an amino acid sequence encoded by SEQ ID NO: 2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modifications are also within the scope of the present disclosure.

[0065] N-linked glycosylation of rhGAA There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are located at the following positions of SEQ ID NO:6: N84, N177, N334, N414, N596, N826, and N869. Similarly, for the full-length amino acid sequence of SEQ ID NO:4, these potential glycosylation sites are located at the following positions: N140, N233, N390, N470, N652, N882, and N925. Other variants of rhGAA may have similar glycosylation sites depending on the position of the asparagine residue. Generally, an Asn-X-Ser or Asn-X-Thr sequence in the protein amino acid sequence indicates a potential glycosylation site, provided that X cannot be His or Pro.

[0066] The rhGAA molecules described herein may have, on average, one, two, three, or four mannose-6-phosphate (M6P) groups on their N-glycans. For example, only one N-glycan on an rhGAA molecule may carry M6P (monophosphorylated or mono-M6P), a single N-glycan may carry two M6P groups (bisphosphorylated or bis-M6P), or two different N-glycans on the same rhGAA molecule may each carry a single M6P group. In some embodiments, the rhGAA molecules described herein have, on average, 3 to 4 moles of M6P groups on their N-glycans per mole of rhGAA. Recombinant human acid α-glucosidase molecules may also have N-glycans that do not carry M6P groups. In another embodiment, the rhGAA contains, on average, greater than 2.5 moles of M6P per mole of rhGAA and greater than 4 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA contains, on average, about 3-3.5 moles of M6P per mole of rhGAA. In some embodiments, the rhGAA contains, on average, about 4-5.4 moles of sialic acid per mole of rhGAA. On average, at least about 3, 4, 5, 6, 7, 8, 9, 10%, or 20% of all N-glycans on rhGAA may be in the form of mono-M6P N-glycans, e.g., about 6.25% of all N-glycans may have a single M6P group, and on average, at least about 0.5, 1, 1.5, 2.0, 2.5, or 3.0% of all N-glycans on rhGAA are in the form of bis-M6P N-glycans, and on average, less than 25% of all rhGAA do not contain phosphorylated N-glycans that bind to CIMPR. In some embodiments, on average, about 10% to about 14% of all N-glycans on rhGAA are monophosphorylated. In some embodiments, on average, about 7% to about 25% of all N-glycans on rhGAA are bisphosphorylated. In some embodiments, on average, rhGAA contains about 1.3 moles of bis-M6P per mole of rhGAA.

[0067] The rhGAA described herein can have an average of 0.5 to 7.0 moles of M6P per mole of rhGAA, or any intermediate value or subrange thereof, including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 moles of M6P per mole of rhGAA. rhGAA can be fractionated to select specific fractions or selectively combine different fractions to provide rhGAA preparations with various average numbers of mono- or bis-M6P-bearing N-glycans, thereby allowing for further customization of rhGAA targeting to lysosomes in target tissues.

[0068] In some embodiments, up to 60% of the N-glycans on rhGAA may be fully sialylated, e.g., up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans may be fully sialylated. In some embodiments, 50% or less of the N-glycans on rhGAA are fully sialylated. In some embodiments, 4% to 20% of the total N-glycans are fully sialylated. In other embodiments, 5%, 10%, 20%, or 30% or less of the N-glycans on rhGAA have sialic acid and a terminal galactose residue (Gal). This range includes all intermediate values ​​and subranges, e.g., 7% to 30% of the total N-glycans on rhGAA may have sialic acid and a terminal galactose residue. In still other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans on the rhGAA have only terminal galactose and no sialic acid, including all intermediate values ​​and subranges within this range, for example, 8% to 19% of the total N-glycans on the rhGAA in the composition have only terminal galactose and no sialic acid.

[0069] In some embodiments, 40% to 60%, 45% to 60%, 50% to 60%, or 55% to 60% of the total N-glycans on rhGAA are complex N-glycans; or 1%, 2%, 3%, 4%, 5%, 6%, or 7% or less of the total N-glycans on rhGAA are hybrid N-glycans; 5%, 10%, 15%, 20%, or 25% or less of the high-mannose N-glycans on rhGAA are non-phosphorylated; at least 5% or 10% of the high-mannose N-glycans on rhGAA are monophosphorylated; and / or at least 1% or 2% of the high-mannose N-glycans on rhGAA are bisphosphorylated. These values ​​include all intermediate values ​​and subranges. The rhGAA may satisfy one or more of the content ranges described above.

[0070] In some embodiments, rhGAA may carry, on average, 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA. This range includes all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles of sialic acid residues per mole of rhGAA. Without being bound by theory, it is believed that the presence of N-glycan units bearing sialic acid residues may prevent non-productive clearance of rhGAA by the asialoglycoprotein receptor.

[0071] In one or more embodiments, rhGAA has a particular N-glycosylation profile at a particular potential N-glycosylation site. In some embodiments, rhGAA has seven potential N-glycosylation sites. In some embodiments, at least 20% of rhGAA is phosphorylated at the first potential N-glycosylation site (e.g., N84 for SEQ ID NO: 6 and N140 for SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of rhGAA may be phosphorylated at the first potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear mono-M6P units at the first potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear bis-M6P units at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.25 moles of mono-M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.2 moles to about 0.3 moles of sialic acid per mole of rhGAA at the first potential N-glycosylation site.In at least one embodiment, the rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6B. In at least one embodiment, the rhGAA comprises a first potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19B or Figure 20B.

[0072] In some embodiments, at least 20% of the rhGAA is phosphorylated at the second potential N-glycosylation site (e.g., N177 for SEQ ID NO: 6 and N223 for SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA may be phosphorylated at the second N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear mono-M6P units at the second N-glycosylation site, hi some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear bis-M6P units at the second N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.5 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the second potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.4 to about 0.6 moles of mono-M6P per mole of rhGAA at the second potential N-glycosylation site. In at least one embodiment, rhGAA contains a second potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation profile as depicted in FIG. 6C. In at least one embodiment, rhGAA contains a second potential N-glycosylation site occupancy as depicted in FIG. 19A and an N-glycosylation profile as depicted in FIG. 19C or FIG. 20B.

[0073] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the third potential N-glycosylation site (e.g., N334 for SEQ ID NO: 6 and N390 for SEQ ID NO: 4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the third potential N-glycosylation site. For example, the third potential N-glycosylation site can have a mixture of non-phosphorylated high-mannose N-glycans, diantennary, triantennary, and tetraantennary complex-type N-glycans, and hybrid-type N-glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site. In at least one embodiment, the rhGAA comprises a third potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6D. In at least one embodiment, the rhGAA comprises a third potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19D or Figure 20B.

[0074] In some embodiments, at least 20% of the rhGAA is phosphorylated at the fourth potential N-glycosylation site (e.g., N414 for SEQ ID NO: 6 and N470 for SEQ ID NO: 4). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA may be phosphorylated at the fourth potential N-glycosylation site. This phosphorylation may be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear a mono-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA bear a bis-M6P unit at the fourth potential N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of the rhGAA is sialylated at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 1.4 moles of M6P (mono-M6P and bis-M6P) per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA contains an average of about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site. In at least one embodiment, rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6E.In at least one embodiment, rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19E or Figure 20B.

[0075] In some embodiments, at least 5% of the rhGAA is phosphorylated at the fifth potential N-glycosylation site (e.g., N596 for SEQ ID NO: 6 and N692 for SEQ ID NO: 4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the fifth potential N-glycosylation site. For example, the fifth potential N-glycosylation site can have fucosylated diantennary complex-type N-glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the fifth potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site. In at least one embodiment, the rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in Figure 6A and an N-glycosylation profile as depicted in Figure 6F. In at least one embodiment, the rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19F or Figure 20B.

[0076] In some embodiments, at least 5% of the rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 for SEQ ID NO: 6 and N882 for SEQ ID NO: 4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site can have a mixture of diantennary, triantennary, and tetraantennary complex N-glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the sixth N-glycosylation site. In some embodiments, rhGAA contains an average of about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.9 moles of acetylated sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least 0.05 moles of glycan species bearing poly-N-acetyl-D-lactosamine (poly-LacNAc) residues per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, greater than 10% of rhGAA contains glycans bearing poly-LacNAc residues at the sixth potential N-glycosylation site. In at least one embodiment, rhGAA contains a sixth potential N-glycosylation site occupancy as depicted in FIG. 6A and an N-glycosylation profile as depicted in FIG. 6G. In at least one embodiment, rhGAA comprises a sixth potential N-glycosylation site occupancy as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19G or Figure 20B.

[0077] In some embodiments, at least 5% of the rhGAA is phosphorylated at the seventh potential N-glycosylation site (e.g., N869 for SEQ ID NO: 6 and N925 for SEQ ID NO: 4). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the seventh potential N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of the rhGAA has any N-glycan at the seventh potential N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of the rhGAA has an N-glycan at the seventh potential N-glycosylation site. In some embodiments, the rhGAA contains, on average, at least 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least 0.8 mol of sialic acid per mol of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of about 0.86 mol of sialic acid per mol of rhGAA at the seventh potential N-glycosylation site. In some embodiments, rhGAA contains an average of at least 0.3 mol of glycan species bearing poly-LacNAc residues per mol of rhGAA at the seventh potential N-glycosylation site. In some embodiments, approximately half of rhGAA contains glycans bearing poly-LacNAc residues at the seventh potential N-glycosylation site. In at least one embodiment, all N-glycans identified at the seventh potential N-glycosylation site are complex N-glycans. In at least one embodiment, rhGAA comprises a seventh potential N-glycosylation site occupancy as depicted in Figure 6A or as depicted in Figure 19A and an N-glycosylation profile as depicted in Figure 19H or Figure 20B.

[0078] In some embodiments, rhGAA contains, on average, 3 to 4 moles of M6P residues per mole of rhGAA and about 4 to about 7.3 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA contains, on average, at least about 0.5 moles of bis-M6P per mole of rhGAA at the first potential N-glycosylation site, about 0.4 to about 0.6 moles of mono-M6P per mole of rhGAA at the second potential N-glycosylation site, about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.4 to about 0.6 moles of bis-M6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.3 to about 0.4 moles of mono-M6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.8 to about 0.9 moles of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. The rhGAA further comprises about 1.5 to about 4.2 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA further comprises, on average, at least 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, the rhGAA further comprises, on average, at least 0.8 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, the rhGAA further comprises, on average, about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In at least one embodiment, the rhGAA comprises seven potential N-glycosylation sites with the occupancy and N-glycosylation profiles as depicted in Figures 6A-6H. In at least one embodiment, rhGAA contains seven potential N-glycosylation sites with occupancy and N-glycosylation profiles as depicted in Figures 19A-19H and 20A-20B.

[0079] Methods for making rhGAA are disclosed in PCT / 2015 / 053252, filed September 30, 2014, U.S. Pat. No. 10,208,299, U.S. Pat. No. 10,961,522, and U.S. Provisional Patent Application No. 62 / 057,842, the entire disclosures of which are incorporated herein by reference.

[0080] Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA formulations have low total levels of mono-M6P and bis-M6P-bearing N-glycans, which in turn results in poor muscle cell targeting, resulting in poor lysosome delivery of rhGAA. The majority of rhGAA molecules in these conventional formulations lack phosphorylated N-glycans and therefore lack affinity for CIMPR. Non-phosphorylated high-mannose N-glycans can also be removed by the mannose receptor, resulting in unproductive clearance of ERT (Figure 2B). In contrast, as shown in Figure 2A, the rhGAA described herein can contain higher amounts of mono-M6P and bis-M6P-bearing N-glycans, leading to productive uptake of rhGAA into specific tissues, such as muscle.

[0081] Preparation and purification of N-linked glycosylated rhGAA As described in PCT / 2015 / 053252, U.S. Patent No. 10,208,299, and U.S. Patent No. 10,961,522 (incorporated herein by reference in their entireties), cells such as Chinese hamster ovary (CHO) cells can be used to produce the rhGAA described herein. Expressing high M6P rhGAA in CHO cells is advantageous compared to post-translationally modifying the glycan profile of rhGAA, at least in part because only the former can be converted by glycan degradation to an optimal glycogenolysis form of rhGAA, which can then enhance therapeutic efficacy.

[0082] In some embodiments, rhGAA is preferably produced by one or more CHO cell lines transformed with a DNA construct encoding rhGAA described herein. Such CHO cell lines may contain multiple copies of the gene, such as 5, 10, 15, or 20 or more copies of the polynucleotide encoding GAA. DNA constructs expressing allelic variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4 or SEQ ID NO: 6, may be constructed and expressed in CHO cells. Those skilled in the art may select alternative vectors suitable for transforming CHO cells to produce such DNA constructs.

[0083] Methods for producing such CHO cell lines are described in PCT / 2015 / 053252, U.S. Pat. No. 10,208,299, and U.S. Pat. No. 10,961,522, which are incorporated herein by reference in their entireties. Briefly, these methods involve transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that stably integrate the DNA encoding GAA into one or more chromosomes and stably express GAA, selecting CHO cells that express GAA with a high content of N-glycans bearing mono-M6P or bis-M6P, and optionally selecting CHO cells with N-glycans with a high sialic acid content and / or low non-phosphorylated high mannose content. The selected CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the CHO cell culture. In some embodiments, rhGAA produced from selected CHO cell lines contains a high content of N-glycans carrying mono-M6P or bis-M6P that target CIMPR. In some embodiments, rhGAA produced as described herein has low levels of complex N-glycans with terminal galactose. In some embodiments, the selected CHO cell line is designated GA-ATB200 or ATB200-X5-14. In some embodiments, the selected CHO cell line includes a subculture or derivative of such a CHO cell culture. In some embodiments, rhGAA produced from selected CHO cell lines is designated ATB200.

[0084] rhGAA produced as described herein may be purified by following the methods described in U.S. Patent No. 10,227,577 and U.S. Provisional Patent Application No. 62 / 506,569, both of which are incorporated herein by reference in their entireties. An exemplary process for the production, capture, and purification of rhGAA produced from a CHO cell line is shown in FIG.

[0085] Briefly, bioreactor 601 contains a culture of cells, such as CHO cells, that express and secrete rhGAA into the surrounding liquid culture medium. Bioreactor 601 may be any bioreactor suitable for culturing cells, such as a perfusion, batch, or fed-batch bioreactor. The culture medium is removed from the bioreactor after a period of time sufficient for the cells to produce rhGAA. Such medium removal may be continuous in a perfusion bioreactor or batch-by-batch in a batch or fed-batch bioreactor. Cells may be removed by filtering the medium through filtration system 603. Filtration system 603 may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, and / or a centrifugal filtration system. In various embodiments, the filtration system utilizes filters having pore sizes of about 10 nanometers to about 2 micrometers.

[0086] After filtration, the filtrate is loaded into a protein capture system 605. The protein capture system 605 may comprise one or more chromatography columns. If more than one chromatography column is used, the columns may be arranged in series so that loading of the next column can begin as the first column is loaded. Alternatively, the media removal process may be stopped while switching columns.

[0087] In various embodiments, protein capture system 605 comprises one or more anion exchange (AEX) columns for direct product capture of rhGAA, particularly rhGAA with high M6P content. The rhGAA captured by protein capture system 605 is eluted from one or more columns by altering the pH and / or salt content of the column. Exemplary conditions for the AEX column are provided in Table 2.

[0088] [Table 5]

[0089] The eluted rhGAA can be subjected to further purification and / or quality assurance steps. For example, the eluted rhGAA can be subjected to a virus kill step 607. Such virus kill step 607 can include one or more of low pH kill, detergent kill, or other techniques known in the art. The rhGAA from virus kill step 607 can be introduced into a second chromatography system 609 to further purify the rhGAA product. Alternatively, the eluted rhGAA from protein capture system 605 can be fed directly to second chromatography system 609. In various embodiments, second chromatography system 609 includes one or more immobilized metal affinity chromatography (IMAC) columns to further remove impurities. Exemplary conditions for the IMAC columns are provided in Table 3 below.

[0090] [Table 6]

[0091] After loading the rhGAA into a second chromatography system 609, the recombinant protein is eluted from the column or columns. The eluted rhGAA can be subjected to a virus kill step 611. Similar to virus kill 607, virus kill 611 can also include one or more of low pH kill, detergent kill, or other techniques known in the art. In some embodiments, only one of virus kill 607 or 611 is used, or virus kill is performed at the same stage in the purification process.

[0092] The rhGAA from the virus killing step 611 may be introduced into a third chromatography system 613 to further purify the recombinant protein product. Alternatively, the eluted recombinant protein from the second chromatography system 609 may be fed directly into the third chromatography system 613. In various embodiments, the third chromatography system 613 includes one or more cation exchange chromatography (CEX) columns and / or size exclusion chromatography (SEC) columns to further remove impurities. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX column are provided below in Table 4.

[0093] [Table 7]

[0094] The rhGAA formulation may also be subjected to further processing steps. For example, a separate filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize a filter with a pore size of 5-50 μm. Other product processing steps may include a product conditioning step 617, in which the recombinant protein product may be sterilized, filtered, concentrated, stored, and / or additional components added for final product formulation.

[0095] Pharmaceutical Composition In various embodiments, pharmaceutical compositions are provided that include rhGAA as described herein, either alone or in combination with other therapeutic agents, and / or a pharmaceutically acceptable carrier.

[0096] In one or more embodiments, the pharmaceutical compositions described herein include pharmaceutically acceptable salts.

[0097] In some embodiments, the pharmaceutically acceptable salt used herein is a pharmaceutically acceptable acid addition salt, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and organic acids, including, but not limited to, acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphate, hemisulfonic acid, acid), hexanoic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, and undecanoic acid.

[0098] In some embodiments, the pharmaceutically acceptable salt used herein is a pharmaceutically acceptable base addition salt. Pharmaceutically acceptable base addition salts can include, but are not limited to, hydroxides, carbonates, or bicarbonates of ammonia or ammonium or metal cations such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, primary, secondary, and tertiary amines, quaternary amine compounds, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, Examples of salts include arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, N,N'-dibenzylethylenediamine, and polyamine resins.

[0099] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof may be formulated as a pharmaceutical composition suitable for intravenous administration. In some embodiments, the pharmaceutical composition is a solution in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. The components of the pharmaceutical composition may be supplied separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the composition is to be administered by infusion, it may be dispensed in an infusion bottle containing sterile pharmaceutical-grade water, saline, or dextrose / water. In some embodiments, the infusion may be performed in a hospital or clinic. In some embodiments, the infusion may be performed outside of a hospital or clinic setting, for example, at the subject's home. If the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed prior to administration.

[0100] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof is administered every two weeks as an intravenous infusion lasting about four hours. In some embodiments, the total infusion volume is determined by the patient's weight. If a patient experiences an IAR, the infusion rate can be reduced and the infusion duration can be increased.

[0101] In some embodiments, the initial infusion rate is 1 mg / kg / hour. In some embodiments, the infusion rate is gradually increased by 2 mg / kg / hour every 30 minutes until a maximum rate of 7 mg / kg / hour is reached in the absence of signs of infusion-related reactions (IARs); the infusion rate is then maintained at 7 mg / kg / hour until the infusion is complete. In some embodiments, the approximate total infusion duration is 4 hours.

[0102] The infusion should be administered stepwise using an infusion pump. The infusion rate can be increased every 30 minutes ± 5 minutes based on the dosage and patient weight as shown in the table below, from the initial rate until the maximum rate is reached.

[0103] [Table 8]

[0104] [Table 9]

[0105] [Table 10]

[0106] The most serious tolerability issue with rhGAA or a pharmaceutically acceptable salt thereof is the occurrence of infusion-related reactions (IARs), which in some cases can include life-threatening anaphylaxis or other severe allergic reactions. In some embodiments, prior to administration of rhGAA or a pharmaceutically acceptable salt thereof, pretreatment with an antihistamine, antipyretic, and / or corticosteroid is administered. If prior treatment was used with previous enzyme replacement therapy (ERT), prior to administration of rhGAA or a pharmaceutically acceptable salt thereof, pretreatment with an antihistamine, antipyretic, and / or corticosteroid is administered.

[0107] In some embodiments, rhGAA or a pharmaceutically acceptable salt thereof may be formulated for oral administration. Orally administered compositions may be in the form of tablets, capsules, suppositories, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders to be reconstituted with water or other suitable vehicles before use, optionally with flavorings and colorants, and may be formulated for immediate release, delayed release, modified release, sustained release, pulsed release, or controlled release applications. Solid compositions such as tablets, capsules, lozenges, troches, pills, boli, powders, pastes, granules, bullets, dragees, or premixed preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients, which may be in solid or liquid form. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients, including but not limited to binders, fillers, lubricants, disintegrants, or wetting agents. Suitable pharmaceutically acceptable excipients are known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), sucrose, gelatin, acacia, lactose, microcrystalline cellulose, calcium hydrogen phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato, or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, calcium hydrogen phosphate, glycine croscarmellose sodium, and complex silicates. Tablets can be coated by methods known in the art.

[0108] In some embodiments, the pharmaceutical compositions described herein may be formulated as described in U.S. Pat. No. 10,512,676 and U.S. Provisional Patent Application No. 62 / 506,574 (both of which are incorporated herein by reference in their entireties). For example, in some embodiments, the pH of the pharmaceutical compositions described herein is about 5.0 to about 7.0 or about 5.0 to about 6.0. In some embodiments, the pH ranges from about 5.5 to about 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH may be adjusted to a target pH by using a pH adjuster (e.g., an alkalinizing agent and an acidifying agent), such as sodium hydroxide and / or hydrochloric acid.

[0109] The pharmaceutical compositions described herein may include buffer systems such as citrate systems, phosphate systems, and combinations thereof. The citrate and / or phosphate may be sodium citrate or sodium phosphate. Other salts include potassium and ammonium salts. In one or more embodiments, the buffer comprises citrate. In further embodiments, the buffer comprises sodium citrate (e.g., a mixture of sodium citrate dihydrate and citric acid monohydrate). In one or more embodiments, a citrate-containing buffer solution may include sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

[0110] In some embodiments, the pharmaceutical compositions described herein comprise at least one excipient. An excipient may function as a tonicity agent, a bulking agent, and / or a stabilizer. A tonicity agent is an ingredient that helps ensure that the formulation has an osmotic pressure similar to or the same as that of human blood. A bulking agent is an ingredient that adds bulk to the formulation (e.g., in a lyophilized form) and provides the appropriate structure to the cake. A stabilizer is a compound that can prevent or minimize aggregate formation at the hydrophobic air-liquid interface. A single excipient may simultaneously function as a tonicity agent and a bulking agent. For example, mannitol may function as a tonicity agent and also provide benefits as a bulking agent.

[0111] Examples of tonicity agents include sodium chloride, mannitol, sucrose, and trehalose. In some embodiments, the tonicity agent comprises mannitol. In some embodiments, the total amount of the one or more tonicity agents ranges from about 10 mg / mL to about 50 mg / mL. In further embodiments, the total amount of the one or more tonicity agents ranges from about 10, 11, 12, 13, 14, or 15 mg / mL to about 16, 20, 25, 30, 35, 40, 45, or 50 mg / mL.

[0112] In some embodiments, the excipient comprises a stabilizer. In some embodiments, the stabilizer is a surfactant. In some embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of stabilizer ranges from about 0.1 mg / mL to about 1.0 mg / mL. In further embodiments, the total amount of stabilizer ranges from about 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL. In yet other embodiments, the total amount of stabilizer is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0113] In some embodiments, the pharmaceutical composition comprises one or more of the following excipients: sodium citrate dihydrate, citric acid monohydrate, mannitol, or polysorbate 80.

[0114] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (e.g., ATB200 or sipa glucosidase alfa), (b) at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and (c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, and has a pH of (i) about 5.0 to about 6.0, or (ii) about 5.0 to about 7.0. In some embodiments, the composition further comprises water. In some embodiments, the composition may further comprise an acidifying agent and / or an alkalizing agent.

[0115] In some embodiments, the pharmaceutical composition comprises (a) rhGAA (e.g., ATB200 or sipa glucosidase alfa) at a concentration of about 5-50 mg / mL, about 5-30 mg / mL, or about 15 mg / mL, (b) sodium citrate buffer at a concentration of about 10-100 mM or about 25 mM, (c) mannitol at a concentration of about 10-50 mg / mL or about 20 mg / mL, (d) polysorbate 80 present at a concentration of about 0.1-1 mg / mL, about 0.2-0.5 mg / mL, or about 0.5 mg / mL, and (e) water, and has a pH of about 6.0. In at least one embodiment, the pharmaceutical composition comprises (a) 15 mg / mL rhGAA (e.g., ATB200 or sipa glucosidase alfa), (b) 25 mM sodium citrate buffer, (c) 20 mg / mL mannitol, (d) 0.5 mg / mL polysorbate 80, and (e) water, and has a pH of about 6.0. In some embodiments, the composition may further comprise an acidifying agent and / or an alkalizing agent.

[0116] In some embodiments, a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) is diluted before administration to a subject in need thereof. In some embodiments, the pharmaceutical compositions described herein may be subjected to a lyophilization (freeze-drying) process to provide a cake or powder. Accordingly, in some embodiments, the pharmaceutical compositions described herein relate to rhGAA compositions after lyophilization. The lyophilized mixture may comprise an rhGAA described herein (e.g., ATB200), a buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of trehalose, mannitol, polysorbate 80, and combinations thereof. In some embodiments, other components (e.g., other excipients) may be added to the lyophilized mixture. The pharmaceutical composition comprising the lyophilized formulation may be provided in a vial, which can then be stored, shipped, reconstituted, and / or administered to a patient.

[0117] In some embodiments, a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) as described herein is a lyophilized powder in a glass vial. In some embodiments, each vial may contain approximately 105 mg of lyophilized rhGAA (e.g., ATB200 or sipa glucosidase alfa). This powder may be reconstituted with sterile water prior to administration by intravenous infusion, followed by dilution with 0.9% sodium chloride. In some embodiments, after reconstitution, the resulting concentrate contains 15 mg of rhGAA (e.g., ATB200 or sipa glucosidase alfa) per mL. In some embodiments, after reconstitution with 7.2 mL of diluent, the vial contains a usable volume of 7.0 mL of concentrate containing 15 mg / mL of rhGAA (e.g., ATB200 or sipa glucosidase alfa). In some embodiments, the diluent is sterile water and / or 0.9% sodium chloride. In some embodiments, each vial may contain an overfill to compensate for liquid loss during preparation. In some embodiments, the present disclosure provides a vial (e.g., a glass vial) containing 105 mg of a lyophilized rhGAA (e.g., ATB200 or sipa glucosidase alfa) composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa), sodium citrate dihydrate, citric acid monohydrate, mannitol, and polysorbate 80 (the amount / concentration of each component may be selected from those described herein).

[0118] In some embodiments, the number of vials to reconstitute is based on the patient's weight and the fact that each vial contains 105 mg of rhGAA (e.g., ATB200 or sipa glucosidase alfa). Thus, in some embodiments, patient dose (mg) = subject's weight (kg) x dose (mg / kg); and number of vials needed = patient dose (in mg) / 105 (mg per vial). In some embodiments, fractional vials are rounded up to the next whole number.

[0119] For example, for a 65 kg patient receiving 20 mg / kg, patient dose (mg) = 65 kg x 20 mg / kg = 1300 mg total dose; number of vials needed = 1300 / 105 mg per vial = 12.381 rounded up to 13 dispensing vials. The patient dose is 20 mg / kg, so the first 12 vials would have their entire volume drawn, but for the 13th vial, 2.7 ml would be drawn and added to the infusion bag.

[0120] For example, for a 40 kg patient receiving a dose of 25 mg / kg: Patient dose (mg) = 40 kg x 25 mg / kg = 1000 mg total dose; Number of vials needed = 1000 / 105 mg each = 9.524, rounded up to 10 vials for dispensing. The patient dose is 25 mg / kg, so the first 9 vials will have the full volume drawn, but for the 10th vial, 3.7 ml will be drawn and added to the infusion bag.

[0121] For example, for a 20 kg patient receiving a 30 mg / kg dose: Patient dose (mg) = 20 kg x 30 mg / kg = 600 mg total dose; number of vials needed = 600 / 105 mg each = 5.714, rounded up to 6 vials for dispensing. The patient dose is 30 mg / kg, so the first 5 vials will have the full volume drawn, but for the 6th vial, 5.0 ml will be drawn and added to the infusion bag.

[0122] In some embodiments, the reconstitution method comprises or consists essentially of the following steps: (1) reconstitute each vial by slowly injecting 7.2 mL of sterile water for injection into the interior wall of each vial, rather than directly into the lyophilized cake; (2) gently rock, do not invert, swirl, or shake each vial; (3) immediately after reconstitution, dilute an amount of reconstituted rhGAA with 0.9% sodium chloride for injection based on the patient's weight to equal the total injection volume in terms of patient weight; (4) remove air from the 0.9% sodium chloride for injection bag before adding the reconstituted rhGAA, so that the total volume is equal to the reconstituted volume; (5) dispense the reconstituted solution into the syringe, taking care to avoid foaming in the syringe. (6) Slowly withdraw from each vial while stirring; (6) Slowly add the reconstituted rhGAA solution directly to the 0.9% sodium chloride injection bag, avoiding foaming in the infusion bag (do not add directly to any voids that may remain in the infusion bag) (alternatively, dilution of the formulation may be achieved by using an appropriately sized empty infusion bag and adding the required volume of 0.9% sodium chloride injection and reconstituted rhGAA based on the patient's weight. Use of prefilled 0.9% sodium chloride injection bags is recommended); (7) Gently invert or massage the infusion bag to mix; and (8) Cover the infusion bag and tubing to protect the drug from light. After reconstitution, each vial will yield a concentration of 15 mg / mL. The total dose that can be withdrawn per vial is 105 mg per 7 mL.

[0123] In some embodiments, only the correct amount of reconstituted rhGAA is diluted based on the patient's weight. In some embodiments, a portion is withdrawn from the reconstituted vial by rounding up or down to the nearest tenth. In some embodiments, the infusion bag is not mixed by shaking, or a pneumatic tube is not used to transport the infusion bag. In some embodiments, the reconstituted and diluted solution contains particles in the form of thin white threads or translucent fibers after initial preparation, which may increase over time.

[0124] The present disclosure also provides pharmaceutical compositions comprising a chaperone. In some embodiments, the pharmaceutical compositions described herein comprise a chaperone. In some embodiments, the chaperone is miglustat or a pharmaceutically acceptable salt thereof. In other embodiments, the chaperone is duvoglustat or a pharmaceutically acceptable salt thereof.

[0125] In some embodiments, the rhGAA described herein is formulated in one pharmaceutical composition, while a chaperone such as miglustat is formulated in a separate pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising miglustat is based on the formulation commercially available as ZAVESCA® (Actelion Pharmaceuticals). In some embodiments, the pharmaceutical composition comprising miglustat comprises microcrystalline cellulose, pregelatinized starch, Emprove® sucralose powder, magnesium stearate, and / or colloidal silicon dioxide. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration comprising about 65 mg of miglustat, microcrystalline cellulose, pregelatinized starch, Emprove® sucralose powder, magnesium stearate, and / or colloidal silicon dioxide.

[0126] In some embodiments, the pharmaceutical composition comprising miglustat comprises 20% to 40% miglustat by weight, such as 30% to 35% miglustat by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 40% to 60% microcrystalline cellulose by weight, such as 45% to 55% microcrystalline cellulose by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 5% to 25% pregelatinized starch by weight, such as 10% to 20% pregelatinized starch by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1% to 5% sucralose by weight, such as 0.2% to 1% sucralose by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1% to 5% magnesium stearate by weight, such as 0.2% to 1% magnesium stearate by weight. In some embodiments, the pharmaceutical composition comprising miglustat further comprises 0.1% to 5% by weight colloidal silicon dioxide, such as 0.2 to 1% by weight colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in a hard gelatin capsule for oral administration. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration comprising about 20% to 40% by weight (e.g., 30 to 35%) miglustat, 40% to 60% by weight (e.g., 45 to 55%) microcrystalline cellulose, 5% to 25% by weight (e.g., 10 to 20%) pregelatinized starch, 0.1% to 5% by weight (e.g., 0.2% to 1%) sucralose, 0.1% to 5% by weight (e.g., 0.2% to 1%) magnesium stearate, and / or 0.1% to 5% by weight (e.g., 0.2% to 1%) colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in an oral liquid dosage form, such as an oral solution, dispersion, or suspension.In some embodiments, the pharmaceutical composition is an oral liquid dosage form comprising about 20% to 40% by weight (e.g., 30 to 35%) miglustat, 40% to 60% by weight (e.g., 45 to 55%) microcrystalline cellulose, 5% to 25% by weight (e.g., 10 to 20%) pregelatinized starch, 0.1% to 5% by weight (e.g., 0.2% to 1%) sucralose, 0.1% to 5% by weight (e.g., 0.2% to 1%) magnesium stearate, and / or 0.1% to 5% by weight (e.g., 0.2% to 1%) colloidal silicon dioxide.

[0127] In some embodiments, the pharmaceutical composition comprising miglustat comprises about 50 to about 100 mg miglustat, such as about 65 mg miglustat. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 50 to about 150 mg microcrystalline cellulose, such as about 75 to about 125 mg microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 20 to about 50 mg pregelatinized starch, such as about 30 to about 40 mg pregelatinized starch. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg sucralose, such as about 0.5 to about 2 mg sucralose. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg magnesium stearate, such as about 0.5 to about 2 mg magnesium stearate. In some embodiments, the pharmaceutical composition comprising miglustat comprises about 0.1 to about 5 mg colloidal silicon dioxide, such as about 0.2 mg to about 1 mg colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in a hard gelatin capsule for oral administration. In some embodiments, the pharmaceutical composition is a hard gelatin capsule for oral administration containing about 50 to about 100 mg (e.g., 65 mg) of miglustat, about 50 to about 150 mg (e.g., 75 mg to about 125 mg) of microcrystalline cellulose, about 20 to about 50 mg (e.g., 30 mg to about 40 mg) of pregelatinized starch, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of sucralose, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of magnesium stearate, and / or about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) of colloidal silicon dioxide. In some embodiments, the pharmaceutical composition comprising miglustat is provided in an oral liquid dosage form, such as an oral solution, dispersion, or suspension.In some embodiments, the pharmaceutical composition is an oral liquid dosage form comprising about 50 to about 100 mg (e.g., 65 mg) miglustat, about 50 to about 150 mg (e.g., 75 mg to about 125 mg) microcrystalline cellulose, about 20 to about 50 mg (e.g., 30 mg to about 40 mg) pregelatinized starch, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) sucralose, about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) magnesium stearate, and / or about 0.1 to about 5 mg (e.g., 0.5 mg to about 2 mg) colloidal silicon dioxide.

[0128] In some embodiments, the pharmaceutical composition comprising miglustat is provided in a hard gelatin capsule for oral administration comprising about 65 mg miglustat, about 100 mg microcrystalline cellulose, about 32.6 mg pregelatinized starch, about 1 mg sucralose powder, about 1 mg magnesium stearate, and about 0.4 mg colloidal silicon dioxide.

[0129] In some embodiments, a pharmaceutical composition comprising miglustat is provided in an oral solution, dispersion, or suspension comprising about 65 mg miglustat, about 100 mg microcrystalline cellulose, about 32.6 mg pregelatinized starch, about 1 mg sucralose powder, about 1 mg magnesium stearate, and about 0.4 mg colloidal silicon dioxide.

[0130] In some embodiments, a pharmaceutical composition comprising miglustat is provided in an oral solution, dispersion, or suspension comprising about 130 mg miglustat, about 200 mg microcrystalline cellulose, about 65.2 mg pregelatinized starch, about 2 mg sucralose powder, about 2 mg magnesium stearate, and about 0.8 mg colloidal silicon dioxide.

[0131] In some embodiments, the pharmaceutical composition comprising miglustat is provided in an oral solution, dispersion, or suspension comprising about 195 mg miglustat, about 300 mg microcrystalline cellulose, about 97.8 mg pregelatinized starch, about 3 mg sucralose powder, about 3 mg magnesium stearate, and about 1.2 mg colloidal silicon dioxide.

[0132] In some embodiments, a pharmaceutical composition comprising miglustat is provided in an oral solution, dispersion, or suspension comprising about 260 mg miglustat, about 400 mg microcrystalline cellulose, about 130.4 mg pregelatinized starch, about 4 mg sucralose powder, about 4 mg magnesium stearate, and about 1.6 mg colloidal silicon dioxide.

[0133] In some embodiments, patients may have difficulty swallowing hard gelatin capsules. Thus, in some embodiments, a pharmaceutical composition comprising miglustat is provided in an oral liquid. In some embodiments, the oral liquid is flavored.

[0134] In some embodiments, the pharmaceutical composition comprising miglustat is provided as an oral liquid containing one or more excipients, ie, a preservative, a buffer, a thickener (or thickener), a sweetener, a flavor, and / or a vehicle.

[0135] In some embodiments, the pharmaceutical composition comprising miglustat is at 1.0-10.0%, 1.5-8%, 2.0-5.0%, 2.0-6.5%, 2-7%, 2.5-5.0%, 2.5-6.5%, 2.5-7%, 3.0-5.0%, 3-6.5%, 3-7%, 3.5-5.0%, 3.5-6%, 3.5-7%, 4.0-5.0%, 4-6.5%, 4-7%, 4.5-5.0%, 4.5-6%, 4.5-7% by weight The oral liquid contains 2.0-4.5%, 2.5-4.5%, 3.0-4.5%, 3.5-4.5%, 4.0-4.5%, 2.0-4.0%, 2.5-4.0%, 3.0-4.0%, or 3.5-4.0% miglustat. In some embodiments, the pharmaceutical composition containing miglustat is an oral liquid containing about 5% or less miglustat by weight. In some embodiments, the pharmaceutical composition containing miglustat is an oral liquid containing 1-200 mg / mL, such as 10-100 mg / mL, 10-50 mg / mL, or 20-60 mg / mL. Exemplary miglustat concentrations include about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0136] In some embodiments, the pharmaceutical composition comprising miglustat is an oral solution comprising a buffer system, such as a citrate system, a phosphate system, or a combination thereof. The citrate and / or phosphate may be a sodium salt (e.g., sodium citrate or sodium phosphate). Other salts include potassium and ammonium salts. In one or more embodiments, the buffer comprises citrate. In further embodiments, the buffer comprises sodium citrate (e.g., a mixture of sodium citrate dihydrate and citric acid monohydrate). In one or more embodiments, the citrate-containing buffer solution may comprise sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

[0137] In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid formulation comprising a thickening agent or a viscosifying agent. In some embodiments, the thickening agent is selected from hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), gelatin, or microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid formulation comprising 0.5-5%, 1.0-3.0%, or 1.0-2.0% by weight of the thickening agent. In some embodiments, the thickening agent is hydroxypropyl methylcellulose (HPMC).

[0138] In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid containing a sweetener. In some embodiments, the sweetener is selected from sucrose or sucralose. In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid containing 0.1 to 1%, 0.1 to 0.5%, 0.1 to 0.3%, or 0.15 to 0.25% sweetener by weight. In some embodiments, the sweetener is sucralose.

[0139] In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid containing a preservative. In some embodiments, the preservative is potassium sorbate. In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid containing 0.1-1%, 0.1-0.5%, 0.1-0.3%, or 0.15-0.25% by weight of a preservative.

[0140] In some embodiments, the pharmaceutical composition comprising miglustat is an oral liquid containing a flavoring agent, ie, 0.1 to 0.5%, 0.1 to 0.3%, or 0.15 to 0.25% by weight of the flavoring agent.

[0141] In some embodiments, the balance of the miglustat-containing oral solution is water, hi some embodiments, the water comprises 80%, 90%, 91%, 92%, 93%, 94%, or 95% or more water.

[0142] In some embodiments, the pH of the oral solution containing miglustat is adjusted with a strong acid (e.g., hydrochloric acid) and / or a strong base (e.g., sodium hydroxide). In some embodiments, the pH of the oral solution containing miglustat is adjusted to 5.0±0.5. In other words, in some embodiments, the pH of the oral solution containing miglustat is in the range of 4.5 to 5.5, 4.7 to 5.3, 4.9 to 5.1, or about 5.0.

[0143] In some embodiments, the oral liquid containing miglustat is an oral liquid containing 1.0-10.0%, 2.0-5.0%, 2.5-5.0%, 3.0-5.0%, 3.5-5.0%, 4.0-5.0%, 4.5-5.0%, 2.0-4.5%, 2.5-4.5%, 3.0-4.5%, 3.5-4.5%, 4.0-4.5%, 2.0-4.0%, 2.5-4.0%, 3.0-4.0%, or 3.5-4.0% miglustat by weight. In some embodiments, the oral liquid contains about 4% or less miglustat by weight.

[0144] In some embodiments, the oral solution containing miglustat is an oral solution containing 1 to 200 mg / mL, such as 10 to 100 mg / mL, 10 to 50 mg / mL, or 20 to 60 mg / mL. Exemplary miglustat concentrations include about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0145] In some embodiments, the oral solution containing miglustat contains a buffering agent. In some embodiments, the buffering agent is sodium citrate. In some embodiments, the oral solution containing miglustat contains 0.5-1%, 0.5-0.55%, 0.55-0.6%, 0.6-0.65%, 0.65-0.7%, 0.7-0.8%, 0.8-0.85%, 0.85-0.9%, or 0.9-0.95% sodium citrate by weight. In some embodiments, the oral solution contains less than, greater than, or equal to about 0.65% sodium citrate by weight. In some embodiments, the oral solution contains less than, greater than, or equal to about 0.84% ​​sodium citrate by weight. In some embodiments, the oral solution comprises a sodium citrate concentration of 0.1 to 100 mg / mL, such as 0.5 to 50 mg / mL, 1 to 20 mg / mL, or 5 to 15 mg / mL. Exemplary sodium citrate concentrations include 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or 50 mg / mL.

[0146] In some embodiments, the oral solution containing miglustat contains a buffering agent. In some embodiments, the buffering agent is citric acid. In some embodiments, the oral solution containing miglustat contains 0.1-0.5%, 0.1-0.2%, 0.2-0.3%, 0.25-0.3%, 0.3-0.35%, 0.35-0.4%, or 0.4-0.5% citric acid by weight. In some embodiments, the oral solution contains less than, greater than, or equal to about 0.27% citric acid by weight. In some embodiments, the oral solution contains less than, greater than, or equal to about 0.35% citric acid by weight. In some embodiments, the oral solution contains a citric acid concentration of 0.1-100 mg / mL, such as 0.5-50 mg / mL, 1-20 mg / mL, or 5-15 mg / mL. Exemplary citrate concentrations include 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or 50 mg / mL.

[0147] In some embodiments, the oral solution containing miglustat contains a thickening agent. In some embodiments, the thickening agent is hypromellose. In some embodiments, the oral solution containing miglustat is an oral solution containing the thickening agent at 1-10 mg / mL, 10-20 mg / mL, 1-5 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL. In some embodiments, the oral solution contains less than, greater than, or equal to about 15 mg / mL of the thickening agent. In some embodiments, the oral solution containing miglustat is an oral solution containing 1-5%, 1.5-2%, 1-1.5%, 1-2%, 2.5-3%, 3.5-4%, 4.5-5%, or 5.5-6% by weight of a thickening agent, hi some embodiments, the oral solution contains less than, greater than, or equal to about 1.5% by weight of a thickening agent.

[0148] In some embodiments, the oral solution containing miglustat contains a sweetener. In some embodiments, the sweetener is sucralose. In some embodiments, the oral solution containing miglustat is an oral solution containing the sweetener at 1-5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL. In some embodiments, the oral solution contains less than, more than, or equal to about 2 mg / mL of sweetener. In some embodiments, the oral solution containing miglustat comprises 0.1-0.2%, 0.15-0.2%, 0.1-0.5%, 0.1-0.25%, 0.15-0.25%, 0.2-0.25%, 0.25-0.3%, 0.25-0.35%, 0.3-0.4%, or 0.4-0.5% sweetener by weight. In some embodiments, the oral solution comprises less than, greater than, or equal to about 0.2% sweetener by weight.

[0149] In some embodiments, the oral solution containing miglustat contains a preservative. In some embodiments, the preservative is potassium sorbate. In some embodiments, the oral solution containing miglustat is an oral solution containing the preservative at 1-5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL. In some embodiments, the oral solution contains less than, more than, or equal to about 2 mg / mL of the preservative. In some embodiments, the oral solution containing miglustat comprises 0.1-0.2%, 0.15-0.2%, 0.1-0.5%, 0.1-0.25%, 0.15-0.25%, 0.2-0.25%, 0.25-0.3%, 0.25-0.35%, 0.3-0.4%, or 0.4-0.5% by weight of a preservative. In some embodiments, the oral solution comprises less than, greater than, or equal to about 0.2% by weight of a preservative.

[0150] In some embodiments, the oral liquid containing miglustat contains a flavor. In some embodiments, the flavor is mixed berry. In some embodiments, the oral liquid containing miglustat is an oral liquid containing flavor at 1-5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL. In some embodiments, the oral liquid contains less than, more than, or equal to about 2 mg / mL of flavor. In some embodiments, the oral liquid containing miglustat comprises 0.1-0.2%, 0.15-0.2%, 0.1-0.5%, 0.1-0.25%, 0.15-0.25%, 0.2-0.25%, 0.25-0.3%, 0.25-0.35%, 0.3-0.4%, or 0.4-0.5% flavor by weight. In some embodiments, the oral liquid comprises less than, greater than, or equal to about 0.2% flavor by weight.

[0151] Some exemplary pharmaceutical compositions containing miglustat in an oral solution are provided below.

[0152] [Table 11]

[0153] Treatment method A. Treatment of Disease Another aspect of the present disclosure relates to a method for treating a disease or disorder associated with dysregulated glycogen storage by administering rhGAA or a pharmaceutical composition described herein. In some embodiments, the disease is Pompe disease (also known as acid maltase deficiency (AMD) and glycogen storage disease type II (GSD II)). In some embodiments, the rhGAA is ATB200 or sipa glucosidase alfa. In some embodiments, the pharmaceutical composition comprises rhGAA (e.g., ATB200 or sipa glucosidase alfa). Also provided herein is the use of rhGAA or ATB200 or sipa glucosidase alfa for treating Pompe disease.

[0154] In some embodiments, the subject treated by the methods disclosed herein is a pediatric patient. In some embodiments, the subject may be 0 to 18 years of age, or any intermediate value or subrange therein, including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months of age or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age.

[0155] In some embodiments, the subject treated by the methods disclosed herein is an ERT-experienced patient. In some embodiments, the ERT-experienced patient is currently receiving an approved ERT (e.g., MYOZYME®, LUMIZYME®, or NEXVIAZYME®). In some embodiments, the ERT-experienced patient is currently on treatment but is declining. In some embodiments, the methods disclosed herein are initiated about 2 weeks after the last ERT dose. In some embodiments, the subject treated by the methods disclosed herein is an ERT-naive patient.

[0156] In some embodiments, patients may be expected to return every three months to ensure clinical benefit and thus continue treatment. This frequency of visits may be required until the drug is commercially available. In some embodiments, patients' treatment response is evaluated periodically based on evaluation of key disease diagnostic and clinical laboratory parameters. In some embodiments, rhGAA (such as sipa glucosidase alfa) and miglustat as described herein are administered every two weeks. In some embodiments, the dosage of rhGAA, such as sipa glucosidase alfa, is about 20 mg / kg, or about 25 mg / kg, or about 30 mg / kg body weight given as a 4-hour infusion. In some embodiments, if the infusion is delayed, it should not begin more than three hours after oral administration of miglustat.

[0157] In some embodiments, subjects receive miglustat capsules (or a liquid dispersion prepared from miglustat capsules) about 1 hour (i.e., 1 hour ± 10 minutes) before the sipa glucosidase alfa injection. The number of capsules, amount of water used, and amount of dispersion administered are summarized in the table below. For example, a subject receiving 260 mg or 195 mg of miglustat would be orally administered 4 or 3 capsules of miglustat, respectively.

[0158] In some embodiments, if a subject is unable to swallow a miglustat capsule, the capsule may be opened and the contents may be poured into water. In some embodiments, a subject is administered a liquid dispersion prepared from a miglustat capsule approximately 1 hour (i.e., 1 hour ± 10 minutes) prior to the injection of sipa glucosidase alfa. The number of capsules used, the amount of water, and the amount of dispersion administered are summarized in the table below. For example, for a subject receiving 260 mg of miglustat, 20 mL of water would be used to disperse 4 capsules of miglustat, resulting in 20 mL being administered.

[0159] [Table 12]

[0160] In some of the above embodiments, rhGAA (such as sipa glucosidase alfa) is administered by infusion every two weeks.

[0161] In some of the above embodiments, a patient weighing 40-50 kg is administered a dosage of three 65 mg capsules (195 mg total) of miglustat. In some of the above embodiments, rhGAA (such as sipa glucosidase alfa) is administered by infusion every two weeks.

[0162] In some embodiments, miglustat is administered as an oral solution as described herein.As described above, exemplary oral solutions may include miglustat together with one or more excipients, such as preservatives, buffers, thickeners (or viscosifiers), sweeteners, flavors, and / or vehicles.In some embodiments, the oral solution includes miglustat and one or more of sodium citrate, citric acid, hypromellose, sucralose, potassium sorbate, flavors, and / or water.

[0163] The rhGAA or pharmaceutical composition described herein is administered by an appropriate route. In one embodiment, the rhGAA or pharmaceutical composition is administered intravenously. In some embodiments, the rhGAA or pharmaceutical composition is administered intravenously using an infusion pump. In some embodiments, when administered intravenously, the infusion bag and tubing are covered to protect from light. In other embodiments, the rhGAA or pharmaceutical composition is administered by direct administration to a target tissue, such as the heart or skeletal muscle (e.g., intramuscularly), or the nervous system (e.g., intracerebral; intracerebroventricular; or direct intrathecal injection). In some embodiments, the rhGAA or pharmaceutical composition is administered orally. If necessary, two or more routes may be used simultaneously.

[0164] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein may be assessed based on one or more of the following criteria: (1) cardiac status (e.g., an increase in end-diastolic volume and / or end-systolic volume, or a reduction, improvement, or prevention of the progressive cardiomyopathy typically seen in GSD-II); (2) pulmonary function (e.g., an increase in crying vital capacity compared to baseline volume and / or normalization of oxygen desaturation during crying); (3) neurodevelopment and / or motor skills (e.g., an increase in AIMS score); and (4) a reduction in glycogen levels in tissues of an individual affected by the disease.

[0165] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the subject's cardiac condition improves by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to that of a vehicle-treated subject or that of the subject before treatment. The subject's cardiac condition may be assessed by measuring end-diastolic and / or end-systolic volumes and / or by clinically assessing cardiomyopathy. In some embodiments, the subject's pulmonary function improves by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) after administration of one or more dosages of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) compared to that of a vehicle-treated subject or that of the subject before treatment. In certain embodiments, improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) of administration. In certain embodiments, rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) improves and / or stabilizes a subject's lung function after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) of administration.

[0166] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the subject's pulmonary function improves by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to that of a vehicle-treated subject or that of the subject before treatment. The subject's pulmonary function may be assessed by normalization of crying vital capacity and / or oxygen desaturation during crying compared to baseline. In some embodiments, after administration of one or more dosages of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa), the subject's pulmonary function improves by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to that of a vehicle-treated subject or that of the subject before treatment. In certain embodiments, improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) of administration. In certain embodiments, rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) improves pulmonary function in a subject after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any time period in between) of administration.

[0167] In some embodiments, after administration of one or more dosages of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or rhGAA (e.g., ATB200 or sipa glucosidase alfa) pharmaceutical compositions described herein, a subject's neurodevelopment and / or motor skills improve by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to that of a vehicle-treated subject or that of the subject before treatment. A subject's neurodevelopment and / or motor skills can be assessed by determining an AIMS score. The AIMS is a 12-item anchored scale administered and scored by a clinician (see Rush JA Jr., Handbook of Psychiatric Measures, American Psychiatric Association, 2000, 166-168). Items 1-10 are scored on a 5-point anchored scale. Items 1-4 assess orofacial movements. Items 5-7 address limb and trunk dyskinesias. Items 8-10 address the overall severity, as judged by the examiner, and the patient's awareness of movement and associated pain. Items 11-12 are yes / no questions regarding dental and / or denture problems (such problems may lead to misdiagnosis of dyskinesias). In some embodiments, after administration of one or more doses of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa), the subject's neurodevelopmental and / or motor skills improve by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to those of a vehicle-treated subject or those of a subject prior to treatment. In certain embodiments, improvement is realized after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) following administration. In certain embodiments, rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) improves and / or stabilizes a subject's neurodevelopment and / or motor skills after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) of administration.

[0168] In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the glycogen level of a specific tissue of a subject is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared with that of a vehicle-treated subject or that of a subject before treatment. In some embodiments, the tissue is muscle, such as quadriceps, triceps, and gastrocnemius. The glycogen level of tissue can be analyzed using methods known in the art. The determination of glycogen level based on amyloglucosidase digestion is well known and is described in publications such as Amalfitano et al. (1999), Proc Natl Acad Sci USA, 96:8861-8866. In some embodiments, after administration of one or more dosages of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa), the subject's muscle glycogen levels are reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to those of a vehicle-treated subject or those of a subject prior to treatment. In certain embodiments, the reduction is achieved 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration. In certain embodiments, rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) reduces the subject's muscle glycogen levels 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration.

[0169] B. Biomarkers A biomarker of glycogen accumulation in a subject, such as urinary hexose tetrasaccharide (Hex4), may be used to assess and compare the therapeutic efficacy of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on glycogen accumulation is assessed by measuring the urinary Hex4 level in the subject.

[0170] Biomarkers of muscle injury or damage, such as creatine kinase (CK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), may be used to evaluate and compare the therapeutic effects of enzyme replacement therapy in subjects with Pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is evaluated by measuring the subject's CK, ALT, and / or AST levels. In at least one embodiment, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle damage is evaluated by measuring the subject's CK levels.

[0171] Biomarkers such as LAMP-1, LC3, and dysferlin may also be used to evaluate and compare the therapeutic effects of rhGAA or the pharmaceutical compositions described herein. In Pompe disease, GAA is unable to hydrolyze lysosomal glycogen, leading to the abnormal accumulation of large glycogen-filled lysosomes in some tissues (Raben et al., JBC 273:19086-19092, 1998). Mouse model studies of Pompe disease have shown that enlarged lysosomes in skeletal muscle do not adequately explain the impaired mechanical performance, and that the presence of large inclusions containing degraded myofibrils (i.e., autophagy buildup) contributes to muscle dysfunction (Raben et al., Human Mol Genet 17:3897-3908, 2008). Reports also suggest that impaired autophagic flux is associated with poor treatment outcomes in patients with Pompe disease (Nascimbeni et al., Neuropathology and Applied Neurobiology doi:10.1111 / nan.12214, 2015; Fukuda et al., Mol Ther 14:831-839, 2006). In addition, late-onset Pompe disease is commonly observed in unclassified limb-girdle muscular dystrophies (LGMDs) (Preisler et al., Mol Genet Metab 110:287-289, 2013), a genetically heterogeneous group of neuromuscular disorders with over 30 genetically defined subtypes of varying severity. IHC analysis revealed substantially elevated sarcoplasmic abundance of dysferlin in skeletal muscle fibers of Gaa KO mice.

[0172] Various known methods can be used to measure the gene expression levels and / or protein levels of such biomarkers. For example, a tissue sample, particularly a muscle biopsy, can be obtained from a subject treated with rhGAA or a pharmaceutical composition described herein. In some embodiments, the sample is a muscle biopsy from the subject. In some embodiments, the muscle is selected from the quadriceps, triceps, and gastrocnemius. The sample obtained from the subject may be stained with one or more antibodies or other detection agents that detect such biomarkers, or may be identified and quantified by mass spectrometry. The sample may also, or alternatively, be processed to detect the presence of nucleic acids, such as mRNA, encoding the biomarkers, for example, by RT-qPCR.

[0173] In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from individuals before and after treatment with rhGAA or pharmaceutical compositions described herein. In some embodiments, gene expression levels and / or protein levels of one or more biomarkers are measured in muscle biopsies obtained from vehicle-treated individuals. In some embodiments, after administration of one or more dosages of rhGAA or pharmaceutical compositions described herein, the gene expression levels and / or protein levels of one or more biomarkers are reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage therebetween) compared to those of vehicle-treated subjects or those of subjects before treatment. In some embodiments, after administration of one or more dosages of rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa), the gene expression level and / or protein level of one or more biomarkers is reduced by 10%, 20%, 30%, 40%, or 50% (or any percentage in between) compared to that of a vehicle-treated subject or that of a subject prior to treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) after administration. In certain embodiments, rhGAA (e.g., ATB200 or sipa glucosidase alfa) or a pharmaceutical composition comprising rhGAA (e.g., ATB200 or sipa glucosidase alfa) reduces the gene expression levels and / or protein levels of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more (or any period in between) following administration.

[0174] C. rhGAA dosage The pharmaceutical formulation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dosage that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease). The therapeutically effective amount in treating a disease may depend on the nature and extent of the disease's effects. Additionally, in vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. In at least one embodiment, rhGAA or pharmaceutical compositions comprising rhGAA described herein are administered at a dose of about 1 mg / kg to about 100 mg / kg, such as about 5 mg / kg to about 50 mg / kg, typically about 20 mg / kg to about 30 mg / kg. In at least one embodiment, rhGAA or pharmaceutical compositions described herein are administered at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In some embodiments, rhGAA is administered at a dose of 5 mg / kg, 10 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 50 mg / kg, 75 mg / kg, or 100 mg / kg. In at least one embodiment, rhGAA or the pharmaceutical composition is administered at a dose of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mg / kg. In at least one embodiment, rhGAA or the pharmaceutical composition is administered at a dose of about 30 mg / kg. In at least one embodiment, rhGAA or the pharmaceutical composition is administered at a dose of about 25 mg / kg. In at least one embodiment, rhGAA or the pharmaceutical composition is administered at a dose of about 20 mg / kg.

[0175] In at least one embodiment, the dosage of rhGAA depends on the age of the patient. In at least one embodiment, the dosage of rhGAA depends on the weight of the patient. In at least one embodiment, the dosage of rhGAA depends on the age and weight of the patient. In any of the above embodiments, the total volume of rhGAA injected is determined by the weight of the subject. In at least one embodiment, the dosage of rhGAA is provided below in Tables 6A-6C.

[0176] In at least one embodiment, the dosages of rhGAA and / or miglustat are provided in Tables 5A-C and / or 6A-C provided herein. In some embodiments, rhGAA or the pharmaceutical composition is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the pharmacological chaperone is miglustat. In at least one embodiment, miglustat is administered as an oral dose ranging from about 20 mg to about 260 mg, or any intermediate or subrange therein, including 20, 40, 65, 85, 115, 130, 175, 195, or 260 mg, depending on the patient's age and weight. The effective dose for a particular individual may also be modified (e.g., increased or decreased) over time, depending on the individual's needs. For example, the amount of rhGAA and / or miglustat can be adjusted during physical illness or stress, if anti-acid α-glucosidase antibodies become present or increase, or if disease symptoms worsen.

[0177] In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition described herein includes improved and / or stabilized motor function, improved and / or stabilized muscle strength (upper body, lower body, or total body), improved and / or stabilized pulmonary function, reduced fatigue, decreased levels of at least one biomarker for muscle injury, decreased levels of at least one biomarker for glycogen accumulation, or a combination thereof. In some embodiments, the therapeutic effect of the rhGAA or pharmaceutical compositions described herein includes reversal of lysosomal lesions in muscle fibers, a faster and / or more effective reduction in glycogen content in muscle fibers, an increase in 6-minute walk test distance, a decrease in timed up-and-go test time, a decrease in 4-stair climb test time, a decrease in 10-meter walk test time, a decrease in Walk-Stairs-Gowers-Chair score, an increase in upper limb strength, improved and / or stabilization of shoulder adduction, improved and / or stabilization of shoulder abduction, improved and / or stabilization of elbow flexion, improved and / or stabilization of elbow extension, improved and / or stabilization of upper body strength, improved and / or stabilization of lower body strength, improved and / or stabilization of total body strength, an improvement in standing (sitting) forced vital capacity, an improvement and / or stabilization of maximum expiratory pressure, an improvement and / or stabilization of maximum inspiratory pressure, a decrease in fatigue severity scale score, a decrease in urinary hexose tetrasaccharide levels, a decrease in creatine kinase levels, a decrease in alanine aminotransferase levels, a decrease in aspartate aminotransferase levels, or any combination thereof.

[0178] In some embodiments, the rhGAA or pharmaceutical compositions described herein achieve a desired therapeutic effect faster than conventional rhGAA formulations when administered at the same dose. The therapeutic effect may be assessed based on one or more of the criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). For example, if a single dose of a conventional rhGAA formulation reduces glycogen levels in the tissues of a treated individual by 10% in one week, administration of the same dose of the rhGAA or pharmaceutical composition described herein may achieve the same degree of reduction in less than one week. In some embodiments, the rhGAA or pharmaceutical composition described herein may achieve a desired therapeutic effect at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more times faster than conventional rhGAA formulations when administered at the same dose.

[0179] In some embodiments, a therapeutically effective amount of rhGAA (or a composition or medicament comprising rhGAA) is administered two or more times. In some embodiments, rhGAA or a pharmaceutical composition described herein is administered continuously at regular intervals depending on the nature and extent of the disease effects. Administration at "regular intervals," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In certain embodiments, rhGAA is administered bimonthly, monthly, biweekly, weekly, twice weekly, or daily. In some embodiments, rhGAA is administered intravenously twice weekly, weekly, or biweekly. The administration interval for a single individual need not be a fixed interval and can vary over time depending on the needs of the individual. For example, the interval between doses can be reduced during physical illness or stress, if anti-rhGAA antibodies become present or increase, or if disease symptoms worsen.

[0180] In some embodiments, when used at the same dose, the rhGAA or pharmaceutical composition described herein may be administered less frequently than a conventional rhGAA formulation and still be capable of producing the same or better therapeutic effect as a conventional rhGAA formulation. For example, if a conventional rhGAA formulation is administered at 20 mg / kg weekly, the rhGAA or pharmaceutical composition described herein, when administered at 20 mg / kg, may produce the same or better therapeutic effect as a conventional rhGAA formulation, even if the rhGAA or pharmaceutical composition is administered less frequently, for example, every other week or every month. The therapeutic effect may be evaluated based on one or more criteria discussed above (e.g., cardiac status, glycogen level, or biomarker expression). In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition described herein is longer than that of a conventional rhGAA formulation. In some embodiments, the interval between two doses of the rhGAA or pharmaceutical composition is at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more times longer than that of a conventional rhGAA formulation.

[0181] In some embodiments, under the same therapeutic conditions (e.g., the same dose administered at the same intervals), the rhGAA or pharmaceutical composition described herein provides a therapeutic effect of a degree greater than that provided by conventional rhGAA formulations. The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., cardiac status, glycogen levels, or biomarker expression). For example, compared with a conventional rhGAA formulation administered at 20 mg / kg weekly, rhGAA or a pharmaceutical composition administered at 20 mg / kg weekly may result in a greater reduction in glycogen levels in the tissues of a treated individual. In some embodiments, when administered under the same therapeutic conditions, the rhGAA or pharmaceutical composition described herein provides a therapeutic effect that is at least about 1.25, 1.5, 1.75, 2.0, 3.0, or more greater than that of a conventional rhGAA formulation.

[0182] D. Two-component therapy In one or more embodiments, the rhGAA or pharmaceutical composition comprising rhGAA described herein is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the rhGAA or pharmaceutical composition is administered by a different route than the pharmacological chaperone. For example, the pharmacological chaperone may be administered orally, while the rhGAA or pharmaceutical composition is administered intravenously.

[0183] In some embodiments, the two-component therapy is administered in combination with an additional therapy, such as gene therapy or substrate reduction therapy. Exemplary gene therapy candidates include those encoding the GAA gene or variants thereof, with optional tags such as insulin-like growth factor II (IGF2), and / or binding immunoglobulin protein (Bip) or variants thereof. Exemplary substrate reduction therapy strategies include inhibiting glycogen synthesis using small molecules or gene inhibition. In some embodiments, the two-component therapy includes an ERT or pharmacological chaperone as the first component of the therapy, and gene therapy or substrate reduction therapy as the second component of the therapy. Such substrate reduction therapies include, for example, small molecule inhibition of glycogenin (GYG) or glycogen synthase (GYS) (e.g., GYS1 small molecule inhibitor in human clinical trial NCT05249621) or genetic approaches (e.g., antisense oligonucleotide-mediated suppression of muscle GYS1 synthesis).

[0184] In various embodiments, the pharmacological chaperone is miglustat. Without wishing to be bound by any theory, it is believed that co-administration of miglustat stabilizes rhGAA (e.g., ATB200 or sipa glucosidase alfa) from degradation in the systemic circulation, thereby enhancing delivery of the active ingredient rhGAA (e.g., ATB200 or sipa glucosidase alfa) to lysosomes.

[0185] In some embodiments, miglustat is administered at an oral dose of about 10 mg to about 600 mg. In at least one embodiment, miglustat is administered at an oral dose of about 20 mg to about 300 mg, or at an oral dose of about 20 mg, about 40 mg, about 65 mg, about 85 mg, about 115 mg, about 130 mg, about 175 mg, about 195 mg, or about 260 mg. Exemplary oral doses of miglustat also include about 15 mg to about 25 mg, about 30 mg to about 50 mg, about 50 mg to about 80 mg, about 70 mg to about 100 mg, about 100 mg to about 130 mg, about 110 mg to about 150 mg, about 150 mg to about 200 mg, about 170 mg to about 220 mg, and about 200 mg to about 300 mg.

[0186] In at least one embodiment, the dosage of miglustat depends on the age of the patient. In at least one embodiment, the dosage of miglustat depends on the weight of the patient. In at least one embodiment, the dosage of miglustat depends on the age and weight of the patient.

[0187] In at least one embodiment, dosages of rhGAA and / or miglustat are provided in the table below.

[0188] [Table 13]

[0189] [Table 14]

[0190] [Table 15]

[0191] Miglustat exhibits linear pharmacokinetics, with the area under the plasma concentration-time curve (AUC) and maximum concentration (C) decreasing with increasing dose from 130 mg (0.5 times the recommended dose of 260 mg in patients weighing 50 kg or more) to 260 mg. max) increased approximately proportionally. At the recommended dose of 260 mg, the mean C max The mean AUC was approximately 3 mcg / mL, and the mean AUC was approximately 25 mcg·hr / mL. The mean time to peak concentration ranged from 2 to 3 hours.

[0192] In some embodiments, rhGAA is administered intravenously at a dose of about 5 mg / kg to about 40 mg / kg, and miglustat is administered orally at a dose of about 10 mg to about 600 mg. In some embodiments, the dose of the recombinant human acid α-glucosidase (rhGAA) molecule population is in the range of about 15 mg / kg to about 40 mg / kg, and the dose of the pharmacological chaperone is in the range of about 15 mg to about 300 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 25 mg / kg to about 40 mg / kg, and miglustat is administered orally at a dose of about 15 mg to about 180 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 20 mg / kg to about 30 mg / kg, and miglustat is administered orally at a dose of about 180 mg to about 220 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 15 mg / kg to about 25 mg / kg, and miglustat is administered orally at a dose of about 240 mg to about 280 mg.

[0193] In some embodiments, miglustat and rhGAA are administered simultaneously. For example, miglustat may be administered within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes before or after administration of rhGAA. In some embodiments, miglustat is administered within 5, 4, 3, 2, or 1 minutes before or after administration of rhGAA.

[0194] In some embodiments, miglustat and rhGAA are administered sequentially. In at least one embodiment, miglustat is administered prior to administration of rhGAA. In at least one embodiment, miglustat is administered less than 3 hours before administration of rhGAA. In at least one embodiment, miglustat is administered about 2 hours before administration of rhGAA. In at least one embodiment, miglustat is administered within a range of 50 to 90 minutes before administration of rhGAA. For example, miglustat may be administered about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes before administration of rhGAA. In at least one embodiment, miglustat is administered about 1 hour before administration of rhGAA. In some embodiments, miglustat is orally administered about 1 hour before administration of rhGAA.

[0195] In some embodiments, miglustat is administered after administration of rhGAA. In at least one embodiment, miglustat is administered within 3 hours after administration of rhGAA. In at least one embodiment, miglustat is administered within 2 hours after administration of rhGAA. For example, miglustat can be administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes after administration of rhGAA.

[0196] In some embodiments, the subject fasts for at least 2 hours before administration of miglustat. In some embodiments, the subject fasts for at least 2 hours after administration of miglustat. In some embodiments, the subject fasts for at least 2 hours before administration of miglustat and at least 2 hours after administration of miglustat.

[0197] In some embodiments, the subject fasts for at least 2 hours before and at least 2 hours after administration of miglustat, and miglustat is administered about 1 hour before administration of rhGAA. In some embodiments, fasting, miglustat administration, and rhGAA administration follow the following administration timeline:

[0198] In some embodiments, a dual-component therapy according to the present disclosure improves one or more disease symptoms in a subject with Pompe disease compared to (1) baseline, or (2) a control treatment comprising administering alglucosidase alfa and a pharmacological chaperone placebo, where a placebo is administered in place of the pharmacological chaperone.

[0199] Provided herein are methods for improving motor and / or pulmonary function in a subject with Pompe disease over a period of at least 24 months, or at least 36 months, comprising co-administering or sequentially administering to the subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules together with a pharmacological chaperone; wherein each rhGAA molecule comprises seven potential N-glycosylation sites; 40% to 60% of the N-glycans on the rhGAA molecules are complex N-glycans; the rhGAA molecules comprise at least 0.5 moles of bis-mannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site, as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); and the method improves the subject's motor and / or pulmonary function compared to baseline.

[0200] In some embodiments, the subject treated with the dual therapy is an ERT-previous patient. In some embodiments, the ERT-previous subject has been previously treated with alglucosidase alfa. In some embodiments, the ERT-previous subject has been previously treated with alglucosidase alfa for about 2 to about 6 years. In some embodiments, the ERT-previous subject has been previously treated with alglucosidase alfa for at least about 7 years. In some embodiments, the ERT-previous subject is non-ambulatory. In some embodiments, the ERT-previous subject is ambulatory. In some embodiments, the subject is ERT-naive.

[0201] In some embodiments, the dual-component therapy disclosed herein improves and / or stabilizes a subject's motor function as measured by the 6-minute walk test (6MWT). In some embodiments, compared to baseline, the subject's 6-minute walk distance (6MWD) increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, the subject's 6MWD increases by at least 20 meters or by at least 5% after 52 weeks of treatment. In some embodiments, the subject's 6MWD improves by at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments of the methods of treating a subject with a history of ERT, motor function is measured by the 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 15, 16, 17, 18, 19, 20, or 21 meters 24 months after initiation of treatment. In some embodiments of the methods for treating ERT-naive subjects, motor function is measured by the 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 35, 40, 41, 42, 43, 44, 45, 46, or 47 meters 36 months after initiation of treatment. In some embodiments of the methods for treating ERT-naive subjects, motor function is measured by the 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 55, 56, 57, 58, 59, or 60 meters 24 months after initiation of treatment. In some embodiments of the methods for treating ERT-naive subjects, motor function is measured by the 6-minute walk test; and the improvement from baseline in 6-minute walk distance (6MWD) is at least 34, 35, 40, 41, 42, or 43 meters 36 months after initiation of treatment.In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0202] In some embodiments, the dual therapy disclosed herein stabilizes a subject's lung function as measured by forced vital capacity (FVC) testing. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, the subject's percent predicted FVC either increases compared to baseline or decreases by less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent predicted FVC decreases by less than 1% compared to baseline. In some embodiments, the subject's percent predicted FVC significantly improves and / or stabilizes after treatment compared to a control treatment. In some embodiments, the subject's percent-predicted FVC significantly improves by at least 0.5%, 1%, 2%, 3%, 4%, 5%, or 6% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to control treatment. In some embodiments, the subject's percent-predicted FVC significantly improves by at least 3% after 52 weeks of treatment, compared to control treatment. In some embodiments of the methods of treating ERT-experienced subjects, pulmonary function is measured by a seated forced vital capacity (FVC) test, and the subject's percent-predicted FVC is stable compared to baseline 24 or 36 months after initiation of treatment. In some embodiments of the methods of treating ERT-naive subjects, pulmonary function is measured by a seated forced vital capacity (FVC) test; and the improvement from baseline in the subject's percent-predicted FVC is at least 6.4, 6.5, 6.6, 6.7, or 6.8% 24 months after initiation of treatment. In some embodiments of the method of treating an ERT-naive subject, pulmonary function is measured by a seated forced vital capacity (FVC) test; and the subject's percent-to-predicted FVC improvement from baseline is at least 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2% 24 months after initiation of treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.In some embodiments, the subject has a baseline FVC of less than 50%. In some embodiments, the subject has a baseline FVC of 50% or greater.

[0203] In some embodiments, a dual-component therapy according to the present disclosure improves and / or stabilizes a subject's muscle strength as measured by manual muscle testing (MMT). In some embodiments, compared to baseline, the subject's MMT leg score improves as indicated by an increase of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, or 3.0 points after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to a control treatment, the subject's MMT leg score significantly improves and / or stabilizes after treatment. In some embodiments of the method of treating a subject with a history of ERT, muscle strength is measured by MMT; and the improvement from baseline in the MMT leg score is at least 1.9, 2, 2.1, 2.2, or 2.3 points 24 months after initiation of treatment. In some embodiments of the method for treating an ERT-naive subject, muscle strength is measured by MMT; and the improvement from baseline in MMT leg score is at least 1.5, 1.6, 1.7, 1.8, or 1.9 points 36 months after initiation of treatment. In some embodiments of the method for treating an ERT-naive subject, muscle strength is measured by MMT; and the improvement from baseline in MMT leg score is at least 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 points 24 months after initiation of treatment. In some embodiments of the method for treating an ERT-naive subject, muscle strength is measured by MMT; and the improvement from baseline in MMT leg score is at least 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 points 36 months after initiation of treatment. In some embodiments, the subject has a baseline MMT leg score of less than 25 points. In some embodiments, the subject has a baseline MMT leg score of 25 points or greater.

[0204] In some embodiments, the dual-component therapy disclosed herein improves and / or stabilizes a subject's motor function as measured by the Walking, Stairs, Gowers, and Chair (GSGC) test. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to baseline. In some embodiments, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment, compared to baseline. In some embodiments, the subject's GSGC score significantly improves after treatment, compared to a control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to the control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 1.0 point after 52 weeks of treatment, compared to the control treatment.

[0205] In some embodiments, a dual-component therapy according to the present disclosure reduces the level of at least one marker of muscle damage after treatment. In some embodiments, the at least one marker of muscle damage includes creatine kinase (CK). In some embodiments, compared to baseline, the subject's CK levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to baseline, the subject's CK levels are reduced by at least 20% after 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK levels are significantly reduced after treatment. In some embodiments, compared to a control treatment, the subject's CK levels are significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, the subject's CK levels are significantly reduced by at least 30% after 52 weeks of treatment compared to a control treatment.

[0206] In some embodiments, a dual-component therapy according to the present disclosure reduces the level of at least one marker of glycogen accumulation after treatment. In some embodiments, the at least one marker of glycogen accumulation includes urinary hexose tetrasaccharide (Hex4). In some embodiments, compared to baseline, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to baseline, the subject's urinary Hex4 level is reduced by at least 30% after 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's urinary Hex4 level is significantly reduced after treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment, compared to a control treatment.

[0207] In some embodiments, a dual-component therapy according to the present disclosure improves and / or stabilizes one or more disease symptoms in a Pompe disease patient subject with a history of ERT compared to (1) baseline or (2) a control treatment comprising administering alglucosidase alfa and a placebo for the pharmacological chaperone.

[0208] In some embodiments, the dual-component therapy for a subject with Pompe disease who has a history of ERT improves and / or stabilizes the subject's motor function as measured by 6MWT. In some embodiments, compared to baseline, the subject's 6MWD increases by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or 50 meters or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, the subject's 6MWD increases by at least 15 meters or at least 5% after 52 weeks of treatment. In some embodiments, compared to control treatment, the subject's 6MWD significantly improves after treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 10, 12, 14, 15, 16, 18, 20, 30, 40, or 50 meters after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to a control treatment. In some embodiments, the subject's 6MWD is significantly improved by at least 15 meters after 52 weeks of treatment, compared to a control treatment. In some embodiments, the subject has a baseline 6MWD of less than 300 meters. In some embodiments, the subject has a baseline 6MWD of 300 meters or greater.

[0209] In some embodiments, a dual-component therapy for a subject with Pompe disease who has a history of ERT improves and / or stabilizes the subject's lung function as measured by FVC testing. In some embodiments, after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, the subject's percent-predicted FVC increases by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% compared to baseline. In some embodiments, after 52 weeks of treatment, the subject's percent-predicted FVC increases by at least 0.1% compared to baseline. In some embodiments, the subject's percent-predicted FVC significantly improves and / or stabilizes after treatment compared to a control treatment. In some embodiments, the subject's percent-predicted FVC, when compared to a control treatment, is significantly improved by at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, or 10% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, the subject's percent-predicted FVC, when compared to a control treatment, is significantly improved by at least 4% after 52 weeks of treatment. In some embodiments, the subject has a baseline FVC of less than 55%. In some embodiments, the subject has a baseline FVC of 55% or greater.

[0210] In some embodiments, the dual-component therapy for a subject with Pompe disease who has a history of ERT improves and / or stabilizes the subject's motor function as measured by the GSGC test. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 points after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to baseline, the subject's GSGC score improves as indicated by a decrease of at least 0.5 points after 52 weeks of treatment. In some embodiments, compared to control treatment, the subject's GSGC score significantly improves and / or stabilizes after treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 0.3, 0.5, 0.7, 1.0, 1.5, 2.5, or 5 points after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to the control treatment. In some embodiments, the subject's GSGC score significantly improves as indicated by a decrease of at least 1.0 point after 52 weeks of treatment, compared to the control treatment.

[0211] In some embodiments, a dual-component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one marker of muscle damage after treatment. In some embodiments, the at least one marker of muscle damage includes CK. In some embodiments, compared to baseline, the subject's CK levels are reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to baseline, the subject's CK levels are reduced by at least 15% after 52 weeks of treatment. In some embodiments, compared to a control treatment, the subject's CK levels are significantly reduced after treatment. In some embodiments, compared to a control treatment, the subject's CK levels are significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, or 50% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, the subject's CK levels are significantly reduced by at least 30% after 52 weeks of treatment compared to a control treatment.

[0212] In some embodiments, a dual-component therapy for a subject with Pompe disease who has a history of ERT reduces the level of at least one marker of glycogen accumulation after treatment. In some embodiments, the at least one marker of glycogen accumulation includes urinary Hex4. In some embodiments, compared to baseline, the subject's urinary Hex4 level is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment. In some embodiments, compared to baseline, the subject's urinary Hex4 level is reduced by at least 25% after 52 weeks of treatment. In some embodiments, compared to control treatment, the subject's urinary Hex4 level is significantly reduced after treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% after 12, 26, 38, or 52 weeks of treatment, or after 18, 24, 30, or 36 months of treatment, compared to a control treatment. In some embodiments, the subject's urinary Hex4 levels are significantly reduced by at least 40% after 52 weeks of treatment, compared to a control treatment.

[0213] E. Kit Another aspect of the present disclosure relates to kits suitable for carrying out the rhGAA therapy described herein. In one or more embodiments, the kits include a container (e.g., a vial, tube, bag, etc.) containing rhGAA or a pharmaceutical composition (either pre-lyophilized or post-lyophilized) and instructions for reconstitution, dilution, and administration. In one or more embodiments, the kits include a container (e.g., a vial, tube, bag, etc.) containing a pharmaceutical composition (either pre-lyophilized or post-lyophilized) comprising a pharmacological chaperone (e.g., miglustat) and rhGAA, and instructions for reconstitution, dilution, and administration of the rhGAA with the pharmacological chaperone. [Example]

[0214] The present invention is further illustrated by the following non-limiting examples.

[0215] Example 1: Preparation of CHO cells producing rhGAA with high mono- or bis-M6P-bearing N-glycan content. DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA. This DNA construct is shown in Figure 4. After transfection, CHO cells harboring a stably integrated GAA gene were selected in hypoxanthine / thymidine-free (-HT) medium. GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

[0216] Cell pools with high GAA expression were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were grown on semi-solid media plates, picked using the ClonePix system, and transferred to 24-deep-well plates. These individual clones were assayed for GAA enzyme activity to identify clones with high GAA expression. Conditioned medium to determine GAA activity was prepared using 4-MU-α-glucosidase substrate. Clones with high GAA production, as measured by the GAA enzyme assay, were further characterized for viability, growth potential, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans were isolated using this procedure, including the CHO cell line GA-ATB200.

[0217] Example 2: Purification of rhGAA Multiple batches of rhGAA according to the present disclosure were produced in shake flasks and perfusion bioreactors using the CHO cell line GA-ATB200. This product is designated "ATB200." Weak anion exchange ("WAX") liquid chromatography was used to fractionate ATB200 rhGAA based on terminal phosphate and sialic acid. ERT was eluted with increasing amounts of salt to generate elution profiles. These profiles were monitored by UV (A280 nm). Similar CIMPR receptor binding profiles (at least approximately 70%) were observed for purified ATB200 rhGAA from different production batches (Figure 5), demonstrating that ATB200 rhGAA can be consistently produced.

[0218] Example 3: Characterization of oligosaccharides of ATB200 rhGAA ATB200 rhGAA was analyzed for site-specific N-glycan profiles using different LC-MS / MS analytical techniques. Results from the first two LC-MS / MS methods are shown in Figures 6A-6H. Results from the third LC-MS / MS method with 2-AA glycan mapping are shown in Figures 19A-19H, 20A-20B, and Table 7.

[0219] For the initial LC-MS / MS analysis, proteins were denatured, reduced, alkylated, and digested prior to LC-MS / MS analysis. For protein denaturation and reduction, 200 μg of protein sample, 5 μL of 1 mol / L Tris-HCl (final concentration 50 mM), 75 μL of 8 mol / L guanidine HCl (final concentration 6 M), 1 μL of 0.5 mol / L EDTA (final concentration 5 mM), 2 μL of 1 mol / L DTT (final concentration 20 mM), and Milli-Q® water were added to a 1.5 mL tube to provide a total volume of 100 μL. The sample was mixed and incubated at 56°C in a dry bath for 30 minutes. For alkylation, the denatured and reduced protein sample was mixed with 5 μL of 1 mol / L iodoacetamide (IAM, final concentration 50 mM) and then incubated in the dark at 10–30°C for 30 minutes. After alkylation, 400 μL of pre-chilled acetone was added to the sample, and the mixture was frozen at -80°C for 4 hours. The sample was then centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. 400 μL of pre-chilled acetone was added to the pellet, which was then centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. The sample was then air-dried on ice in the dark to remove residual acetone. Forty microliters of 8 M urea and 160 μL of 100 mM NH4HCO3 were added to the sample to dissolve the protein. For trypsin digestion, 50 μg of protein was then added with trypsin digestion buffer to a final volume of 100 μL, and 5 μL of 0.5 mg / mL trypsin (protein to enzyme ratio 20 / 1 w / w) was added. The solution was mixed thoroughly and incubated at 37°C overnight (16 ± 2 hours). The reaction was quenched by adding 2.5 microliters of 20% TFA (final concentration 0.5%), and the samples were then analyzed using a Thermo Scientific™ Orbitrap Velos Pro™ mass spectrometer.

[0220] In a second LC-MS / MS analysis, ATB200 samples were prepared by a similar denaturation, reduction, alkylation, and digestion procedure, except that iodoacetic acid (IAA) was used instead of IAM as the alkylating reagent, and then analyzed using a Thermo Scientific™ Orbitrap Fusion™ Lumos Tribid™ mass spectrometer.

[0221] The results of the first and second analyses are shown in Figures 6A-6H. In Figures 6A-6H, the results of the first analysis are represented by the left bar (dark gray), and the results of the second analysis are represented by the right bar (light gray). The symbolic nomenclature of glycan representations follows Varki, A., Cummings, RD, Esko JD, et al., Essentials of Glycobiology, 2nd edition (2009).

[0222] As can be seen from Figures 6A-6H, these two analyses provided similar results, although there was some variability between the results. This variability can be attributed to several factors, including the instrumentation used and the completeness of the N-glycan analysis. For example, if some species of phosphorylated N-glycans were not identified and / or quantified, then the total number of phosphorylated N-glycans may be underestimated, and the proportion of rhGAA bearing phosphorylated N-glycans at that site may be underestimated. As another example, if some species of non-phosphorylated N-glycans were not identified and / or quantified, then the total number of non-phosphorylated N-glycans may be underestimated, and the proportion of rhGAA bearing phosphorylated N-glycans at that site may be overestimated.

[0223] Figure 6A shows the N-glycosylation site occupancy of ATB200. As can be seen from Figure 6A, the first, second, third, fourth, fifth, and sixth N-glycosylation sites were nearly occupied, and both analyses detected that around 90% or more, up to about 100%, of the ATB200 enzyme had detected N-glycans at each of the potential N-glycosylation sites. However, for the seventh potential N-glycosylation site, only about half were N-glycosylated.

[0224] Figure 6B shows the N-glycosylation profile of the first potential N-glycosylation site, N84. As can be seen from Figure 6B, the major N-glycan species is bis-M6P N-glycan. Both the first and second analyses detected that more than 75% of ATB200 had bis-M6P at the first site, which corresponded to an average of about 0.8 mol of bis-M6P per mol of ATB200 at the first site.

[0225] Figure 6C shows the N-glycosylation profile of the second potential N-glycosylation site, N177. As can be seen from Figure 6C, the major N-glycan species are mono-M6P N-glycan and non-phosphorylated high-mannose N-glycan. Both the first and second analyses detected that more than 40% of ATB200 had mono-M6P at the second site, which corresponded to an average of about 0.4 to about 0.6 mol of mono-M6P per mol of ATB200 at the second site.

[0226] Figure 6D shows the N-glycosylation profile of the third potential N-glycosylation site, N334. As can be seen from Figure 6D, the major N-glycan species are non-phosphorylated high-mannose N-glycans, diantennary, triantennary, and tetraantennary complex-type N-glycans, and hybrid-type N-glycans. Both the first and second analyses detected that more than 20% of ATB200 had sialic acid residues at the third site, which corresponded to an average of about 0.9 to about 1.2 mol of sialic acid per mol of ATB200 at the third site.

[0227] Figure 6E shows the N-glycosylation profile of the fourth potential N-glycosylation site, N414. As can be seen from Figure 6E, the major N-glycan species are bis-M6P and mono-M6P N-glycans. Both the first and second analyses detected that more than 40% of ATB200 had bis-M6P at the fourth site, which corresponded to an average of about 0.4 to about 0.6 mol of bis-M6P per mol of ATB200 at the fourth site. Both the first and second analyses also detected that more than 25% of ATB200 had mono-M6P at the fourth site, which corresponded to an average of about 0.3 to about 0.4 mol of mono-M6P per mol of ATB200 at the fourth site.

[0228] Figure 6F shows the N-glycosylation profile of the fifth potential N-glycosylation site, N596. As can be seen from Figure 6F, the major N-glycan species is a fucosylated diantennary complex N-glycan. Both the first and second analyses detected that more than 70% of ATB200 had sialic acid residues at the fifth site, which corresponded to an average of about 0.8 to about 0.9 mol of sialic acid per mol of ATB200 at the fifth site.

[0229] Figure 6G shows the N-glycosylation profile of the sixth potential N-glycosylation site, N826. As can be seen from Figure 6G, the major N-glycan species are diantennary, triantennary, and tetraantennary complex N-glycans. In both the first and second analyses, more than 80% of ATB200 was detected to have sialic acid residues at the sixth site, which corresponded to an average of about 1.5 to about 1.8 mol of sialic acid per mol of ATB200 at the sixth site.

[0230] Analysis of N-glycosylation at the seventh site, N869, revealed approximately 40% N-glycosylation, with the most prevalent N-glycans being A4S3S3GF (12%), A5S3G2F (10%), A4S2G2F (8%), and A6S3G3F (8%).

[0231] Figure 6H shows a summary of phosphorylation at each of the seven potential N-glycosylation sites. As can be seen from Figure 6H, both the first and second analyses detected high phosphorylation levels at the first, second, and fourth potential N-glycosylation sites. Both analyses detected that more than 80% of ATB200 was mono- or bis-phosphorylated at the first site, more than 40% of ATB200 was mono-phosphorylated at the second site, and more than 80% of ATB200 was mono- or bis-phosphorylated at the fourth site.

[0232] Another N-glycosylation analysis of ATB200 was performed by the LC-MS / MS method described below, which produced an average N-glycosylation profile of ATB200 across 10 lots (Figures 19A-H, 20A-B).

[0233] N-linked glycans from ATB200 were enzymatically released with PNGase-F and labeled with 2-anthranilic acid (2-AA). The 2-AA-labeled N-glycans were further processed by solid-phase extraction (SPE) to remove excess salts and other contaminants. The purified 2-AA N-glycans were dissolved in acetonitrile / water (20 / 80; v / v), and 10 micrograms were loaded onto an aminopolymer analytical column (apHera™, Supelco) for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and high-resolution mass spectrometry (HRMS) analysis.

[0234] Liquid chromatographic (LC) separation was performed under normal-phase conditions using a gradient elution mode with mobile phase A (2% acetic acid in acetonitrile) and mobile phase B (5% acetic acid; 20 mM ammonium acetate in water adjusted to pH 4.3 with ammonium hydroxide). The initial mobile phase composition was 70% A / 30% B. For fluorescence detection, the detector parameters (RF-20Axs, Shimadzu) were excitation (Ex): 320 nm; emission (Em): 420 nm. HRMS analysis was performed using a quadrupole time-of-flight mass spectrometer (Sciex X500B QTOF) operated in independent data acquisition (IDA) mode. The acquired data files were converted to mzML files using MSConvert in ProteoWizard and then subjected to a glycan database search using GRITS Toolbox 1.2 Morning Blend software (UGA). The identified N-glycans were subsequently annotated. N-glycans were identified using both precursor monoisotopic mass (m / z) and product ion m / z. Experimental product ion and fragmentation patterns were confirmed in silico using the GlycoWorkbench 2 application.

[0235] To determine the relative quantification of N-linked glycans from ATB200, the data obtained from the HPLC-FLD-QTOF MS / MS experiment were processed as follows: All N-glycan peaks in the FLD chromatogram were integrated, and each peak was assigned a percentage of the total area of ​​all peaks in the FLD chromatogram. The fluorescence signal, expressed as the peak area, is a quantitative measure of the amount of each N-glycan in the sample. However, in most cases, multiple N-glycan species were contained in the same FLD peak. Therefore, mass spectrometry data was also required to obtain the relative quantification of each N-glycan species (Table 7). The ion intensity signal of each N-glycan was "extracted" from the data to create chromatographic peaks called extracted ion chromatograms (XICs). XICs aligned with the FLD chromatographic peaks were specific to only one N-glycan species. The XIC peaks created from the ion intensity signals were then integrated. This peak area is a relative quantitative measure of the amount of glycan present. Using both the FLD peak area and the mass spectrometer XIC peak area allowed for relative quantification of all N-linked glycan species of ATB200 reported herein.

[0236] The results of this LC-MS / MS analysis are provided in Table 7 below. Symbolic nomenclature for glycan representations is described in Wopereis W, et al. 2006. "Abnormal glycosylation with hypersialylated O-glycans in patients with sialuria." Biochimica et Biophysica Acta. 1762:598-607; Gornik O, et al. 2007. "Changes of serum glycans during sepsis and acute pancreatitis." Glycobiology. 17:1321-1332; Kattla JJ, et al. 2011. "Biologic protein glycosylation." In: Murray Moo-Young (ed.), Comprehensive Biotechnology, Second Edition, 3:467-486; Tharmalingam-Jaikaran T, et al. al. "N-glycan profiling of bovine follicular fluid at key dominant follicle developmental stages." 2014. Reproduction. 148:569-580; Clerc F, et al. "Human plasma protein N-glycosylation." 2015. Glycoconj J. DOI 10.1007 / s10719-015-9626-2; and Blackler RJ, et al. 2016."Single-chain antibody fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner." Glycobiology. 26-2:181-192.

[0237] [Table 16]

[0238] [Table 17]

[0239] Based on this 2-AA and LC-MS / MS analysis, and as further summarized, the tested ATB200 has an average M6P content (accounting for both mono-M6P and bis-M6P) of 3-5 mol per mol of ATB200 and a sialic acid content of 4-7 mol per mol of ATB200.

[0240] As shown in Figures 19A-19H and summarized in Figure 20B, the first potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, accounted for by an average mono-M6P content of about 0.25 mol mono-M6P / mol ATB200 and an average bis-M6P content of about 0.56 mol bis-M6P / mol ATB200; the second potential N-glycosylation site of ATB200 has an average M6P content of about 0.5 mol M6P / mol ATB200, where the predominant phosphorylated N-glycan species is mono-M6P N-glycan; and the third potential N-glycosylation site of ATB200 has an average M6P content of about 1 mol sialic acid / mol the fourth potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 mol M6P / mol ATB200, accounted for by an average mono-M6P content of about 0.35 mol mono-M6P / mol ATB200 and an average bis-M6P content of about 0.52 mol bis-M6P / mol ATB200; the fifth potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / mol ATB200; the sixth potential N-glycosylation site of ATB200 has an average sialic acid content of about 4.2 mol sialic acid / mol ATB200; and the seventh potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mol sialic acid / mol ATB200.

[0241] Furthermore, according to the 2-AA and LC-MS / MS analytical techniques, on average about 65% of the N-glycans at the first potential N-glycosylation site of ATB200 were high mannose-type N-glycans, about 89% of the N-glycans at the second potential N-glycosylation site of ATB200 were high mannose-type N-glycans, more than half of the N-glycans at the third potential N-glycosylation site of ATB200 were sialylated (nearly 20% were fully sialylated), and about 85% of the N-glycans at the third potential N-glycosylation site of ATB200 were complex-type N-glycans, about 84% of the N-glycans at the fourth potential N-glycosylation site of ATB200 were high mannose-type N-glycans, and about 85% of the N-glycans at the fifth potential N-glycosylation site of ATB200 were complex-type N-glycans. About 70% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are sialylated (about 26% are fully sialylated), and about 100% of the N-glycans at the 5th potential N-glycosylation site of ATB200 are complex-type N-glycans, about 85% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are sialylated (nearly 27% are fully sialylated), and about 98% of the N-glycans at the 6th potential N-glycosylation site of ATB200 are complex-type N-glycans, and about 87% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are sialylated (nearly 8% are fully sialylated), and about 100% of the N-glycans at the 7th potential N-glycosylation site of ATB200 are complex-type N-glycans.

[0242] Example 4: Analytical Comparison of ATB200 and MYOZYME® / LUMIZYME® Purified ATB200 and LUMIZYME® N-glycans were analyzed by MALDI-TOF to determine the distinct N-glycan structures found in each ERT. LUMIZYME® was obtained from a commercial source. As shown in Figure 7, ATB200 exhibited four prominent peaks that eluted to the right of LUMIZYME®. Because this determination is based on terminal charge rather than CIMPR affinity, this confirms that ATB200 was more highly phosphorylated than LUMIZYME®. As summarized in Figure 8, the ATB200 sample was found to contain lower amounts of non-phosphorylated high-mannose N-glycans than LUMIZYME®.

[0243] To determine the ability of conventional rhGAA in MYOZYME® and LUMIZYME® to interact with CIMPR, two conventional rhGAA formulations were injected onto a CIMPR affinity column (which binds rhGAA bearing M6P groups) and the flow-through was collected. The bound material was eluted with a free M6 gradient. Fractions were collected in a 96-well plate and assayed for GAA activity with 4MU-α-glucosidase substrate. The relative amounts of unbound rhGAA (flow-through) and bound rhGAA (eluted M6P) were determined based on GAA activity and reported as a percentage of total enzyme. Figures 9A and 9B show the binding profiles of rhGAA in MYOZYME® and LUMIZYME®: 73% of rhGAA in MYOZYME® (Figure 9B) and 78% of rhGAA in LUMIZYME® (Figure 9A) did not bind to CIMPR. In fact, only 27% of rhGAA in MYOZYME® and 22% of rhGAA in LUMIZYME® contained M6P, which may be productive in targeting it to CIMPR on muscle cells. In contrast, as shown in Figure 5, under the same conditions, more than 70% of rhGAA in ATB200 was found to bind to CIMPR.

[0244] In addition to the high percentage of rhGAA capable of binding to CIMPR, it is important to understand the quality of that interaction. LUMIZYME® and ATB200 receptor binding was determined using a CIMPR plate binding assay. Briefly, GAA was captured using a CIMPR-coated plate. Various concentrations of rhGAA were applied to the immobilized receptor, and unbound rhGAA was washed away. The amount of remaining rhGAA was determined by GAA activity. As shown in Figure 10A, ATB200 bound to CIMPR significantly better than LUMIZYME®. Figure 10B shows the relative content of bis-M6P N-glycans in LUMIZYME® (a conventional rhGAA formulation) and ATB200 of the present invention. For LUMIZYME®, on average, only 10% of the molecules have bis-phosphorylated N-glycans. In contrast, for ATB200, on average, every rhGAA molecule has at least one bis-phosphorylated N-glycan.

[0245] Overall, the higher content of M6P N-glycans in ATB200 compared to LUMIZYME® indicates that a higher proportion of rhGAA molecules are able to target muscle cells with ATB200. As shown above, the high proportion of monophosphorylated and bisphosphorylated structures determined by MALDI is consistent with the CIMPR profile, which explains the significantly greater binding of ATB200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule possesses at least one natural bis-M6P N-glycan structure. This higher bis-M6P N-glycan content in ATB200 is consistent with the higher affinity binding (K) to CIMPR in the M6P receptor plate binding assay. D This directly correlates with the ATP concentration (approximately 2-4 nM).

[0246] The relative cellular uptake of ATB200 and LUMIZYME® rhGAA was compared using normal and Pompe disease fibroblast cell lines. Comparisons involved 5-100 nM of the disclosed ATB200 and 10-500 nM of the conventional rhGAA formulation, LUMIZYME®. After 16 hours of incubation, external rhGAA was inactivated with Tris base, and cells were washed three times with PBS before harvesting. Internalized GAA was measured by 4MU-α-glucoside hydrolysis and plotted against total cellular protein. Results are shown in Figures 11A-11C.

[0247] ATB200 was also shown to be efficiently internalized by cells. As illustrated in Figures 11A-11B, ATB200 was internalized by both normal and Pompe disease fibroblasts to a greater extent than the conventional rhGAA formulation, LUMIZYME®. ATB200 saturates cell receptors at approximately 20 nM, while approximately 250 nM of LUMIZYME® is required to saturate cell receptors. The uptake efficiency constant (K) estimated from these results is uptake ) is 2-3 nM for ATB200 and 56 nM for LUMIZYME®, as shown in Figure 11C. These results suggest that ATB200 is a well-targeted treatment for Pompe disease.

[0248] Example 5: ATB200 and pharmacological chaperones The stability of ATB200 in acidic or neutral pH buffers was determined in a thermal stability assay using SYPRO Orange, which shows an increase in dye fluorescence upon protein denaturation. As shown in Figure 12, the addition of miglustat stabilized ATB200 at pH 7.4 in a concentration-dependent manner, which was comparable to the stability of ATB200 at pH 5.2, a condition that mimics the acidic environment of lysosomes. As summarized in Table 10, the addition of miglustat increased the melting temperature (T m ) rose by almost 10°C.

[0249] [Table 18]

[0250] Example 6: Co-administration of ATB200 and miglustat in Gaa KO mice The therapeutic effects of ATB200 and miglustat were evaluated and compared with alglucosidase alfa in Gaa KO mice. Male Gaa KO (3-4 months old) and age-matched wild-type (WT) mice were used in this study. Alglucosidase alfa was administered via bolus intravenous (IV) injection into the tail vein. In the co-administration regimen, miglustat was administered via oral gavage (PO) 30 minutes before the IV injection of ATB200. Treatments were given every other week. 14 days after the final administration, treated mice were sacrificed, and various tissues were harvested for further analysis. The study design is summarized in Table 11.

[0251] [Table 19]

[0252] Tissue glycogen content of tissue samples was determined using amyloglucosidase digestion as discussed above. As shown in Figure 13, the combination of 20 mg / kg ATB200 and 10 mg / kg miglustat significantly reduced glycogen content in four different tissues (quadriceps, triceps, gastrocnemius, and heart) when compared to the same dosage of alglucosidase alfa.

[0253] Tissue samples were also analyzed by the methods described in Khanna R, et al. (2012), "The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease," Plos One 7(7):e40776; and Khanna, R et al. (2014), "The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid α-Glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease." Biomarker changes were also analyzed according to the methods discussed in "The Journal of Neuropathic Neuropathy and Neuropathological Disease," PLoS ONE 9(7):e102092. As shown in Figure 14, compared to WT, muscle fibers from Gaa KO animals exhibited a significant increase in LAMP1-positive vesicles, an indication of lysosomal proliferation, and their hypertrophy. Co-administration of ATB200 / miglustat led to more fibers with normalized LAMP1 levels, while the remaining LAMP1-positive vesicles also decreased in size (inset).

[0254] Similarly, prominent LC3-positive aggregates in muscle fibers from untreated Gaa KO mice indicate the presence of autophagic regions and autophagic buildup. LC3-positive aggregates (red) were preferentially reduced in mice treated with ATB200 / miglustat co-administration compared with mice treated with alglucosidase alfa (Figure 15A). Similar observations were made when LC3 expression was assessed using Western blot. As shown in Figure 15B, the majority of animals treated with ATB200 / miglustat exhibited significantly reduced levels of LC3 II, a lipidated form associated with autophagosomes, suggesting improved autophagic flux. In comparison, alglucosidase alfa had a less pronounced effect on autophagy.

[0255] Dysferlin, a protein involved in membrane repair whose deficiency / mistranslocation is associated with several muscular dystrophies, was also evaluated. As shown in Figure 16, dysferlin (brown) severely accumulated in the sarcoplasm of Gaa KO mice. Compared with alglucosidase alfa, ATB200 / miglustat was able to restore dysferlin to the sarcolemma of a greater number of muscle fibers.

[0256] These data are consistent with the cellular improvements demonstrated in human Pompe disease patients treated with ATB200 and miglustat (e.g., these patients exhibit reduced levels of biomarkers of glycogen accumulation and muscle damage), leading to not only an effective treatment for Pompe disease but also to reversal of disease progression. Clinical data in human Pompe disease patients are summarized in Examples 8 and 9 below.

[0257] Example 7: Single Fiber Analysis As shown in Figure 17, the majority of vehicle-treated mice exhibited significantly enlarged lysosomes (green) (see, e.g., "B") and the presence of clumped autophagic buildup (red) (see, e.g., "A"). MYOZYME®-treated mice did not show any significant differences when compared to vehicle-treated mice. In contrast, most fibers isolated from ATB200-treated mice showed a dramatic reduction in lysosome size (see, e.g., "C"). Furthermore, there was also a variable reduction in the extent of autophagic buildup (see, e.g., "C"). As a result, a large proportion (36-60%) of analyzed muscle fibers from ATB200-treated mice appeared normal or near-normal. Table 12 below summarizes the single fiber analysis shown in Figure 17.

[0258] [Table 20]

[0259] Overall, this data indicates that ATB200, with its high M6P content, both alone and further stabilized with the pharmacological chaperone miglustat at the neutral pH of blood, exhibits more efficient tissue targeting and lysosomal transport when administered to Gaa KO mice than alglucosidase alfa, consistent with the stabilization of ATB200 by miglustat as shown in Figure 18. As a result, ATB200 administration and ATB200 / miglustat coadministration were more effective than alglucosidase alfa in correcting some of the disease-associated pathologies, such as glycogen accumulation, lysosomal proliferation, and the formation of autophagic areas. These positive therapeutic effects suggest that ATB200 administration and ATB200 / miglustat coadministration may help muscle fibers recover from injury and even reverse damage by removing glycogen accumulated in cells due to a lack of optimal GAA activity. Similar to Example 6, these data are also consistent with the cellular improvements demonstrated in human Pompe disease patients following administration of ATB200 and miglustat, leading to both effective treatment of Pompe disease and reversal of disease progression. Clinical data from human Pompe disease patients are summarized in Examples 8 and 9 below.

[0260] Example 8: ATB200-02 Clinical Trial A Phase 1 / 2 (ATB200-02, NCT-02675465) open-label, fixed-sequence, dose-escalation clinical trial was conducted to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary efficacy of intravenous infusion of ATB200 and miglustat in adult subjects with Pompe disease. The data were reported in WO 2020 / 163480, the disclosure of which is incorporated herein by reference.

[0261] Example 9: ATB200-03 Clinical Trial: A Phase 3 In-Human Study of ATB200 / Miglustat in Patients with Pompe Disease The ATB200-03 trial was a phase 3, double-blind, randomized, multicenter, international study of ATB200 / miglustat compared with alglucosidase alfa / placebo in adult subjects with late-onset Pompe disease (LOPD) who had previously received enzyme replacement therapy with alglucosidase alfa (i.e., ERT-experienced) or had never received ERT (i.e., ERT-naive).

[0262] Study design As shown in Figure 21, the trial consisted of a screening period of up to 30 days, a treatment period of 12 months, and a safety follow-up period of 30 days. Eligible subjects were randomized in a 2:1 ratio to receive ATB200 / miglustat or alglucosidase alfa / placebo and stratified by ERT status (ERT-experienced, ERT-naive) and baseline 6-minute walk distance (6MWD) (≥75 meters <150 meters, ≥150 meters <400 meters, ≥400 meters).

[0263] Efficacy assessments (i.e., functional assessments) included assessment of gait function (6MWT), motor function tests (Galliarts-Stair-Gowers-Chair Graduation (GSGC) test and Timed Up-and-Go (TUG) test), muscle strength (manual muscle testing and quantitative muscle testing), and pulmonary function tests (FVC, SVC, MIP, MEP, and SNIP). Patient-reported outcomes (Rasch-built Pompe-specific Activity (R-Pact) scale, EuroQol 5 Dimensions 5 Level instrument (EQ-5D-5L), Patient-Reported Outcomes Measurement Information System (PROMIS®) instrument for physical function, fatigue, dyspnea, and upper limbs, and Subject's Global Impression of Change) were recorded. Physician's Global Impression of Change was also performed.

[0264] Pharmacodynamic evaluation included measurement of biomarkers for muscle injury (creatine kinase (CK)) and disease substrates (urinary hexose tetrasaccharide (Hex4)). Low-density blood samples for population PK analysis were collected in ERT-experienced subjects to determine plasma total GAA protein levels and miglustat concentrations. In ERT-naive subjects, time-series blood sampling was performed to characterize the PK profiles of total GAA protein and miglustat.

[0265] Safety assessments included monitoring for adverse events (AEs), including infusion-related reactions (IARs), clinical tests (chemistry, hematology, and urinalysis), vital signs, physical examination including weight, electrocardiogram (ECG), and immunogenicity. Concomitant medications and non-pharmacological therapies were also recorded.

[0266] Selecting a target Subjects enrolled in the study met all of the following inclusion criteria and did not meet any exclusion criteria. Overall, 122 subjects participated in the ATB200-03 clinical trial. Of these, 85 subjects (ERT-experienced: 65; ERT-naïve: 20) received ATB200 / miglustat treatment, and 37 subjects (ERT-experienced: 30; ERT-naïve: 7) received alglucosidase alfa / placebo treatment. As shown in Figure 22, baseline 6MWD and FVC data were representative of the population and generally similar between the two treatment groups.

[0267] Inclusion Criteria: 1. Subjects provided signed informed consent before any study-related procedures were performed. 2. Male and female subjects were 18 years of age or older and weighed 40 kg or more at the time of screening. 3. Female and male subjects of childbearing potential agreed to use medically accepted methods of contraception during this study and for 90 days after the final administration of the study drug. 4. Subject had a documented diagnosis of LOPD with one of the following: A deficiency of the GAA enzyme b.GAA genotyping 5. Subjects were classified into one of the following with respect to ERT status: a. ERT-previous, defined as having received standard of care ERT (alglucosidase alfa) at the recommended dose and regimen (i.e., 20 mg / kg every 2 weeks) for ≥ 24 months Specifically for Australia, ERT-experienced is defined as having received standard of care ERT (alglucosidase alfa) at a dose of 20 mg / kg of lean body mass or ideal body mass every 2 weeks at the recommended dose and regimen. b. ERT-naive, defined as never having received investigational or over-the-counter ERT 6. Subjects had a sitting FVC at screening that was 30% or greater than predicted for healthy adults (National Health and Nutrition Examination Survey III). 7. Subject performed two 6MWTs at screening that were valid as determined by the clinical assessor and met all of the following criteria: a. Both screening values ​​for 6MWD were above 75 meters. b. Both 6MWD screening values ​​were below 90% of the predicted value for healthy adults c. The lower 6MWD value was within 20% of the higher 6MWD value

[0268] Exclusion criteria: 1. Subjects had received, or were expected to receive during the study, any investigational therapy or pharmacological treatment for Pompe disease other than alglucosidase alfa within the 30 days prior to Day 1 or 5 half-lives of that therapy or treatment, whichever was longer. 2. Subject had previously received gene therapy for Pompe disease. 3. Subject was taking any of the following prohibited drugs within the 30 days prior to Day 1: Miglitol Miglustat Acarbose Voglibose 4. Subject required the use of invasive or non-invasive ventilatory support for more than 6 hours per day while awake. 5. Subject had a hypersensitivity to ATB200, alglucosidase alfa, or any of the excipients in miglustat. 6. Subject had a medical condition or any other extenuating circumstances that, in the opinion of the Investigator or Medical Monitor, posed an undue safety risk to the subject or impaired or adversely affected the subject's ability to comply with the protocol requirements. This included clinical depression with uncontrolled or poorly controlled symptoms (as diagnosed by a psychiatrist or other mental health professional). 7. Subject, if female, was pregnant or lactating at the time of screening. 8. Subjects, regardless of gender, were planning to become pregnant during the study. 9. Subject refused to undergo genetic testing.

[0269] Investigational drug, dosage, and method of administration Subjects were randomized in at least a 2:1 randomization ratio to receive either ATB200 / miglustat or alglucosidase alfa / placebo. Table 13 below summarizes the treatments of enrolled subjects.

[0270] [Table 21]

[0271] Data analysis and statistical considerations The primary efficacy outcome was the change in 6MWD from baseline to week 52. The primary outcome was tested for superiority of ATB200 / miglustat over alglucosidase alfa / placebo using a mixed-effects model for repeated measures (MMRM) and pre-specified non-parametric tests when normality was violated.

[0272] Key secondary efficacy endpoints, in prespecified order of hierarchical importance, were: These secondary endpoints were analyzed using an analysis of covariance (ANCOVA) model with missing value imputation by imputing the value of the most recent observation (ITT LOCF). Change from baseline to week 52 in sitting FVC (% predicted) Change in lower limb manual muscle test score from baseline to week 52 Change in PROMIS-Physical Function Total Score from Baseline to Week 52 Change in PROMIS-Fatigue total score from baseline to week 52 Change in GSGC total score from baseline to week 52

[0273] Other secondary efficacy endpoints were: Changes in the following motor function variables from baseline to week 52: - Time taken to complete the 10-meter walk of the GSGC test (i.e., gait assessment) - Time taken to complete the 4-step stair climb in the GSGC test - Time taken to complete the Gowers movement in the GSGC test - Time taken to stand up from a chair as part of the GSGC test - Time taken to complete the TUG test Change from baseline to week 52 in the following muscle strength variables: - Upper limb manual muscle test score - Manual Muscle Test Total Score - Upper limb quantitative muscle strength test value (kg) - Lower limb quantitative muscle strength test value (kg) - Quantitative muscle strength test total value (kg) Change from baseline to week 52 in the following variables from patient-reported outcome measures: - PROMIS-dyspnea total score - PROMIS-Upper Extremity Total Score - R-PAct scale total score - EQ-5D-5L health status Subject's actual functional status (improved, stable, or worsened) regarding the effect of the study drug in the following life domains at Week 52, as measured by the Subject's Global Impression of Change: - General physical health - breathing effort - Muscle strength - Muscle function - Ability to move around - Daily life activities - Vitality level - Muscle pain level Actual subject functional status (improved, stable, or worsening) at Week 52 as measured by Physician's Global Impression of Change Change from baseline to week 52 in the following lung function measures: - Sitting FVC (% predicted) - MIP (cmH2O) - MIP (% of predicted value) - MEP (cmH2O) - MEP (% of predicted value) - SNIP (cmH2O)

[0274] Pharmacodynamic endpoints were as follows: Change in serum CK levels from baseline to week 52 Change in urinary Hex4 levels from baseline to week 52

[0275] For ERT-experienced subjects, pharmacokinetic endpoints from the population PK analysis of total GAA protein levels and miglustat concentrations were collected. For ERT-naive subjects, plasma total GAA protein concentrations and miglustat PK parameters were calculated.

[0276] The safety profile of ATB200 / miglustat was characterized using the incidence of treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and AEs leading to discontinuation of study drug, the frequency and severity of immediate and delayed IARs, and any abnormalities observed in other safety assessments. The impact of immunogenicity to ATB200 and alglucosidase alfa on safety and efficacy was also evaluated.

[0277] Statistical methods included the following considerations for sample randomization, sample size calculation, efficacy analysis, and safety analysis.

[0278] Randomization. The following two factors were identified as design stratification variables: 1. Baseline 6MWD (≥75 <150 meters, ≥150 <400 meters, ≥400 meters); and 2. ERT status (ERT-experienced, ERT-naive). These two factors formed six factor combinations (i.e., levels, strata). Balancing the above risk factors using a centralized block randomization procedure 1) reduced bias and increased precision of statistical inferences, and 2) enabled various planned and unplanned subset analyses. A block randomization scheme was implemented for each of the six strata. The randomization ratio was fixed at 2:1: ATB200 / miglustat vs. alglucosidase alfa / placebo.

[0279] Sample Size Calculation. A two-sample t-test with a two-sided significance level of 0.05 and a 2:1 randomization scheme (66 subjects in the ATB200 / miglustat group and 33 subjects in the alglucosidase alfa / placebo group, for a total sample size of 99 subjects) was determined to have approximately 90% power to detect a standardized effect size of 0.7 between these two samples in a superiority test. This calculation was performed using Nquery 8©. Assuming a 10% dropout rate, the sample size was approximately 110 subjects.

[0280] Efficacy Analysis. A parametric analysis of covariance (ANCOVA) model was used to analyze the primary efficacy endpoint (i.e., change in 6MWD from baseline to week 52) and compare new treatment with control. This model typically adjusted for baseline 6MWD (as a continuous covariate) and stratified randomization using two factors: ERT status (ERT-naive vs. ERT-experienced) and baseline 6MWD (75 to < 150 meters, 150 to < 400 meters, ≥ 400 meters). However, baseline 6MWD could not be used twice in the model (as both a continuous and categorical variable) due to the expected high point-biserial correlation between them. Therefore, the 6MWD continuous variable remained in the model, but the categorical 6MWD was removed. The ANCOVA model then had columns for treatment, baseline 6MWD (continuous), and ERT status (categorical).

[0281] In addition, potential treatment × covariate interactions (i.e., treatment × ERT status and treatment × baseline 6MWD continuous) were examined. If an interaction term was statistically significant (e.g., p<0.10, two-sided) and had a logical biological interpretation, it could then be added to the final ANCOVA model, which would potentially be used for the primary endpoint analysis. The data were then analyzed based on the ANCOVA model, providing all relevant estimates (e.g., LS means for each treatment group, LS means of the differences, 95% confidence intervals (CIs) for the LS mean differences, and p-values ​​for comparisons between the two treatment groups).

[0282] To support interpretation of clinical benefit, a composite subject-level response was defined based on the aggregate treatment outcome data. Subjects were classified according to an ordinal response variable consisting of significant improvement, moderate improvement, or slight improvement / no improvement based on treatment outcome.

[0283] Key secondary endpoints were analyzed in a hierarchical order using a stepwise closed testing procedure to control the type I error rate. Key secondary and other secondary endpoints were analyzed separately using methods similar to those used for the primary endpoint analysis.

[0284] Safety Analysis. Safety data were summarized using counts and percentages for categorical data and descriptive statistics (mean, standard deviation, median, minimum, maximum) for continuous data.

[0285] Efficacy results of the ATB200-03 clinical trial In the overall population, ATB200 / miglustat treatment demonstrated improvement in 6MWD and stabilization of percent-predicted FVC compared to baseline at week 52 (FIG. 23A) and over time (FIG. 23B). Compared with alglucosidase alfa / placebo, ATB200 / miglustat treatment demonstrated greater improvement in 6MWD in the overall population at week 52 (FIG. 23A). Furthermore, as shown in FIG. 23A, ATB200 / miglustat treatment demonstrated clinically significant improvement in percent-predicted FVC in the overall population at week 52 compared with alglucosidase alfa / placebo.

[0286] In the ERT-experienced population, ATB200 / miglustat treatment demonstrated improvement in 6MWD and stabilization of percent-predicted FVC at week 52 compared to baseline ( FIG. 24 ). Compared to alglucosidase alfa / placebo, ATB200 / miglustat treatment demonstrated improvement in 6MWD over time and stabilization of percent-predicted FVC over time in the ERT-experienced population ( FIG. 25 ). Furthermore, as shown in FIG. 24 , ATB200 / miglustat treatment demonstrated clinically significant improvements in both 6MWD and percent-predicted FVC at week 52 in the ERT-experienced population compared to alglucosidase alfa / placebo.

[0287] As shown in Figures 26A and 26B, in the smaller ERT-naive population (n=27), ATB200 / miglustat treatment demonstrated improvement in 6MWD and stabilization of percent-predicted FVC compared to baseline at week 52 (Figure 26A) and over time (Figure 26B). There was greater variability between the two treatment groups, with no clinically meaningful improvements in 6MWD or percent-predicted FVC (Figure 26A).

[0288] As shown in Figure 28, in the overall population and the population with a history of ERT, lower limb MMT was more favorable with ATB200 / miglustat treatment compared to alglucosidase alfa / placebo.

[0289] There was a numerical improvement in mean change (SD) from baseline to week 52 in lower extremity MMT scores of 1.6 (3.78) in subjects treated with coadministered sipaglucosidase alfa and miglustat compared with 0.9 (2.58) in subjects treated with alglucosidase alfa and placebo (p=0.191).

[0290] As shown in Figure 29, in the overall population and the ERT-experienced population, ATB200 / miglustat treatment demonstrated clinically significant improvements in GSGC at week 52 compared to alglucosidase alfa / placebo.

[0291] The mean change from baseline to week 52 in GSGC total score was -0.53 (2.5) in subjects treated with coadministered sipa glucosidase alfa and miglustat compared with 0.77 (1.8) in subjects treated with alglucosidase alfa and placebo (p=0.009).

[0292] As shown in Figure 30, PROMIS physical function scores were better with ATB200 / miglustat treatment compared to alglucosidase alfa / placebo in the overall population and in the ERT-experienced population.

[0293] As shown in Figure 31, PROMIS fatigue improved to a similar extent between the two treatment groups in the overall population and in the ERT-experienced population.

[0294] Biomarker results of the ATB200-03 clinical trial In the overall population and the ERT-experienced population, ATB200 / miglustat treatment demonstrated improvements in biomarkers of muscle damage (CK) and disease substrate (Hex4) over time (Figures 32 and 33). Furthermore, as shown in Figures 32 and 33, in the overall population and the ERT-experienced population, the reductions in CK and urinary Hex4 were significantly greater with ATB200 / miglustat treatment compared to alglucosidase alfa / placebo at week 52.

[0295] In patients with a history of ERT, baseline mean urinary Glc(4) concentrations were 4.6 mmol / mol and 7.2 mmol / mol in the ATB200 / miglustat-treated and alglucosidase alfa vs. placebo-treated groups, respectively. At week 52, mean urinary Glc(4) concentrations were 2.9 mmol / mol and 9.1 mmol / mol in the ATB200 / miglustat-treated and alglucosidase alfa vs. placebo-treated groups, respectively.

[0296] As summarized in Figure 34, ATB200 / miglustat treatment was consistently superior to alglucosidase alfa / placebo in the overall population and in the ERT-experienced population for motor function, pulmonary function, muscle strength, patient-reported outcomes (PROs), and biomarker endpoints. Furthermore, 16 of the 17 efficacy and biomarker endpoints evaluated were superior to alglucosidase alfa / placebo.

[0297] Safety results of the ATB200-03 clinical trial As shown in Figure 35, the overall safety profile of the ATB200 / miglustat treatment group was similar to that of the alglucosidase alfa / placebo group.

[0298] Figures 36-40 describe additional aspects of the ATB200-03 trial.

[0299] Example 10: Results of the PROPEL Phase 3 Clinical Trial AT-GAA demonstrated clinically meaningful and significant improvements in both musculoskeletal and respiratory measures of late-onset Pompe disease compared with standard of care in the pivotal Phase 3 PROPEL trial. PROPEL is also referred to as "ATB200-03." See Example 9.

[0300] Patients who switched from the approved standard of care ERT (alglucosidase alfa) to AT-GAA walked an average of 17 meters more (p=0.046).

[0301] Patients who switched to AT-GAA also showed improvement in percent-predicted forced vital capacity (FVC), the most important measure of respiratory function in Pompe disease, compared with deterioration in patients treated with alglucosidase alfa (FVC difference 4.1%; p=0.006).

[0302] AT-GAA demonstrated a nominally statistically significant and clinically meaningful difference in the first key secondary endpoint of FVC superiority compared with patients treated with alglucosidase alfa (FVC difference 3.0%; p=0.023).

[0303] In the combined study population of ERT-converted and ERT-naive patients, AT-GAA outperformed alglucosidase alfa by 14 m on the primary endpoint (21 m vs. 7 m), although the superiority was not statistically significant (p=0.072).

[0304] Improvement in two key biomarkers of Pompe disease (Hex-4 and CK) for the combined study population was significantly better with AT-GAA compared with alglucosidase alfa (p<0.001).

[0305] PROPEL was a 52-week, double-blind, randomized, international trial designed to evaluate the efficacy, safety, and tolerability of AT-GAA compared with the current standard of care, enzyme replacement therapy (ERT), alglucosidase alfa. The trial enrolled 123 adult Pompe disease patients who were still able to walk and breathe without mechanical ventilation and was conducted at 62 clinical sites in 24 countries on five continents. This was the largest controlled clinical trial ever conducted in a lysosomal disorder.

[0306] Patients enrolled in PROPEL were randomized 2:1, so that for every two patients randomized to treatment with AT-GAA, one was randomized to treatment with alglucosidase alfa. Of the Pompe disease patients enrolled in PROPEL, 77% had been treated with alglucosidase alfa immediately prior to enrollment (n=95), and 23% had never received any ERT treatment (n=28). 117 patients completed the PROPEL trial, and all 117 willingly enrolled in the long-term extension study and are currently being treated for their Pompe disease exclusively with AT-GAA.

[0307] Prespecified analyses of 6-minute walk distance (6MWD) and percent-predicted forced vital capacity (FVC) in a combined ERT conversion and ERT-naive study population: The primary endpoint of this study was the mean change in 6-minute walk distance compared to baseline measurements at 52 weeks in the ERT-switched and ERT-naive combination patient populations. In this combination population, patients taking AT-GAA (n=85) walked an average of 21 meters more at 52 weeks compared to 7 meters in patients treated with alglucosidase alfa (n=37) (Table 14). This primary endpoint in the combination population was evaluated for superiority, and although numerically large, statistical significance for superiority in this combination population was not achieved for the AT-GAA arm when compared to the alglucosidase alfa arm (p=0.072).

[0308] According to the hierarchy of the statistical analysis plan, the first key secondary endpoint of this study was the mean change in percent-predicted FVC at 52 weeks in the combination population. In this combination population, patients taking AT-GAA demonstrated a nominally statistically significant and clinically meaningful difference in superiority over patients treated with alglucosidase alfa. AT-GAA significantly slowed the rate of respiratory deterioration in patients after 52 weeks. Patients treated with AT-GAA demonstrated a 0.9% absolute worsening of percent-predicted FVC compared with a 4.0% absolute worsening in the alglucosidase alfa arm (p=0.023) (Table 15). Percent-predicted FVC is the most important measure of respiratory muscle function in Pompe disease and was the basis for the approval of alglucosidase alfa.

[0309] [Table 22]

[0310] [Table 23]

[0311] Pre-specified analysis of 6-minute walk distance (6MWD) and percent-predicted forced vital capacity (FVC) in the ERT conversion trial population (n=95): PROPEL-converted patients who had been treated with alglucosidase alfa for at least two years were enrolled in the study. Over two-thirds (67%+) of these patients had been on ERT for more than five years (mean 7.4 years) before enrolling in the PROPEL study.

[0312] A prespecified analysis of 6-minute walk distance in patients who switched from alglucosidase alfa showed that AT-GAA-treated patients (n=65) walked 16.9 meters more than their baseline 52 weeks after switching compared with 0.0 meters for patients randomized to remain on alglucosidase alfa (n=30) (p=0.046) (Table 16).

[0313] A prespecified analysis of percent-predicted FVC in patients who switched from alglucosidase alfa showed that AT-GAA-treated patients stabilized and slightly improved their respiratory function on this important measure, while patients who remained on alglucosidase alfa continued to experience a significant deterioration in respiratory muscle function. AT-GAA patients showed an absolute increase of 0.1% in percent-predicted FVC over 1 year, while alglucosidase alfa patients showed an absolute deterioration of 4.0% (p=0.006) (Table 17).

[0314] [Table 24]

[0315] [Table 25]

[0316] Pre-specified analysis of 6-minute walk distance (6MWD) and percent-predicted forced vital capacity (FVC) in an ERT-naive population (n=28): A prespecified analysis of 6-minute walk distance in patients never previously treated with any ERT showed that after 52 weeks, AT-GAA-treated patients (n=20) walked 33 meters more than their baseline. Alglucosidase alfa-treated patients (n=7) walked 38 meters more than their baseline. The difference between the two groups was not statistically significant (p=0.60) (Table 18).

[0317] A prespecified analysis of patients never previously treated with any ERT showed a similar deterioration in percent-predicted forced vital capacity (FVC) at 52 weeks: -4.1% for AT-GAA-treated patients and -3.6% for alglucosidase alfa-treated patients (Table 19). The difference between the two groups was not statistically significant (p=0.57).

[0318] [Table 26]

[0319] [Table 27]

[0320] Pre-specified analyses of other key secondary and biomarker endpoints in the ERT-switched and ERT-naive overall study populations: Musculoskeletal and other key secondary endpoints: GSGC (Gait, Stairs, Gowers, Chair): GSGC is a widely used outcome measure capturing muscle strength, coordination, and mobility important in Pompe disease. AT-GAA-treated patients demonstrated statistically significant improvement in scores on this important assessment compared with worsening scores in alglucosidase alfa-treated patients in the overall population (p<0.05).

[0321] Lower limb MMT (manual muscle testing), PROMIS physical function: For both of these validated measures of muscle strength and patient-reported outcomes, AT-GAA-treated patients improved numerically more than alglucosidase alfa-treated patients, but the results were not statistically significant.

[0322] PROMIS Fatigue: Fatigue as measured by this scale was slightly better in AT-GAA-treated patients than in alglucosidase alfa-treated patients.

[0323] Biomarkers of disease treatment response: Urinary Hex-4: For both ERT-converted and ERT-naive patients in the combined study population, patients receiving AT-GAA demonstrated substantial improvement in this biomarker, with a mean decrease in Hex-4 of -31.5% after 52 weeks compared with a +11.0% increase (i.e., worsening) in Hex-4 in alglucosidase alfa-treated patients (p=<0.001). Urinary Hex-4 is a commonly used biomarker in Pompe disease and is used as an indirect measure of the extent of skeletal glycogen clearance in Pompe disease patients receiving ERT. Glycogen is the substrate that accumulates in the muscle lysosomes of Pompe disease patients.

[0324] CK (creatine kinase): After 52 weeks, AT-GAA-treated patients showed a similarly substantial improvement in this biomarker, with a mean CK reduction of -22.4% compared with a +15.6% increase (i.e., worsening) in alglucosidase alfa-treated patients (p<0.001). CK is an enzyme that leaks from damaged muscle cells and is elevated in Pompe disease patients.

[0325] AT-GAA demonstrated a safety profile similar to alglucosidase alfa. Two patients (2.4%) receiving AT-GAA discontinued treatment due to adverse events compared with one (2.6%) unrelated to treatment for alglucosidase alfa. Infusion-related reactions (IARs) were reported in 25% of AT-GAA participants and 26% of alglucosidase alfa patients.

[0326] Post-hoc subgroup analysis: Baseline 6MWD and FVC Categories: ERT-Naive Population (n = 27): Three patients had a baseline 6MWD < 300 m and three had a baseline FVC < 55%; CFBL analyses were not performed in these subgroups due to small patient numbers. Baseline 6MWD ≥ 300 m: Both the AT-GAA (n = 18) and alglucosidase alfa / placebo (n = 6) groups showed similar improvements over time (mean [SE] CFBL by week 52: +34.4 [12.1] m and +30.8 [9.6] m, respectively). Baseline FVC ≥ 55% worsened over time in both the sipaglucosidase alfa / miglustat (n = 19) and sipaglucosidase alfa / placebo (n = 5) groups (mean [SE] CFBL to week 52: -3.7 [1.5]% and -3.3 [2.6]%, respectively). As shown in Figures 41A and 41B, in the overall and ERT-experienced populations, outcomes were consistently better with sipaglucosidase alfa / miglustat in patients with baseline 6MWD < 300 m and ≥ 300 m and FVC < 55% and ≥ 55%.

[0327] In the full study population, including ERT-naive and ERT-experienced patients, sipaglucosidase alfa / miglustat demonstrated positive trends or clinically meaningful improvements in exercise and respiratory function compared with approved ERT, independent of baseline 6MWD and %FVC assessments, and in both pre-specified and post-hoc subgroup analyses.

[0328] Sipaglucosidase alfa / miglustat demonstrated a safety profile similar to alglucosidase alfa / placebo (Figure 42).

[0329] About AT-GAA AT-GAA is an investigational two-component therapy consisting of a unique recombinant human acid alpha-glucosidase (rhGAA) enzyme, sipa-glucosidase alfa (ATB200), with an optimized carbohydrate structure, specifically bis-phosphorylated mannose-6-phosphate (bis-M6P) glycan, for enhanced cellular uptake, administered in conjunction with miglustat (AT2221), a stabilizer of sipa-glucosidase alfa. In preclinical studies, AT-GA was associated with increased levels of the mature lysosomal form of GAA and reduced levels of glycogen in muscle, alleviating autophagy defects, and improving muscle strength.

[0330] About Pompe disease Pompe disease is an inherited lysosomal disorder caused by a deficiency of the enzyme acid alpha-glucosidase (GAA). It is believed that reduced or absent GAA levels lead to the accumulation of glycogen in cells, resulting in the clinical symptoms of Pompe disease. The disease can be debilitating and is characterized by severe muscle weakness that worsens over time. Pompe disease ranges from a rapidly fatal infantile form with significant impact on cardiac function to a more slowly progressing, late-onset form that primarily affects skeletal muscle. It is estimated that approximately 5,000 to 10,000 people worldwide suffer from Pompe disease.

[0331] Example 11: Results of an open-label Phase I / II study (ATB200-02) ATB200-02 (NCT02675465) is an open-label, Phase I / II clinical trial designed to determine the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of sipaglucosidase alfa / miglustat in adults with Pompe disease. Sipaglucosidase alfa / miglustat is an investigational, dual-component therapy for late-onset Pompe disease (LOPD) consisting of intravenous sipaglucosidase alfa, a rhGAA, administered in combination with oral miglustat, an enzyme stabilizer.

[0332] Figure 43 shows the study design for the Phase I / II ATB200-02 trial. The trial will be conducted at 16 sites across five countries. The ATB200-02 trial enrolled four cohorts of Pompe disease patients: Cohort 1: Patients with a history of ERT, age 18-65 years, receiving ERT with 20 mg / kg alglucosidase alfa every 2 weeks for 2-6 years (n=11) Cohort 2: Non-ambulatory patients with a history of ERT, aged 18-65 years, receiving ERT with 20 mg / kg alglucosidase alfa every 2 weeks for ≥2 years (n=6) Cohort 3: ERT-naive, age 18–65 (n=6) Cohort 4: Patients with a history of ERT, aged 18-75 years, receiving ERT with alglucosidase alfa at 20 mg / kg every 2 weeks for 7 years or more (n=6)

[0333] Eligible ambulatory patients had a 6-minute walk distance (6MWD) of at least 200 m (Cohorts 1 and 3) or 75 m (Cohort 4) and a standing forced vital capacity (FVC) of 30-80% of normal predicted value.

[0334] 6MWD, %-predicted sitting FVC, manual muscle testing (MMT), and changes from baseline (CFBL) in the biomarkers urinary glucose tetrasaccharide (Hex4) and serum creatine kinase (CK) were assessed periodically. Data are provided herein for ERT-experienced and ERT-naive patients with up to 36 months of follow-up. Figure 44 shows an overview of the reported endpoints and cohorts. Figure 45 shows baseline characteristics and patient disposition. Due to lags in patient enrollment, the number of patients with currently available data will decrease at later time points in this ongoing study.

[0335] Patients with a history of ERT demonstrated sustained mean improvements from baseline in 6MWD up to 48 months. After 12, 24, 36, and 48 months of follow-up, 13 / 16, 9 / 13, 6 / 12, and 6 / 9 patients with a history of ERT had numerical improvements from baseline in 6MWD, respectively (Figure 46A). The mean increases were 33 meters (m) by 12 months, 25 m by 24 months, 9 m by 36 months, and 20 m by 48 months.

[0336] For patients with a history of ERT, change from baseline in FVC (CFBL) was generally stable for up to 48 months of follow-up. After 12, 24, 36, and 48 months of follow-up, FVC improved (>3%) or remained stable (±3%) from baseline in 7 / 16, 8 / 13, 4 / 10, and 4 / 6 patients, respectively (Figure 47A). The mean changes were -1.2% by 12 months, +1.0% by 24 months, -0.3% by 36 months, and +1.0% by 48 months.

[0337] In patients with a history of ERT, the mean change in MMT leg scores improved numerically from baseline, and the improvement was maintained over follow-up up to 48 months (Figure 48A). The mean increases were 3.1 points by 12 months, 2.1 points by 24 months, 2.5 points by 36 months, and 3.5 points by 48 months.

[0338] ERT-naive patients demonstrated sustained mean improvements from baseline in 6MWD up to 48 months. After 12, 24, 36, and 48 months of follow-up, 6 / 6, 6 / 6, 4 / 5, and 4 / 4 ERT-naive patients had numerical improvements from baseline in 6MWD, respectively (Figure 46B). The mean increases were 57 m by 12 months, 54 m by 24 months, 43 m by 36 months, and 52 m by 48 months.

[0339] In ERT-naive patients, the mean CFBL of FVC improved numerically from baseline during follow-up periods up to 48 months. After 12, 24, 36, and 48 months of follow-up, FVC improved (>3%) from baseline or remained stable (±3%) in 4 / 6, 5 / 6, 5 / 5, and 4 / 4 patients, respectively (Figure 47B). As indicated by an asterisk in Figure 47B, one patient in the ERT-naive cohort experienced a significant decline in %-predicted FVC at 21 months, which returned to previous levels at the next visit (month 24). The mean changes were 3.2% by 12 months, 4.7% by 24 months, 6.2% by 36 months, and 8.3% by 48 months.

[0340] In ERT-naive patients, the mean change in MMT leg scores improved numerically from baseline, and the improvement was maintained over follow-up up to 48 months (Figure 48B). The mean increases were 2.8 points by 12 months, 3.0 points by 24 months, 3.3 points by 36 months, and 1.0 point by 48 months.

[0341] During 48 months of follow-up, sipaglucosidase alfa / miglustat was generally associated with a mean decrease from baseline in urinary Hex4, with the decrease being greater in ERT-naive patients. After 12, 24, 36, and 48 months of follow-up, 16 / 16, 11 / 14, 11 / 12, and 6 / 9 ERT-experienced patients and 5 / 6, 5 / 6, 4 / 5, and 4 / 5 ERT-naive patients, respectively, had numerically decreased Hex4 levels from baseline (Figure 49A).

[0342] During 48 months of follow-up, sipaglucosidase alfa / miglustat was associated with either stable plasma CK levels or a mean decrease from baseline, with the decrease being greater in ERT-naive patients. After 12, 24, 36, and 48 months of follow-up, 13 / 15, 14 / 15, 9 / 11, and 8 / 9 ERT-experienced patients and 6 / 6, 6 / 6, 5 / 5, and 4 / 5 ERT-naive patients, respectively, had numerically decreased CK levels from baseline (Figure 49B).

[0343] Figure 50 shows an overview of treatment-emergent adverse events (TEAEs) with an onset date at or after the first dose of study drug in Study ATB200-02. The mean (SD) duration of treatment was 37.2 months (14.48), 19.9 months (4.13), and 36.9 months (12.14) in Cohorts 1 (2–6 years of prior ERT), 4 (7+ years of prior ERT), and 3 (ERT-naive), respectively. The most common TEAEs included falls, nasopharyngitis, arthralgia, headache, and diarrhea; most TEAEs were mild or moderate in severity and did not lead to study withdrawal.

[0344] Results from the ATB200-02 study of sipa glucosidase alfa plus miglustat in ambulatory patients with up to 48 months of follow-up indicate the following: Patients with prior ERT demonstrated sustained mean improvements from baseline in motor function, an improvement compared with the expected deterioration in many patients receiving long-term ERT, which persisted over up to 48 months of follow-up, while respiratory function remained stable and was maintained over the same period. ERT-naive patients demonstrated sustained mean improvements from baseline in motor and respiratory function, which persisted over up to 48 months of follow-up. Mean levels of two biomarkers, Hex4 and CK, either remained stable or declined from baseline over up to 48 months of follow-up, with the declines being most pronounced in the ERT-naive cohort. The safety profile of sipa glucosidase alfa plus miglustat was similar to that reported for sipa glucosidase alfa.

[0345] Figure 55 shows the endpoints reported for Cohort 2 (non-ambulatory patients with prior ERT) and cohort overview. Figure 56 shows baseline characteristics and patient disposition for Cohort 2.

[0346] For these non-ambulatory patients with a history of ERT, change from baseline in FVC (CFBL) improved and / or remained numerically stable from baseline for up to 48 months of follow-up (Figure 57). The mean changes were +2.5% by 12 months, +2.0% by 24 months, -2.0% by 36 months, and -1.0% by 48 months.

[0347] In these non-ambulatory patients with a history of ERT, the mean change in MMT total score (upper body) improved and / or remained numerically stable from baseline for up to 36 months of follow-up (Figure 57). The mean change was +1.3 points by 12 months, +2.0 points by 24 months, and -0.8 points by 36 months.

[0348] During 48 months of follow-up in these non-ambulatory patients with a history of ERT, sipa glucosidase alfa / miglustat was generally associated with mean decreases from baseline in urinary Hex4 and plasma CK (Figure 57). After 12, 24, 36, and 48 months of follow-up, Hex4 levels numerically declined from baseline with mean decreases of -15.6%, -34.1%, -36.5%, and -4.0%, respectively. After 12, 24, 36, and 48 months of follow-up, plasma CK levels numerically declined from baseline with mean decreases of -20.8%, -25.3%, -27%, and -23.7%, respectively.

[0349] Figure 58 shows a summary of treatment-emergent adverse events (TEAEs) with an onset date at or after the first dose of study drug or Cohort 2 (among non-ambulatory patients with prior ERT) in Study ATB200-02. The mean (SD) duration of treatment was 46.3 months (22.86 months). The most common TEAEs included nasopharyngitis and diarrhea (both occurring in 3 patients); most TEAEs were mild or moderate in severity and did not lead to study withdrawal.

[0350] Results from the ATB200-02 study of sipa glucosidase alfa plus miglustat in non-ambulatory patients with a history of ERT, with up to 48 months of follow-up, indicate that: These patients demonstrated sustained mean improvement and / or stabilization from baseline in motor and pulmonary function, an improvement over the expected deterioration in many patients receiving long-term ERT, which persisted over up to 48 months of follow-up. Mean levels of two biomarkers, Hex4 and CK, either remained stable or declined from baseline up to 48 months of follow-up. Sipa glucosidase alfa plus miglustat was generally well tolerated in this patient population.

[0351] Example 12: Long-term effects of ERT in patients with Pompe disease Figure 51 shows a comparison of the long-term effects of sipaglucosidase alfa / miglustat and avalglucosidase alfa on the change from baseline in 6MWD and percentage vs. predicted FVC (sitting) in ERT-experienced subjects. Figure 52 shows a comparison of the long-term effects of sipaglucosidase alfa / miglustat and avalglucosidase alfa on the change from baseline in 6MWD and percentage vs. predicted FVC (sitting) in ERT-naive subjects. This comparison shows that ERT-experienced patients who are switched from alglucosidase alfa to avalglucosidase alfa continue to experience a progressive loss of motor function as assessed by 6MWD, similar to the progression shown above for patients continuing to take alglucosidase alfa long-term, while patients who are switched from alglucosidase alfa to sipaglucosidase alfa / miglustat experience a clear change indicative of a progressive gain in motor function as assessed by 6MWD. A similar trend was observed for FVC, where patients switched to avalglucosidase alfa showed a progressive loss of lung function as measured by FVC, whereas patients switched from avalglucosidase alfa to sipaglucosidase alfa / miglustat showed a stable lung function as measured by FVC.

[0352] Figures 53A-B show 6-minute walk test (6MWT) percentage vs. predicted value during treatment with alglucosidase alfa. Figure 53B shows the data from Figure 53A replotted from year 2 onwards. Figure 54 shows FVC percentage vs. predicted value during treatment with alglucosidase alfa. Data from year 2 onwards indicate that deterioration is expected in patients with a history of ERT who continue to take alglucosidase alfa.

[0353] Example 13: Comparison of Alglucosidase Alfa (Alglu), Avalglucosidase Alfa (Aval), and Cipaglucosidase Alfa + Miglustat (Cipa+mig) In the absence of a head-to-head trial comparing Aval with Cipa+mig, indirect treatment comparisons (ITCs) are a suitable approach to better understand the clinical distinctions between the three treatments for LOPD. ITCs are widely required by Health Technology Assessment Agencies (HTAs) to support comparative health economic evaluations.

[0354] An ITC was performed to provide a relative effect estimate in the intended target population (i.e., the LOPD analysis population, which includes a mix of ERT-naive and ERT-experienced subjects, such as those in the pivotal Phase III trial comparing Cipa+mig to Alglu [PROPEL]).

[0355] A systematic literature review (SLR) was conducted to identify relevant published clinical trials of ERT in LOPD. Outcomes evaluated were change from baseline in 6-minute walk distance (6MWD) (m) and forced vital capacity (FVC; % predicted) at week 52, identified as key LOPD clinical trial endpoints by clinicians, HTA organizations, and payers. Summary results for change from baseline in 6MWD and FVC over time and baseline characteristics (age, sex, race, duration of previous ERT, baseline 6MWD, and baseline FVC) were extracted from included studies.

[0356] Multilevel network meta-regression (ML-NMR) was performed. This is an extension of standard network meta-analysis (NMA) that can be applied to any connected network containing any mixture of individual patient-level data (IPD) and aggregate data, taking into account the effects of study-level covariates. ML-NMR is a method generally accepted by the UK National Institute for Health and Care Excellence (NICE) to support cost-effectiveness analyses. Results from single-arm studies were included in the network by matching them to the appropriate comparison arm of controlled studies. Mean treatment differences were calculated with 95% credible intervals (CrIs) for changes from baseline in 6MWD and FVC at week 52.

[0357] A base-case scenario was determined in which all covariates were set for the target population of the PROPEL trial. To test the effect of previous ERT duration on the relative effect, the value of ERT duration was varied while keeping the covariate values ​​unchanged, as in the base-case scenario. A sensitivity analysis was performed by excluding all corresponding single-arm evidence from the network to assess its impact on the results.

[0358] Both fixed-effect (FE) and random-effect (RE) ML-NMR models were applied, and the deviance information criterion (DIC) was used to assess the model fit and identify the appropriate model (FE or RE model). The models were implemented in a Bayesian framework with the help of the R package multinma.

[0359] Seven clinical trials were identified through the SLR. Their baseline characteristics are shown in Figure 59. These trials included, but were not limited to, three randomized clinical trials (LOTS: Alglu vs. placebo; COMET: Aval vs. Alglu; PROPEL: Cipa+mig vs. Alglu). Each shared 6MWD and FVC as key primary or secondary endpoints (see Figures 60 and 61), but the study populations were different (PROPEL is the only randomized controlled trial [RCT] that included both ERT-naive and ERT-experienced subjects). Efficacy results from the included trials are shown in Figure 60.

[0360] For both endpoints, the network was the same and is shown in Figure 61. This network included evidence from the single-arm trials LOTS OLE, NEO-1 / -EXT, COMET OLE, and ATB200-02 by matching the single-arm results to the appropriate comparator results from the head-to-head trials, as shown in the blue boxes.

[0361] In the base case scenario, covariates were set to the baseline characteristics of the target population (i.e., the PROPEL trial; see Table 20) and time was set to 52 weeks.

[0362] [Table 28]

[0363] Based on DIC, the RE model was selected for 6MWD and the FE model was selected for FVC. For both endpoints (Figures 62 and 63): Cipa+mig showed a statistically significant beneficial effect compared to Alglu and Aval; and Cipa+mig showed a numerically beneficial effect compared to placebo.

[0364] Note that the 95% CrIs for the relative effect estimates versus placebo are generally much wider than those for Alglu or Aval, reflecting the greater uncertainty in these estimates due to the fact that placebo data were available only for ERT-naive subjects and the relatively long duration of prior ERT in the base-case scenario (5.7 years).

[0365] Relative effect estimates for different prior ERT durations (prior ERT duration = 0 years [i.e., naive patients], 2.5 years, 5 years, and 9.2 years) are shown in Figure 64 (6MWD) and Figure 65 (FVC).

[0366] Based on the DIC, the RE model was selected for both 6MWD and FVC. Figures 66 and 67 provide a summary of the relative effect estimates for the base case scenario using sensitivity analysis, along with the 95% CrI, showing: Incorporating corresponding single-arm evidence into the network of primary analyses reduces uncertainty in the relative effect estimates Cipa+mig: statistically favorable vs. Alglu; numerically unfavorable vs. Aval; numerically favorable vs. placebo (6MWT and FVC) Aval: Numerically advantageous (6MWT and FVC) compared with Alglu and placebo Alglu: Numerically advantageous compared to placebo

[0367] In conclusion, the ML-NMR comparisons presented here demonstrated that Cipa+mig was statistically significantly advantageous compared with Alglu and Aval for 6MWD and FVC in the base-case scenario of the primary analysis. Cipa+mig was also statistically significantly advantageous compared with Alglu and Aval for 6MWD and FVC across different ERT durations, with one exception: for FVC, Cipa+mig was only numerically advantageous compared with Aval in the ERT-naive setting.

[0368] Sensitivity analyses (incorporating only RCT data) demonstrate that incorporating corresponding single-arm evidence into the network of the primary analysis reduces uncertainty in the relative effect estimates. Overall, these results indicate that Cipa+mig may have a distinct clinical profile compared with other ERTs, particularly for individuals with some level of prior ERT treatment. Further analyses are expected to validate and refine this finding as additional long-term data are published.

[0369] Example 14: Long-term comparison of Cipa glucosidase alfa plus miglustat (Cipa+mig) with alglucosidase alfa (Alglu) The Phase III double-blind PROPEL trial (NCT03729362; ATB200-03), described in Examples 9 and 10 above, compared the investigational dual therapy cipaglucosidase alfa / miglustat (cipa / mig) with alglucosidase alfa / placebo (alglu) over 52 weeks in adults with late-onset Pompe disease (LOPD). The ongoing open-label extension (OLE) of PROPEL (NCT04138277; ATB200-07) will determine the long-term safety and efficacy of cipa / mig. Outcomes include 6-minute walk distance (6MWD), forced vital capacity (FVC), creatine kinase (CK) and hexose tetrasaccharide (Hex4) levels, and safety. Data are reported as change from PROPEL baseline to OLE week 52 (104 weeks after PROPEL baseline). In the OLE (N=119; 91 enzyme replacement therapy [ERT]-experienced patients and 28 ERT-naive patients), 82 / 85 (96.5%) patients previously treated with CIPA / MIG continued CIPA / MIG, and 37 / 38 (97.4%) switched from alglu to CIPA / MIG; 90.8% of patients remained in the OLE study through week 52. The mean change in %-predicted 6MWD was +3.1 (8.07 standard deviation) for CIPA / MIG-CIPA / MIG and -0.5 (7.76) for alglu-CIPA / MIG in ERT-experienced patients and +8.6 (8.57) for CIPA / MIG-CIPA / MIG and +8.9 (11.65) for alglu-CIPA / MIG in ERT-naive patients. The mean change in %-predicted FVC was -0.6 (7.50) for cipa / mig-cipa / mig and -3.8 (6.23) for alglu-cipa / mig in ERT-experienced patients and -4.8 (6.48) and -3.1 (6.66) for ERT-naive patients. The mean decrease in CK (U / L) in ERT-experienced and ERT-naive patients was -132.1 (215.74) and -216.9 (243.66) for cipa / mig-cipa / mig and -161.0 (269.52) and -218.6 (316.47) for alglu-cipa / mig, respectively.The mean decreases in Hex4 (mmol / mol) in ERT-experienced and ERT-naive patients were -1.9 (3.22) and -2.9 (2.45) for cipa / mig-cipa / mig and -2.6 (3.75) and -2.9 (2.22) for alglu-cipa / mig, respectively. During PROPEL through Week 52 of the OLE, 84 (98.8%) cipa / mig-cipa / mig and 36 (97.3%) alglu-cipa / mig patients experienced treatment-emergent adverse events. Three patients discontinued the OLE due to infusion-related reactions (urticaria, urticaria and hypotension, and anaphylaxis, respectively). No new safety signals were identified. Data demonstrate that treatment with CIPA / MIG for up to 104 weeks was well tolerated with durable efficacy, supporting the long-term benefit of this treatment for patients with LOPD.

[0370] Example 15: ATB200-04 Clinical Trial: Safety, Pharmacokinetics, Efficacy, Pharmacodynamics, and Immunogenicity in Pediatric LOPD Subjects The ATB200-04 trial aims to determine the safety and tolerability of coadministration of sipa glucosidase alfa / miglustat. To characterize the pharmacokinetics (PK) of sipa glucosidase alfa / miglustat in Cohort 1 and Cohort 2 To compare plasma sipa glucosidase alfa (total human acid alpha-glucosidase [GAA] protein with signature peptide T09) / plasma miglustat exposure in each pediatric age group with adult exposure, and to confirm or adjust starting dose regimens that are initially simulated from adult exposure. To determine the effect of sipaglucosidase alfa / miglustat on efficacy To determine the effect of sipaglucosidase alfa / miglustat on pharmacodynamics (PD) To determine the effect of sipa glucosidase alfa exposure on efficacy and PD endpoints (exposure-response relationship) To determine the effect of anti-rhGAA antibodies and immunogenicity on rhGAA exposure. We aim to achieve this.

[0371] The study will determine the safety, PK, efficacy, PD, and immunogenicity of sipaglucosidase alfa / miglustat treatment in enzyme replacement therapy (ERT)-experienced and ERT-naive pediatric subjects with late-onset Pompe disease, aged 0 to <18 years. The study will enroll subjects with Pompe disease other than classical IOPD.

[0372] Subjects will be enrolled in one of two cohorts: Cohort 1 will be treated with sipaglucosidase alfa / miglustat and will include at least 10 subjects (ERT-experienced and ERT-naive) aged 12 to less than 18 years at the time of enrollment. Cohort 2 will be treated with sipaglucosidase alfa / miglustat and will include at least 12 subjects (ERT-experienced and ERT-naive) aged 0-12 years at the time of enrollment, with at least 4 subjects aged 6-12 years and at least 4 subjects aged 0-6 years.

[0373] Subjects will receive an oral dose of miglustat every other week, followed approximately one hour later by an intravenous (IV) infusion of sipaglucosidase alfa. Subjects will undergo PK assessments on Day 1, Week 26, and Week 52. If a subject discontinues the study before Week 52, PK assessments will be obtained at the early termination visit.

[0374] Plasma total GAA protein and plasma miglustat exposures from signature peptide T09 will be compared to adult exposures to confirm or adjust starting dose regimens for each pediatric age / weight group based on simulations from adult exposures adjusted for pediatric weight, plasma clearance, and volume of distribution. Plasma total GAA protein and plasma miglustat exposures from signature peptide T09 from adolescents in Cohort 1 will also inform dose setting for younger pediatric age groups in Cohort 2.

[0375] Infusion visits will be scheduled every 2 weeks throughout the study; initial safety monitoring assessments (e.g., laboratory tests) will be conducted at these visits during the first 6 weeks of the study. Study visits, including efficacy, additional safety, and other assessments, will be scheduled approximately every 3 months and may occur over 4 days, except that all study assessments and procedures (except PK sampling) will be performed before administration of study drug.

[0376] Subjects were scheduled for PK assessments of plasma total GAA protein and miglustat at Day 1, Week 26, and Week 52. Blood samples were collected to determine plasma total GAA protein levels and miglustat concentrations for population PK analysis. Sparse PK samples were collected for pediatric subjects (ages 2 to <18 years) at Day 1, Week 26, and Week 52 according to the following schedule: Day 1: Approximately 5 minutes before the start of the sipa glucosidase alfa-ATGA infusion (time 0), and 2, 4, 6, and 8 hours after the start of the sipa glucosidase alfa-ATGA infusion; determine plasma miglustat concentrations from the same blood samples as the sipa glucosidase alfa-ATGA determinations; however, the nominal time of miglustat will be shifted 1 hour before each sipa glucosidase alfa-ATGA infusion to account for the 1-hour difference in miglustat dose administration. Week 26: Approximately 5 minutes before the start of the sipa glucosidase alfa-atga infusion (time 0), and 4, 8, and 24 hours after the start of the sipa glucosidase alfa-atga infusion; if a 24-hour sample is not collected at week 26, it will be collected at week 52 or at any visit during the study. Week 52: Approximately 5 minutes before the start of the sipa glucosidase alfa-atga infusion (time 0), and 4 and 8 hours after the start of the sipa glucosidase alfa-atga infusion.

[0377] Safety assessments included monitoring for adverse events (AEs), clinical laboratory tests (chemistry, hematology, and urinalysis), physical examination, vital signs, 12-lead electrocardiogram (ECG), and detection of sipa glucosidase alfa antibodies.

[0378] The efficacy, PD, and other assessments included in this study are outlined below.

[0379] [Table 29]

[0380] [Table 30]

[0381] Additionally, in Cohort 1, additional efficacy data may be captured (optionally) using wearable devices that monitor physical activity (e.g., total activity and intensity) and smartphone applications that report quality of life (e.g., mood, fatigue, energy, and appetite).

[0382] The study consists of a 30-day screening and 12-month treatment period. Upon completion of the study, subjects may have the opportunity to enroll in a separate long-term extension (LTE) period for continued evaluation of safety and efficacy. The LTE period will last until June 2026 or until the sponsor's termination of the study. For subjects not enrolled in the LTE period, follow-up will consist of a 30-day safety follow-up period and an immunogenicity follow-up period lasting up to 52 weeks after the last dose of study drug for subjects requiring further monitoring for anti-rhGAA antibodies.

[0383] Diagnosis and main inclusion criteria: Subjects in each cohort must meet all inclusion criteria and none of the exclusion criteria to be eligible to participate in the study.

[0384] Inclusion Criteria: About Cohort 1 1. Male and female subjects diagnosed with LOPD aged 12 to 18 years at screening (ERT-naive [never administered rhGAA at any dose] or ERT-experienced [having received rhGAA at least 6 months immediately prior to enrollment, and if ERT dosage has been modified, the modified dosage must have been continued for at least 3 months prior to enrollment]). 2. Subject weighs less than 115 kg. 3. The subject's parent or legally authorized representative is willing and able to provide written informed consent and authorization for the use and disclosure of personal or research-related health information, and the subject will provide consent consistent with institutional and local regulations, if applicable. 4. Subject must have a documented diagnosis of LOPD with at least one of the following: A deficiency of the GAA enzyme b. Genotyping of the gene encoding human acid α-glucosidase (GAA) 5. If of reproductive potential and sexually active, female and male subjects agree to use highly effective methods of contraception throughout the study and until 90 days after their final dose of sipaglucosidase alfa / miglustat. 6. Subjects have a sitting forced vital capacity (FVC) ≥ 30% of predicted value for healthy young adults (Global Lung Function Initiative) at screening. 7. Subject performs one valid 6-minute walk test (6MWT) (≥75 meters) as determined by a clinical assessor at screening, OR About Cohort 2 1. Male and female subjects diagnosed with LOPD aged 0 months to less than 12 years at screening (ERT-naive [never administered rhGAA] or ERT-experienced [having administered rhGAA for at least 6 months immediately prior to enrollment, and if the ERT dose has been modified, the modified dose must have been continued for at least 3 months prior to enrollment]). 2. The subject's parent or legally authorized representative is willing and able to provide written informed consent and authorization for the use and disclosure of personal or research-related health information. 3. Subject must have a documented diagnosis of LOPD with one of the following: A deficiency of the GAA enzyme b.GAA genotyping 4. If of reproductive potential and sexually active, female and male subjects agree to use highly effective methods of contraception throughout the study and until 90 days after their final dose of sipaglucosidase alfa / miglustat. 5. Subjects aged 5 to 12 years who perform a single valid 6MWT (≥40 meters) as determined by a clinical assessor at the time of screening, or Exclusion criteria: About Cohort 1 1. Subject has received any investigational / experimental drug, oral anabolic steroid or derivative, biologic, or device within 30 days or within 5 half-lives of therapy or treatment, whichever is longer, prior to screening 2. Subject has been treated with prohibited medications within 30 days of screening 3. Subject has received any gene therapy at any time 4. The subject has any intercurrent illness or condition at screening or baseline that may prevent the subject from fulfilling the requirements of the protocol or that suggests to the investigator and / or medical monitor that the potential subject may be at unacceptable risk by participating in the study. 5. Subject has hypersensitivity to sipa glucosidase alfa, approved rhGAA, or any of the excipients in miglustat 6. Female subjects are pregnant or lactating at the time of screening 7. Subject requires the use of mechanical ventilatory support for more than 6 hours per day while awake 8. Subject has evidence of moderate to severe hypertrophic cardiomyopathy consistent with classic IOPD 9. In the opinion of the investigator, the parent or legally authorized representative is unlikely or unable to comply with the requirements of the study. 10. Subject has any past history of a disease or condition known to affect motor function, including but not limited to Guillain-Barré syndrome, cerebral palsy, etc. About Cohort 2 1. Subject has received any investigational / experimental drug, oral anabolic steroid or derivative, biologic, or device within 30 days or within 5 half-lives of therapy or treatment, whichever is longer, prior to screening 2. Subject has been treated with prohibited medications within 30 days of screening 3. Subject has received any gene therapy at any time 4. The subject has any intercurrent illness or condition at screening or baseline that may prevent the subject from fulfilling the requirements of the protocol or that suggests to the investigator and / or medical monitor that the potential subject may be at unacceptable risk by participating in the study. 5. Subject has a hypersensitivity to sipa glucosidase alfa, approved rhGAA, or any of the excipients in miglustat. 6. Female subjects are pregnant or lactating at the time of screening 7. Subject requires the use of mechanical ventilatory support for more than 6 hours per day while awake 8. Subject has moderate to severe hypertrophic cardiomyopathy consistent with classic IOPD 9. In the opinion of the investigator, the parent or legally authorized representative is unlikely or unable to comply with the requirements of the study. 10. Subject has any past history of a disease or condition known to affect motor function, including but not limited to Guillain-Barré syndrome, cerebral palsy, etc. 11. Subjects who have been diagnosed with Pompe disease through newborn screening and are asymptomatic (i.e., have no signs or symptoms of Pompe disease)

[0385] In this study, systemic exposure from the start of the pediatric dosing regimen will be determined relative to adult exposure using the concept of extrapolation (i.e., if a given pediatric dosing regimen results in a systemic exposure equivalent to adult exposure, a similar clinical response can be assumed). Dose adjustments will be made if the starting pediatric dosing regimen results in an underestimate or overestimate of adult exposure. These determinations will be made for the first 4-6 subjects within each age group, followed by when all subjects within each age group have completed each PK sampling day: after Day 1, after Week 26, and after all subjects have completed Week 52.

[0386] To mitigate underexposure in pediatric subjects before sufficient subjects are available for analysis, PK samples will be immediately shipped to the respective bioanalytical laboratories. If a pediatric subject demonstrates definitive clinical decline, PK samples will be analyzed to provide data for exposure equivalent determination analysis. If a commensurately lower level of total GAA protein exposure (maximum observed concentration [Cmax] and area under the plasma drug concentration-time curve [AUC]) compared to adult exposure is confirmed in combination with definitive clinical decline, the sipa glucosidase alfa dose will be increased.

[0387] Dose Modifications: Dose adjustment within limits established by juvenile toxicity studies is based on modeling and simulation-based adult exposure equivalent determinations (predicted C max Dose adjustments of sipa glucosidase alfa will be made in 5 mg / kg increments, followed by further PK sampling to determine adult exposure equivalents.

[0388] If a dose adjustment is required, sparse PK sampling (pre-dose, 2, 4, 6, 8, and 24 hours after initiation of the sipa glucosidase alfa infusion) will be performed within 4 to 8 weeks of administering the adjusted dose to assess pediatric exposure relative to the target adult exposure. Additional PK samples (pre-dose, 2, 4, 6, 8, and 24 hours after initiation of the sipa glucosidase alfa infusion) may be collected as needed to ensure achievement of the target exposure. Infants (<2 years of age) will have a special sampling scheme: either 1 and 6 hours or 4 and 7 hours after initiation of the sipa glucosidase alfa infusion. Infants will be assigned to one of two sampling schedules and will remain on that schedule throughout their participation in the study, including if dose adjustments are required during PK collection.

[0389] Additionally, because pediatric subjects are expected to gain weight as they grow, individual miglustat dose adjustments may be made based on changes in weight category. If there are increases in the dose of sipa glucosidase alfa, administration will be escalated in 5 mg / kg increments, with subsequent PK sampling to assess exposure levels compared to adults and ensure those exposures are within the safety limits established by the juvenile toxicity study. Miglustat dose adjustments will be made solely based on changes in age and weight.

[0390] Dosage and Administration: About Cohort 1 Miglustat will be administered 1 hour before the start of the intravenous infusion of sipa glucosidase alfa. Any subject who has difficulty swallowing capsules may receive miglustat as an oral solution by dissolving the powder from the capsule in an appropriate volume of water.

[0391] Subjects are required to fast for at least 2 hours before and 2 hours after oral administration of miglustat.

[0392] About Cohort 2 For Cohort 2, the initial doses of sipaglucosidase alfa and miglustat will be derived from population PK modeling and simulation analyses using all available adult and pediatric study data.

[0393] Sipaglucosidase alfa / miglustat will be co-administered once every other week. An intravenous infusion of sipaglucosidase alfa will begin approximately one hour after miglustat administration, and the infusion will be administered over approximately four hours. Subjects are required to fast for at least two hours before and two hours after oral administration of miglustat. Infants (under 2 years of age) may be given Pedialyte® or a similar dextrose-electrolyte oral solution between fasting intervals.

[0394] During the study, the dose of sipa glucosidase alfa will remain the same or may be increased. Because it is expected that subjects will gain weight as they grow, adjustments to the miglustat dose will be based on changes in weight category and confirmation of the new weight category at two consecutive infusion visits.

[0395] For both cohorts, initial dosing is specified in Table 6 above.

[0396] Judgment criteria: Population PK parameters included plasma total GAA protein concentration and clearance (C estimated from miglustat concentration). L ) and volume of distribution (V). Other PK parameters will be derived for Cohorts 1 and 2 as follows: AUC: Area under the plasma drug concentration-time curve C max : The highest observed concentration obtained directly from the concentration profile

[0397] Safety endpoints for both cohorts were as follows: Incidence of treatment-emergent AEs, serious AEs, infusion-related reactions, and AEs leading to discontinuation of study drug Change in anti-rhGAA antibodies (total, neutralizing, and cross-reactive antibodies) from baseline over time Changes in laboratory test results from baseline over time Changes in vital signs from baseline over time Change in 12-lead ECG values ​​from baseline over time

[0398] Efficacy endpoints were as follows: About Cohort 1 Changes from baseline to week 52 in the following variables related to walking function: -6MWD (6MWT distance walked, in meters) -6MWD (% of predicted value) Changes from baseline to week 52 in the following variables related to motor function: - Total score of the Gait, Stairs, Gowers, and Chair (GSGC) tests - Time taken to complete the 10-meter walk of the GSGC test (i.e., gait assessment) - Time taken to complete the GSGC test of climbing four stairs -Time taken to complete the Gowers movement of the GSGC test - Time taken to stand up from a chair as part of the GSGC test - Timed Up and Go - how long it takes to complete the test - Gross Motor Function Scale-88 (GMFM-88) total score and subdomain scores Time to start using assistive devices Change from baseline to week 52 in the following strength-related variables: - Manual Muscle Testing (MMT) scores for upper and lower limbs -MMT total score Change from baseline to week 52 in the following lung function measures: -FVC locus (% predicted) - Sitting slow expiratory vital capacity (SVC) (% predicted) -Maximum inspiratory pressure (MIP) (cmH2O) -MIP (% of predicted value) -Maximum expiratory pressure (MEP) (cmH2O) -MEP (% of predicted value) - Mechanical ventilation use (hours per day) Ventilator-independent (non-ventilator-dependent subjects only) Use of continuous positive airway pressure (CPAP) / bilevel positive airway pressure (BiPAP) Change from baseline to week 52 in the following variables from patient-reported outcome (PRO) measures: - NeuroQoL (Quality of Life in Neuropathy) Pediatric Lower Limb Function (Motor Function) Score Actual measurement of the subject's functional status (improving, stable, or declining) as measured by Physician's Global Impression of Change (PGIC) Actual measurement of the subject's functional status (improving, stable, or declining) as measured by the Subject's Global Impression of Change (SGIC; as reported by the subject or the subject's caregiver) Other reviews - Activity level (total and intensity) as measured by a wearable device (optional) -Quality of life (e.g., mood, fatigue, energy, appetite) captured by a smartphone application (optional) -Immunogenicity assessment (total antibodies, cross-reactive antibodies, and neutralizing antibodies, anti-rhGAA antibodies) About Cohort 2 Changes in the following variables related to walking function from baseline to week 52 (for subjects aged 5 years and older) -6MWD (6MWT distance walked, in meters) -6MWD (% of predicted value) Changes from baseline to week 52 in the following variables related to motor function: Alberta Infant Motor Development Scale total score (for children aged 18 months and younger) -GMFM-88 total score and subdomain scores (for subjects aged 5 months or older) Time to start using assistive devices Changes in the following muscle strength-related variables from baseline to week 52 (for subjects aged 4 years and older): -MMT scores for lower and upper limbs and neck -MMT total score Change from baseline to week 52 in the following lung function measures (for subjects aged 5 years and older): -FVC locus (% predicted) -SVC locus (% predicted) -MIP (cmH2O) -MIP (% of predicted value) -MEP (cmH2O) -MEP (% of predicted value) - Mechanical ventilation use (hours per day) Ventilator-independent (non-ventilator-dependent subjects only) CPAP / BiPAP use Change from baseline to week 52 in the following variables from PRO scales: -NeuroQOL pediatric lower limb function (motor function) score Actual measurements of the subject's functional status (improvement, stability, or decline) regarding the effect of the study drug on the following life domains, as measured by the SGIC: The actual functional status of the subject (improvement, stability, or decline) as measured by PGIC Other reviews: - Changes in growth charts (weight and height / length percentiles) from baseline to week 52 -Immunogenicity assessment (total antibodies, cross-reactive antibodies, and neutralizing antibodies, anti-rhGAA antibodies) Pharmacodynamic endpoints for both cohorts were as follows: Change from baseline to week 52 in the following PD variables: Serum creatine kinase level -Urinary hexose tetrasaccharide levels

[0399] Data Validity. Since the initial site visit in 2019, eight subjects in Cohort 1 and five subjects in Cohort 2 have been enrolled and treated. After 3 years and 8 months, eight adolescent subjects were evaluable for PK exposure equivalents (seven subjects from Cohort 1 and one subject from Cohort 2 who turned 12 years old shortly after enrollment in the study). One adolescent subject achieved an exposure-equivalent dose level after completing participation in the 12-month treatment period of the study and is excluded from the analysis (the protocol did not include an optional long-term extension at this time). Pharmacokinetic analysis of these eight evaluable subjects supports the confirmation of adult exposure equivalents at the recommended adolescent dosing setting.

[0400] A sample size of 8 adolescent subjects is considered sufficient to determine exposure equivalents based on achieving exposures between the 5th and 95th percentiles for adults. In addition, a sample size of 7 to 10 subjects per age group will achieve least squares geometric mean (LSM) AUC and C values ​​between 80% and 125% to achieve bioequivalence with adult exposures. max It is estimated to provide ratios and 90% confidence intervals.

[0401] Methods for Determining Exposure Equivalents. Pediatric PK data from both the clinical trial and the expanded access program were used to determine the adult PK data exposure equivalents. Data from the expanded access program study provided PK data from eight subjects prior to the initiation of ATB200-04, providing early results to support dose selection. Pharmacokinetic data for these subjects are provided in Table 22. The historical baseline population PK models from sipa glucosidase alfa-atga and miglustat were re-run with the latest dataset. To account for longitudinal changes in body size in pediatric patients, body weight over time was used to estimate the effect of body size on PK parameters in the population PK model. The effect of baseline age on clearance was also determined in this PK model. Individual AUCs for sipa glucosidase alfa and miglustat 0-24 and C max was predicted using the PK empirical Bayes estimator (EBE). Exposure prediction used children's weight over time and baseline age. PK exposure (AUC 0-24 and C max In addition, some subjects had unscheduled PK visits. The current overview provides results for adolescent (ages 12 to <18 years) subjects from Study ATB200-04.

[0402] Adolescent Subject Results. The observed total GAA protein with signature peptide T09 concentration-time profiles for adolescents after administration of sipa glucosidase alfa and miglustat, and for adults after administration of sipa glucosidase alfa alone and in combination with miglustat, are presented in Figure 68. Adult subjects received a 4-hour infusion of 20 mg / kg sipa glucosidase alfa and either 260 mg or, for subjects weighing less than 50 kg, 195 mg miglustat. Adolescent subjects received a 4-hour infusion of either 20 mg / kg (for body weight ≥ 50 kg) or 25 mg / kg (for body weight ≥ 40 kg but < 50 kg) sipa glucosidase alfa and either 260 mg (for body weight ≥ 50 kg) or 195 mg (for body weight ≥ 40 kg but < 50 kg) miglustat as a starting dose. To date, no subjects have been enrolled in the 30 kg to less than 40 kg weight range; however, if any subjects in this weight range were to enroll, the 30 mg / kg dose would be available. The dose may have been adjusted at a later visit to determine equivalent exposure. However, it should be noted that in the early stages of this study, all subjects were started at 20 mg / kg to assess safety. The starting dose may have been modified based on the subject's age and weight once safety has been clearly established. The concentration-time curves for each treatment group, presented as Loessian smoothed curves, indicate that adolescents generally had profiles similar to those of adults receiving sipa glucosidase alfa and miglustat.

[0403] Predicted sipaglucosidase alfa exposure (AUC) for adults (N=100) and adolescents (N=8) 0-24h and C max ) is presented as a boxplot in Figure 69. AUC for adolescents 0-24 and C max Both are well within the 5th to 95th percentile range for adults.

[0404] A summary of the PK parameter analysis of sipa glucosidase alfa for adolescents and adults is presented in Table 21. AUC 0-24 and Cmax The median and mean values ​​are similar to those of adults. The 5th and 95th percentiles of adolescents fall well within the same ranges as adults. An additional analysis of variance (ANOVA) was performed. AUC 0-24 and C max The least squares geometric mean (LSM) ratios and 90% confidence intervals (CI) for were within the acceptable range of 0.80 to 1.25, confirming that adolescent exposures were comparable to those of adults.

[0405] [Table 31]

[0406] A list of individualized predictions of exposure-equivalent sipa glucosidase alfa PK parameters is provided in Table 22. This table includes the most recent treatment and weight, and the exposure-equivalent dose. Subjects were assigned a starting dose of 20 mg / kg sipa glucosidase alfa. If exposure was not equivalent, the dose was increased to 25 mg / kg. Based on the adult population PK model, a weight effect was detected for sipa glucosidase alfa, which is confirmed by the adolescent population, where 6 of 8 adolescents who had their dose increased to 25 mg / kg tended to have lower body weights compared to the 2 subjects who did not receive an increased dose.

[0407] [Table 32]

[0408] Figure 70 presents the miglustat concentration-time profiles observed for adolescents and adults after administration of sipa glucosidase alfa-atga and miglustat. Adult subjects received either 260 mg (for body weight ≥ 50 kg) or 195 mg (for body weight ≥ 40 kg < 50 kg) of miglustat. A small cohort of adults in ATB200-02 received 130 mg of miglustat and 20 mg / kg of sipa glucosidase alfa. Similarly, adolescent subjects received 260 mg (for body weight ≥ 50 kg) or 195 mg (for body weight ≥ 30 kg < 50 kg) of miglustat. The miglustat concentration-time curves for each treatment group, presented as Loessian smoothed curves, indicate that the adolescent profiles were more similar to adults receiving the 260 mg or 195 mg dose than the 130 mg dose.

[0409] Predicted miglustat exposure (AUC 0-24 and C max ) are presented as boxplots in Figure 71. AUC for most adolescents 0-24 and C max are in the 5th to 95th percentile range for adults, well within the overall range of individual adult exposures.

[0410] A summary of the miglustat PK parameter analysis in adolescents and adults is presented in Table 23. Adolescent AUC 0-24 and C max The median and mean values ​​are similar to those of adults. The 5th and 95th percentiles for adolescents fall roughly within the same ranges as adults. An additional analysis of variance (ANOVA) was performed. AUC 0-24 and C max Least-squares geometric mean (LSM) ratios and 90% confidence intervals (CI) were generally within the acceptable range of 0.80 to 1.25, confirming that adolescent exposures were comparable to those of adults.

[0411] [Table 33]

[0412] A list of individual predicted exposure-equivalent miglustat PK parameters is provided in Table 24. This table includes treatment and body weight at the time of the most recent exposure-equivalent dose. This dosing table was provided by modeling and simulation of pooled adult data. As subjects increased in age and weight during the study, the miglustat dose was adjusted accordingly. Based on the adult population PK model, age and body weight effects were detected for miglustat.

[0413] [Table 34]

[0414] Recommended dosage. Based on adult exposure equivalents, Table 25 provides recommended dosing for adolescent and adult patients.

[0415] [Table 35]

[0416] The whole population Ten of 13 (76.9%) subjects reported 70 treatment-emergent adverse events (TEAEs). The most commonly reported TEAEs were nasopharyngitis and vomiting (3 subjects each). Most TEAEs were mild to moderate in severity.

[0417] A total of 4 of 13 (30.8%) subjects reported 13 TEAEs related to the study drug: 2 subjects had diarrhea, and 1 subject each had tachycardia, anaphylactic reaction, urticaria, and hypotension.

[0418] A total of five serious adverse events (SAEs) were reported in two subjects (gunshot wound, jaw fracture, and hemothorax in one subject in Cohort 1, and two anaphylactic reaction events in one subject in Cohort 2).

[0419] One SAE case (anaphylactic reaction) involved a 7-year-old Japanese female LOPD patient with a history of ERT and no prior history of IAR. She was administered IV sipa glucosidase alfa (25 mg / kg) and oral miglustat (80 mg) once weekly (QOW) for three doses without any incidents. She then began receiving sipa glucosidase alfa (30 mg / kg) and oral miglustat (130 mg) QOW and experienced two anaphylactic episodes, one during the fourth sipa glucosidase alfa infusion and one during the fifth sipa glucosidase alfa infusion. The first episode was severe and characterized by vomiting, dyspnea, low oxygen saturation levels (88%), and pallor; it was treated with IV epinephrine, IV hydrocortisone, and oxygen. Symptoms improved immediately, but the subject was admitted for safety follow-up and achieved complete resolution of the event (and discharged from the hospital) the following day. A second episode occurred two weeks later and was mild and characterized by dyspnea, low oxygen saturation levels (86%), pallor, and respiratory distress. The infusion was discontinued, and symptoms improved quickly after treatment with oxygen. Subsequent treatment included IV methylprednisolone and Ringer's acetate solution. The event resolved later the same day. The subject resumed treatment with miglustat (130 mg) and a lower dose of sipa glucosidase alfa (20 mg / kg) administered at a slower rate. Premedication with levocetirizine and corticosteroids was administered before the fifth infusion to prevent IAR. At the time of last report, the subject had received 25 infusions since resuming treatment and was able to resume the 25 mg / kg dose at the usual infusion rate without further IARs. The investigator rated the first anaphylactic episode as serious (hospitalization) and the second episode as serious (major medical event). Both anaphylactic events were assessed by the investigator as definitively related to sipa-glucosidase alfa and unrelated to miglustat.

[0420] Two subjects reported a total of six IARs, including one tachycardia, two anaphylactic reactions, two urticaria, and one hypotension. One subject experienced two episodes of severe anaphylactic reaction, and the other subject experienced two non-severe IARs of urticaria and one non-severe IAR each of tachycardia and hypotension.

[0421] No subjects discontinued due to adverse effects, and there were no fatal adverse effects.

[0422] Youth group. In Cohort 1, a total of 48 TEAEs were reported in 8 of 8 (100%) adolescent subjects. The most commonly reported TEAEs were diarrhea, vomiting, nasopharyngitis, and a positive SARS-CoV-2 test (2 subjects each). Of the overall population, 8 were adolescent subjects included in the PK exposure equivalent determination simulation. A total of 41 TEAEs were reported in 7 of 8 (87.5%) of these adolescent subjects. The most commonly reported TEAEs were diarrhea and a positive SARS-CoV-2 test (2 subjects each).

[0423] In both Cohort 1 and the PK exposure equivalent population, most TEAEs were mild to moderate in severity.

[0424] Two (25.0%) adolescent subjects in Cohort 1 reported seven TEAEs related to study medication (diarrhea). These subjects were also included in the PK exposure equivalent determination population. Only one (12.5%) of these subjects with diarrhea was related to both study medications.

[0425] A total of three SAEs (gunshot wound, jaw fracture, and hemothorax) were reported in one (12.5%) adolescent subject in Cohort 1. This subject was also included in the PK exposure equivalent determination population.

[0426] No IARs were reported in any of the adolescent subjects in Cohort 1 or the PK exposure equivalent population. No subjects discontinued due to adverse effects, and there were no fatal adverse effects in these adolescent subjects.

[0427] Safety Overview The adverse effects reported are consistent with the known safety profile of sipaglucosidase alfa / miglustat and are consistent with the established benefit-risk profile of sipaglucosidase alfa / miglustat in adults with LOPD.

[0428] conclusion These analyses confirmed adolescent exposure equivalent to adult doses for sipaglucosidase alfa and miglustat, thus supporting full extrapolation. Additionally, safety data confirmed that the safety profile in adolescents was comparable to that observed in the adult LOPD population.

[0429] Example 16: Treatment of a male Taiwanese pediatric LOPD patient A male patient was born with hypotonia and muscle weakness. His developmental milestones were delayed, whereas his twin sister had normal muscle tone and developmental milestones. The patient was able to sit at age 1 year and walk at age 1 year and 9 months. At age 2 years and 10 months, low GAA activity was detected in the blood, confirming the diagnosis of Pompe disease.

[0430] Diagnostic Laboratory Test Data 1. GAA enzyme activity: 4.32 μmol / L / hr in a dried blood spot at 2 years and 10 months of age (normal: 19.92 ± 8.49). 2. GAA gene mutation: c.1781G>A(p.R594H) / c.1935C>A(p.D645E)

[0431] Follow-up conditions to keep in mind Previous ERT treatment At the age of 6 years and 6 months, the patient entered elementary school. Due to his weakened condition, he began enzyme replacement therapy (ERT) with a biweekly regimen of 20 mg / kg Myozyme. During ERT, the patient experienced multiple fevers, prompting the initiation of premedication therapy. After premedication therapy was initiated before ERT infusion, the patient no longer experienced fevers. During ERT with premedication, the patient gradually became able to stand, eat without dysphagia, and change positions slowly. The patient was able to walk 800 meters independently with two or three breaks, although he frequently experienced knee locking when walking briskly. The patient participated in biweekly rehabilitation sessions and was temporarily able to stand on one leg. The patient's speech was hypernasal, and there was evidence of hypoventilation, but the patient's family refused continuous positive airway pressure (CPAP).

[0432] At the age of 7 years and 6 months, the patient continued his premedication and ERT treatment with 20 mg / kg Myozyme every other week. At this age, the patient began taking albuterol, and thereafter, he experienced no further complications and felt much better. With this treatment regimen, the patient continued to experience temporary improvements in his ability to stand on one leg, fewer falls, and improved muscle strength as evidenced by his ability to ride a tricycle uphill, as well as improved endurance, requiring only one break to walk 500 meters. However, despite this improvement, the patient still suffered from knee locking when walking briskly and hyperlordosis while standing. The patient was able to change positions, but did so very slowly. His speech remained hypernasal, but he did not snore, and he did not experience any difficulty swallowing or urinating.

[0433] At age 8 years and 5 months, the patient was switched to weekly Myzome treatment at 18.5 mg / kg. At this age, the patient discontinued albuterol treatment for 2 months, after which h...

Claims

1. 1. A method of treating Pompe disease in a subject under the age of 18, comprising administering to said subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules simultaneously or sequentially with a pharmacological chaperone, The method, wherein the dose of the recombinant human acid alpha-glucosidase (rhGAA) molecule population ranges from about 15 mg / kg to about 40 mg / kg, and the dose of the pharmacological chaperone ranges from about 15 mg to about 300 mg.

2. 10. The method of claim 1, wherein the subject has late-onset Pompe disease (LOPD).

3. The method of claim 1 or 2, wherein the subject has a history of enzyme replacement therapy (ERT).

4. 3. The method of claim 1 or 2, wherein the subject is an enzyme replacement therapy (ERT) naive subject.

5. The method of any one of claims 1 to 4, wherein the pharmacological chaperone comprises miglustat.

6. 6. The method of any one of claims 1 to 5, wherein the dose of the rhGAA population is from about 20 mg / kg to about 30 mg / kg.

7. 7. The method of any one of claims 1 to 6, wherein the dose of the rhGAA population is about 25 mg / kg.

8. 8. The method of any one of claims 1 to 7, wherein the dose of the rhGAA population is about 30 mg / kg.

9. The method of any one of claims 1 to 8, wherein the subject is less than 30 days old.

10. 10. The method of any one of claims 1 to 9, wherein the miglustat dose is from about 15 mg to about 25 mg.

11. 11. The method of any one of claims 1 to 10, wherein the miglustat dose is about 20 mg.

12. The method of any one of claims 1 to 11, wherein the subject is between 1 month and less than 6 months old.

13. 13. The method of any one of claims 1 to 12, wherein the miglustat dose is from about 30 mg to about 50 mg.

14. 14. The method of any one of claims 1 to 13, wherein the miglustat dose is about 40 mg.

15. The method of any one of claims 1 to 14, wherein the subject is between 6 months and under 2 years old.

16. 16. The method of any one of claims 1 to 15, wherein the subject weighs less than 10 kg and the miglustat dose is from about 50 mg to about 80 mg.

17. 17. The method of any one of claims 1 to 16, wherein the subject weighs less than 10 kg and the miglustat dose is about 65 mg.

18. 18. The method of any one of claims 1 to 17, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is between about 70 mg and about 100 mg.

19. 19. The method of any one of claims 1 to 18, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 85 mg.

20. 20. The method of any one of claims 1 to 19, wherein the subject is between 2 and under 6 years old.

21. 21. The method of any one of claims 1 to 20, wherein the subject weighs less than 10 kg and the miglustat dose is from about 70 mg to about 100 mg.

22. 22. The method of any one of claims 1 to 21, wherein the subject weighs less than 10 kg and the miglustat dose is about 85 mg.

23. 23. The method of any one of claims 1-22, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is between about 100 mg and about 130 mg.

24. 24. The method of any one of claims 1 to 23, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 115 mg.

25. 25. The method of any one of claims 1 to 24, wherein the subject weighs between 20 kg and less than 30 kg and the miglustat dose is between about 110 mg and about 150 mg.

26. 26. The method of any one of claims 1 to 25, wherein the subject weighs between 20 kg and less than 30 kg and the miglustat dose is about 130 mg.

27. 27. The method of any one of claims 1 to 26, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is between about 150 mg and about 200 mg.

28. 28. The method of any one of claims 1 to 27, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is about 175 mg.

29. 29. The method of any one of claims 1 to 28, wherein the subject is between 6 and under 12 years old.

30. 30. The method of any one of claims 1-29, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is between about 100 mg and about 130 mg.

31. 31. The method of any one of claims 1 to 30, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 115 mg.

32. 32. The method of any one of claims 1-31, wherein the subject weighs between 20 kg and less than 30 kg and the miglustat dose is between about 110 mg and about 150 mg.

33. 33. The method of any one of claims 1 to 32, wherein the subject weighs between 20 kg and less than 30 kg and the miglustat dose is about 130 mg.

34. 34. The method of any one of claims 1 to 33, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is between about 150 mg and about 200 mg.

35. 35. The method of any one of claims 1 to 34, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is about 175 mg.

36. 36. The method of any one of claims 1-35, wherein the subject weighs between 40 kg and less than 50 kg and the miglustat dose is between about 170 mg and about 220 mg.

37. 37. The method of any one of claims 1 to 36, wherein the subject weighs between 40 kg and less than 50 kg and the miglustat dose is about 195 mg.

38. 38. The method of any one of claims 1-37, wherein the subject weighs at least 50 kg and the miglustat dose is from about 200 mg to about 300 mg.

39. 39. The method of any one of claims 1-38, wherein the subject weighs at least 50 kg and the miglustat dose is about 260 mg.

40. 40. The method of any one of claims 1 to 39, wherein the subject is between 12 and under 18 years of age.

41. 41. The method of any one of claims 1 to 40, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is between about 150 mg and about 200 mg.

42. 42. The method of any one of claims 1-41, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is about 175 mg.

43. 43. The method of any one of claims 1-42, wherein the subject weighs between 40 kg and less than 50 kg and the miglustat dose is between about 170 mg and about 220 mg.

44. 44. The method of any one of claims 1 to 43, wherein the subject weighs between 40 kg and less than 50 kg and the miglustat dose is about 195 mg.

45. 45. The method of any one of claims 1-44, wherein the subject weighs at least 50 kg and the miglustat dose is from about 200 mg to about 300 mg.

46. 46. ​​The method of any one of claims 1-45, wherein the subject weighs at least 50 kg and the miglustat dose is about 260 mg.

47. Each rhGAA molecule contains seven potential N-glycosylation sites; 40% to 60% of the N-glycans on the rhGAA molecule are complex N-glycans; the rhGAA molecule contains at least 0.5 moles of bis-mannose-6-phosphate (bis-M6P) per mole of rhGAA at the first potential N-glycosylation site as determined using liquid chromatography tandem mass spectrometry (LC-MS / MS); 47. The method of any one of claims 1 to 46.

48. 48. The method of any one of claims 1-47, wherein the rhGAA and pharmacological chaperone are administered weekly.

49. 49. The method of any one of claims 1 to 48, wherein the miglustat or a pharmaceutically acceptable salt thereof is administered before administration of the population of rhGAA molecules, optionally 1 hour before administration of the population of rhGAA molecules.

50. A method of treating late-onset Pompe disease (LOPD) in a subject under the age of 18, comprising administering to said subject a population of recombinant human acid alpha-glucosidase (rhGAA) molecules, either simultaneously or sequentially, with a pharmacological chaperone. Including, The method, wherein the dose of the recombinant human acid alpha-glucosidase (rhGAA) molecule population ranges from about 20 mg / kg to about 30 mg / kg, and the dose of the pharmacological chaperone ranges from about 15 mg to about 300 mg.

51. 51. The method of claim 50, wherein the pharmacological chaperone comprises miglustat.

52. 52. The method of claim 50 or 51, wherein the subject weighs less than 40 kg and the dose of the rhGAA population is about 30 mg / kg.

53. 53. The method of any one of claims 50 to 52, wherein the subject is less than 30 days old.

54. 54. The method of any one of claims 50-53, wherein the miglustat dose is about 20 mg.

55. 55. The method of any one of claims 50 to 54, wherein the subject is between 1 month and less than 6 months old.

56. 56. The method of any one of claims 50-55, wherein the miglustat dose is about 40 mg.

57. 57. The method of any one of claims 50 to 56, wherein the subject is between 6 months and less than 2 years old.

58. 58. The method of any one of claims 50-57, wherein the subject weighs less than 10 kg and the miglustat dose is about 65 mg.

59. 59. The method of any one of claims 50-58, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 85 mg.

60. 60. The method of any one of claims 50 to 59, wherein the subject is between 2 and under 6 years old.

61. 61. The method of any one of claims 50-60, wherein the subject weighs less than 10 kg and the miglustat dose is about 85 mg.

62. 62. The method of any one of claims 50-61, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 115 mg.

63. 63. The method of any one of claims 50 to 62, wherein the subject is between 6 and under 12 years of age.

64. 64. The method of any one of claims 50-63, wherein the subject weighs between 10 kg and less than 20 kg and the miglustat dose is about 115 mg.

65. 65. The method of any one of claims 50 to 64, wherein the subject is between 12 and under 18 years of age.

66. 66. The method of any one of claims 50-65, wherein the subject weighs between 20 kg and less than 30 kg and the miglustat dose is about 130 mg.

67. 54. The method of any one of claims 53, wherein the subject weighs between 30 kg and less than 40 kg and the miglustat dose is about 175 mg.

68. 53. The method of claim 52, wherein the subject weighs between 40 kg and less than 50 kg, the dose of the rhGAA population is about 25 mg / kg, and the dose of miglustat is about 195 mg.

69. 53. The method of claim 52, wherein the subject weighs at least 50 kg, the dose of the rhGAA population is about 20 mg / kg, and the dose of miglustat is about 260 mg.

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