CN117157095A

CN117157095A - Recombinant human acid alpha-glucosidase and uses thereof

Info

Publication number: CN117157095A
Application number: CN202280027532.7A
Authority: CN
Inventors: H·杜; 拉塞尔·戈乔尔; 亨·査尔; 杰伊·巴斯
Original assignee: Amicus Therapeutics Inc
Current assignee: Amicus Therapeutics Inc
Priority date: 2021-02-11
Filing date: 2022-02-11
Publication date: 2023-12-01

Abstract

Provided herein are methods of treating pompe disease comprising administering a population of recombinant human acid alpha-glucosidase molecules, or a pharmaceutical composition or formulation thereof, and a pharmacological chaperone.

Description

Recombinant human acid alpha-glucosidase and uses thereof

Cross Reference to Related Applications

The present application claims the benefits of U.S. provisional patent application No. 63/162,683 to 2021, month 3, 18, and U.S. provisional patent application No. 63/148,596 to 2021, month 2, 11, the disclosures of each of which are hereby incorporated by reference in their entirety.

Description of electronically submitted text files

The contents of the electronically submitted text file are incorporated herein by reference in their entirety: a computer-readable format copy of the sequence listing (file name: amcs_013_02wo_seqlist_st25.txt, recording date: 2022, 2, 11 days, file size: 45560 bytes).

Technical Field

The present application relates to recombinant human alpha-glucosidase (rhGAA) and to the treatment of pompe disease.

Background

Pompe disease is an inherited lysosomal storage disease resulting from a lack of acid alpha-Glucosidase (GAA) activity. Individuals with pompe disease lack or have reduced levels of acid alpha-Glucosidase (GAA), which breaks down hepatic glucose into glucose, the primary energy source for muscle. This enzyme deficiency causes excess hepatic saccharides to accumulate in lysosomes, which are intracellular organelles that contain enzymes that normally break down hepatic saccharides and other cellular debris or waste. The accumulation of liver glycans in certain tissues (especially muscles) of individuals with pompe disease impairs the ability of the cells to function normally. Under pompe disease, liver glycans are not metabolized properly and accumulate gradually in lysosomes, especially in skeletal muscle cells, and in the infant onset form of the disease, in lysosomes of cardiac muscle cells. The accumulation of liver glycans damages muscle and nerve cells and those in other affected tissues.

Pompe disease is traditionally clinically identified as early stage infant form or late stage onset form depending on age of onset. The age of onset is often parallel to the severity of the genetic mutation that causes pompe disease. The most severe genetic mutation causes complete loss of GAA activity and manifests itself as early onset disease during infancy. Mutations in genes that reduce GAA activity but do not eliminate it are associated with a poincare modality that delays onset and progression. Infant onset pompe is shown soon after birth and is characterized by muscle weakness, respiratory insufficiency and heart failure. Untreated, it is often fatal within two years. Adolescents and adults develop pompe disease that appears later in life and generally progresses more slowly than infants do. Although this form of disease does not generally affect the heart, it can also cause death because skeletal muscles and skeletal muscles involved in breathing weaken.

Current non-palliative treatments for pompe disease involve and are used under the trademarkAnd->Enzyme Replacement Therapy (ERT) with recombinant arabinosidase alpha product sold below. This conventional enzyme replacement therapy attempts to treat pompe disease by administering rhGAA to replace the missing GAA in lysosomes, thus restoring the ability of the cells to break down lysosomal liver sugar. And->rhGAA in its conventional form is produced or sold as a biological agent by Genzyme and approved by the united states food and drug administration and described by Reference to "Physician's Desk Reference" (2014), which is incorporated herein by Reference. Alcosidase alpha is identified by the chemical name [ 199-arginine, 223-histidine]Prepro-alpha-glucosidase (human); molecular formula C ₄₇₅₈ H ₇₂₆₂ N ₁₂₇₄ O ₁₃₆₉ S ₃₅ The method comprises the steps of carrying out a first treatment on the surface of the CAS number 420794-05-0. These products, also known as liver sugar storage type II (GSD-II) or acid maltogenic enzyme deficiency, are administered to individuals suffering from pompe disease.

However, current ERTs provide limited improvements in muscle function, strength, and respiratory function at best for a limited period of time, followed by a slow decline in these parameters (Toscano and Schoser 2013; wyatt et al 2012).

In 2012, a systematic review of all studies conducted in individuals with delayed pompe disease (LOPD) was conducted by Toscano and Schoser 2013. The examination included data from 368 individuals with LOPD from the public study, including 27 adolescent individuals (age range: 2 to 17 years) and 251 adult individuals who received arabinosidase alpha for at least 2 years. The results indicated that >30% of individuals did not exhibit initial improvement during treatment with arasidase a and continued to experience muscle and respiratory tract function degeneration despite treatment. In the group of individuals initially responding to the arabinosidase alpha treatment, several additional long-term studies showed improvement typically lasting only about 2 years. Thereafter, the individual typically reaches a plateau before beginning to descend gradually.

In 2012, as part of the national institutes of health in the united kingdom (Wyatt et al 2012), the british health technical assessment program issued advice on follow-up data review of 81 patients with pompe disease, including infant onset and tardive forms (children and adults), who received the current approved ERT standard care for arosidase a. Key markers for pompe disease progression (forced vital capacity, ventilator dependence, mobility, 6-minute walking test, muscle strength and body mass index) were assessed and treatment time for the arasidase alpha treatment was modeled using. This evaluation indicates that FVC, 6-minute walking test, and improvement in muscle strength in LOPD patients occurred the first 2 years after the onset of ERT with arasidase a, and decreased with continued treatment beyond this time range. In addition, a 3-year study in 38 individuals with LOPD who received arabinosidase a showed that the individuals exhibited improvement in motor function in the first year of treatment, remained generally stable in the second year, and began to decline in the third year (Regnery et al 2012).

In addition, for stage 3(Genzyme Corporation) study 1The 0-year follow-up report shows that after experiencing some improvement in exercise and lung function a few years prior to treatment, the individual begins to slowly decline with ongoing treatment (van der Ploeg et al 2017). In this study, the percentage of 6-minute walking distance at baseline was predicted to drop on average by about 10% from the 3 rd and 6 th years of treatment, with about 80% of individuals experiencing a drop.

The most serious tolerogenic problem for the enzyme alpha is the occurrence of infusion-related reactions (IARs), which in some cases may include life-threatening systemic or other severe allergic reactionsSummary of Product Characteristics, december 2018). Management of these events includes dose reduction, reduced infusion rates, and prolonged infusion times and dose interruption or cessation. Preoperative administration of antihistamines and steroids (prior to infusion) is also often used to prevent or reduce the incidence and severity of IAR and allergic reactions associated with the infusion of arasidase a. Despite these measurements, patients with pompe disease may still experience IAR, and some patients cannot tolerate conventional infusions of currently approved ERTs.

In 2017, a systematic review of the literature by the european poincare alliance (expert network from 11 european countries in poincare) was made (van der Ploeg et al 2017). Evidence of ERT effects at the group level was assessed by the consortium based on data obtained from one clinical study and 43 observed studies, which covered a total of 586 individual adult individuals. The current european poincare alliance is consensus as stopping ERT treatment in the presence of severe IAR or progressive clinical exacerbations of disease symptoms, and in the presence of high neutralizing antibody (Ab) titers, effectively deactivating existing ERT treatments. The european poincare alliance recommendation also includes consideration of reinitiation ERT treatment in cases of disease progression and clinical exacerbation reproduction after ERT has ceased.

Thus, there remains a need to identify improved rhGAA therapies that can effectively treat pompe disease and reduce adverse events.

Cellular uptake of the rhGAA molecule is promoted by the dedicated carbohydrate mannose-6-phosphate (M6P), which binds to the cation-independent mannose-6-phosphate receptor (CIMPR) present on target cells such as muscle cells. After binding, the rhGAA molecule is taken up by the target cell and subsequently transferred into lysosomes within the cell. However, most conventional rhGAA products lack a higher total content of N-glycans carrying single and double M6P (i.e., N-glycans carrying one M6P residue or two M6P residues, respectively), which limit their cellular uptake via CIMPR and lysosomal delivery, thereby making conventional enzyme replacement therapies less effective. For example, while conventional rhGAA products at doses of 20mg/kg or higher do improve some aspects of pompe disease, they fail to adequately (inter alia) (i) treat potential cellular dysfunction, (ii) restore muscle structure, or (iii) reduce liver glucose accumulation in many target tissues, such as skeletal muscle, to reverse disease progression. In addition, higher doses can impose additional burdens on the individual and the medical professional treating the individual, such as extending the infusion time required for intravenous administration of rhGAA.

Glycosylation of GAA or rhGAA can be modified in vitro by enzymes such as phosphotransferase and uncovered enzymes described in U.S. patent No. 6,534,300 to Canfield et al to produce the M6P group. However, enzymatic glycosylation is not adequately controlled and rhGAA with undesirable immunological and pharmacological properties can be produced. The enzyme modified rhGAA may contain only high mannose oligosaccharides, all of which may potentially be enzymatically phosphorylated with phosphotransferase or in vitro uncovered by the enzyme. The glycosylation pattern resulting from in vitro enzymatic treatment of GAA is problematic because the additional terminal mannose residues, particularly the non-phosphorylated terminal mannose residues, negatively affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannose groups increase the inefficient clearance of GAA, increase the uptake of enzymatically modified GAA by immune cells and reduce the efficacy of rhGAA treatment by less GAA reaching the targeted tissue (such as cardiac or skeletal muscle myocytes). For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen that cause rapid clearance of enzymatically modified rhGAA and reduce rhGAA targeting to target tissues. Furthermore, the glycosylation pattern of enzymatically modified GAA with high mannose N-glycans containing terminal non-phosphorylated mannose residues is similar to enzymatically modified rhGAA in terms of glycoproteins produced with yeast and mold, and increased risk of triggering immune or allergic reactions such as severe allergy (allergy/allergy) or hypersensitivity of both life and danger.

In contrast to conventional recombinant rhGAA products and in vitro phosphorylated rhGAA, the rhGAA used in the two-component treatment according to the invention has an optimized N-glycan profile for increased biodistribution and lysosomal absorption, thereby minimizing non-productive clearance of the rhGAA after administration. The present disclosure provides stable or declining pompe patients with effective treatments to reverse disease progression at the cellular level, including more effective clearance of lysosomal hepatic glucose than current standard care. Patients treated with the bi-component of the present disclosure comprising rhGAA and a pharmacological partner (e.g., miglutide) exhibit significant health improvements, including improvement in muscle strength, motor function, and/or lung function, and/or include reversal of disease progression, as demonstrated by the various efficacy results from clinical studies (e.g., examples 8 and 9).

Disclosure of Invention

Provided herein is a method of treating a disease or disorder, such as pompe disease, in an individual comprising administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules and a pharmacological chaperone (e.g., miglutide).

The rhGAA molecules described herein can be expressed in Chinese Hamster Ovary (CHO) cells and comprise seven potential N-glycosylation sites. In some embodiments, the N-glycosylation pattern of a population of rhGAA molecules as described herein is determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). In some embodiments, the rhGAA molecule comprises an average of 3-4 moles of mannose-6-phosphate (M6P) residues per mole of rhGAA. In some embodiments, the rhGAA molecule comprises on average about at least 0.5 moles of bisphosphorylated N-glycan groups (bis M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an amino acid sequence having at least 95% identity to SEQ ID NO. 4 or SEQ ID NO. 6. In some embodiments, the rhGAA comprises an amino acid sequence that is identical to SEQ ID NO. 4 or SEQ ID NO. 6. In some embodiments, at least 30% of the molecules of the rhGAA comprise one or more N-glycan units carrying one or two M6P residues. In some embodiments, the rhGAA molecule comprises an average of about 0.5 moles to about 7.0 moles of N-glycan units bearing one or two M6P residues per mole of rhGAA. In some embodiments, the rhGAA molecule comprises an average of 2.0 to 8.0 moles of sialic acid per mole of rhGAA. In some embodiments, the rhGAA molecule comprises an average of at least 2.5 moles of M6P residues per mole of rhGAA and at least 4 moles of sialic acid residues per mole of rhGAA. In some embodiments, the rhGAA molecule comprising an average of 3-4 moles of M6P residues per mole of rhGAA and an average of at least about 0.5 moles of bis M6P per mole of rhGAA at the first potential N-glycosylation site further comprises an average of about 0.4 to about 0.6 moles of mono-phosphorylated N-polysaccharide (mono M6P) per mole of rhGAA at the second 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, and about 0.3 to about 0.4 moles of mono M6P per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA molecule further comprises an average of about 4 to about 7.3 moles of sialic acid residues per mole of rhGAA, including about 0.9 to about 1.2 moles of sialic acid per mole of rhGAA at the third 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 1.5 to about 4.2 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, the population of rhGAA molecules is formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising a population of rhGAA molecules further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof. In some embodiments, the pH of the pharmaceutical composition is about 5.0 to about 7.0, about 5.0 to about 6.0, or about 6.0. In some embodiments, the pharmaceutical composition further comprises water, an acidulant, an alkalizing agent, or a combination thereof. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 5-50mg/mL rhGAA molecules, about 10-100mM sodium citrate buffer, about 10-50mg/mL mannitol, about 0.1-1mg/mL polysorbate 80, and water, and optionally an acidifying and/or basifying agent. In some embodiments, the pharmaceutical composition has a pH of 6.0 and comprises a population of about 15mg/mL rhGAA molecules, about 25mM sodium citrate buffer, about 20mg/mL mannitol, about 0.5mg/mL polysorbate 80, and water, and optionally an acidifying and/or basifying agent.

In some embodiments, the population of rhGAA molecules is administered at a dose of about 1mg/kg to about 100mg/kg or about 5mg/kg to about 20 mg/kg. In some embodiments, the population of rhGAA molecules is administered at a dose of about 20 mg/kg. In some embodiments, the population of rhGAA molecules is administered for two months, monthly, biweekly, weekly, twice weekly, or daily, e.g., every two weeks. In some embodiments, the population of rhGAA molecules is administered intravenously.

In some embodiments, the population of rhGAA molecules is administered simultaneously or sequentially with a pharmacological chaperone, such as miglutide (also known as AT 2221), or a pharmaceutically acceptable salt thereof. In some embodiments, the meglumine (miglustat) or a pharmaceutically acceptable salt thereof is administered orally, e.g., at a dose of about 50mg to about 200mg or about 200mg to about 600mg, and optionally about 130mg, about 195mg, or about 260 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and the meglumine or pharmaceutically acceptable salt thereof is administered orally at a dose of about 233mg to about 500 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and the meglumine or pharmaceutically acceptable salt thereof is administered orally at a dose of about 50mg to about 200 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 20mg/kg and the meglumine or pharmaceutically acceptable salt thereof is administered orally at a dose of about 260 mg. In some embodiments, the population of rhGAA molecules is administered intravenously at a dose of about 20mg/kg and the meglumine or pharmaceutically acceptable salt thereof is administered orally at a dose of about 195 mg. In some embodiments, the meglumine or a pharmaceutically acceptable salt thereof is administered prior to administration of the population of rhGAA molecules (e.g., about one hour prior to administration of the population of rhGAA molecules). In at least one embodiment, the subject is fasted at least two hours prior to administration of the meglumine or a pharmaceutically acceptable salt thereof and at least two hours after administration of the meglumine or a pharmaceutically acceptable salt thereof.

Embodiments of the present invention exhibit the efficacy of the bi-component therapies described herein to treat and reverse disease progression in individuals with pompe disease. In some embodiments, the individual has experienced a patient of ERT. In some embodiments, the subject is an ERT untreated patient.

In some embodiments, a two-component treatment according to the invention ameliorates one or more disease symptoms in an individual with pompe disease compared to (1) baseline, or (2) a control treatment comprising administration of an arabinosidase a and a placebo for a pharmacological chaperone. In such control treatments, a placebo, but not a pharmacological chaperone, is administered.

In some embodiments, the two-component treatment according to the invention improves motor function of the individual as measured by the 6-minute walk test (6 MWT). In some embodiments, the subject's 6-minute walking distance (6 MWD) 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 compared to baseline. In some embodiments, the individual's 6MWD increases by at least 20 meters or 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 as compared to control treatment. In some embodiments, the subject's 6MWD improves by at least 13 meters after 52 weeks of treatment compared to control treatment. In some embodiments, the individual has a baseline 6MWD of less than 300 meters. In some embodiments, the individual has a baseline 6MWD of greater than or equal to 300 meters.

In some embodiments, the two-component treatment according to the invention stabilizes the pulmonary function of the individual as measured by the Forced Vital Capacity (FVC) test. In some embodiments, the predicted FVC percentage of the individual increases from 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% from baseline after 12, 26, 38 or 52 weeks of treatment. In some embodiments, the predicted FVC percentage of the individual is reduced by less than 1% from baseline after 52 weeks of treatment. In some embodiments, the individual's predicted FVC percentage is significantly improved after treatment compared to control treatment. In some embodiments, the predicted FVC percentage of the individual is significantly improved by at least 0.5%, 1%, 2%, 3%, 4%, 5% or 6% after 12, 26, 38 or 52 weeks of treatment as compared to control treatment. In some embodiments, the predicted FVC percentage of the individual is significantly improved by at least 3% after 52 weeks of treatment compared to control treatment. In some embodiments, the individual has a baseline FVC of less than 55%. In some embodiments, the individual has a baseline FVC of greater than or equal to 55%.

In some embodiments, the two-component treatment according to the invention improves the motor function of the individual as measured by gait, stair, golgi, chair (GSGC) tests. In some embodiments, the GSGC score of the individual is improved compared to baseline, 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. In some embodiments, the GSGC score of the individual is improved compared to baseline, as indicated by a decrease of at least 0.5 point after 52 weeks of treatment. In some embodiments, the GSGC score of the individual is significantly improved after treatment compared to control treatment. In some embodiments, the GSGC score of the individual is significantly improved compared to a control treatment, 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. In some embodiments, the GSGC score of the individual is significantly improved compared to the control treatment, as indicated by a decrease of at least 1.0 point after 52 weeks of treatment.

In some embodiments, the two-component treatment according to the present invention reduces the content of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle injury marker comprises Creatine Kinase (CK). In some embodiments, the CK content of the individual is reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38 or 52 weeks of treatment as compared to baseline. In some embodiments, the CK content of the subject is reduced by at least 20% after 52 weeks of treatment, as compared to baseline. In some embodiments, the CK content of the individual is significantly reduced after treatment compared to control treatment. In some embodiments, the CK content of the individual is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38, or 52 weeks of treatment as compared to control treatment. In some embodiments, the CK content of the subject is significantly reduced by at least 30% after 52 weeks of treatment compared to control treatment.

In some embodiments, a two-component treatment according to the invention reduces the amount of at least one liver glucose accumulation marker after treatment. In some embodiments, the at least one liver sugar accumulation marker comprises urine hexose tetraose (Hex 4). In some embodiments, the individual's urinary Hex4 content is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to baseline. In some embodiments, the individual's urine Hex4 content is reduced by at least 30% after 52 weeks of treatment as compared to baseline. In some embodiments, the individual has a significantly reduced urine Hex4 content after treatment as compared to a control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 40% after 52 weeks of treatment as compared to a control treatment.

In some embodiments, the two-component treatment according to the present invention ameliorates one or more disease symptoms in an individual of a patient suffering from pompe disease who has experienced ERT compared to (1) baseline, or (2) a control treatment comprising administration of an arabinosidase a and a placebo for a pharmacological chaperone.

In some embodiments, the two-component treatment of an individual experiencing ERT with pompe disease improves the motor function of the individual, as measured by 6 MWT. In some embodiments, the individual'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 compared to baseline. In some embodiments, the individual's 6MWD increases by at least 15 meters or at least 5% after 52 weeks of treatment. In some embodiments, the individual's 6MWD is significantly improved after treatment compared to control treatment. In some embodiments, the individual'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 compared to control treatment. In some embodiments, the subject's 6MWD is improved by at least 15 meters after 52 weeks of treatment compared to control treatment. In some embodiments, the individual has a baseline 6MWD of less than 300 meters. In some embodiments, the individual has a baseline 6MWD of greater than or equal to 300 meters.

In some embodiments, the two-component treatment of an individual experiencing ERT with pompe disease improves the lung function of the individual as measured by the FVC test. In some embodiments, the predicted FVC percentage of the individual increases by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4% or 5% from baseline after 12, 26, 38 or 52 weeks of treatment. In some embodiments, the predicted FVC percentage of the individual increases by at least 0.1% from baseline after 52 weeks of treatment. In some embodiments, the individual's predicted FVC percentage is significantly improved after treatment compared to control treatment. In some embodiments, the predicted FVC percentage of the individual is significantly improved by at least 1%, 2%, 3%, 4%, 5%, 6%, 8% or 10% after 12, 26, 38 or 52 weeks of treatment compared to control treatment. In some embodiments, the predicted FVC percentage of the individual is significantly improved by at least 4% after 52 weeks of treatment compared to control treatment. In some embodiments, the individual has a baseline FVC of less than 55%. In some embodiments, the individual has a baseline FVC of greater than or equal to 55%.

In some embodiments, the two-component treatment of an individual experiencing ERT with pompe disease improves the motor function of the individual as measured by the GSGC test. In some embodiments, the GSGC score of the individual is improved after 12, 26, 38, or 52 weeks of treatment as compared to baseline, as indicated by at least a 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 point decrease. In some embodiments, the GSGC score of the individual is improved after 52 weeks of treatment as compared to baseline, as indicated by at least a 0.5 point decrease. In some embodiments, the GSGC score of the individual is significantly improved after treatment compared to control treatment. In some embodiments, the GSGC score of the individual is significantly improved compared to a control treatment, 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. In some embodiments, the GSGC score of the individual is significantly improved compared to the control treatment, as indicated by a decrease of at least 1.0 point after 52 weeks of treatment.

In some embodiments, the two-component treatment for an individual experiencing ERT with pompe disease reduces the content of at least one muscle damage marker after treatment. In some embodiments, the at least one muscle damage marker comprises CK. In some embodiments, the CK content of the individual is reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38 or 52 weeks of treatment as compared to baseline. In some embodiments, the CK content of the subject is reduced by at least 15% after 52 weeks of treatment, as compared to baseline. In some embodiments, the CK content of the individual is significantly reduced after treatment compared to control treatment. In some embodiments, the CK content of the individual is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40% or 50% after 12, 26, 38, or 52 weeks of treatment as compared to control treatment. In some embodiments, the CK content of the subject is significantly reduced by at least 30% after 52 weeks of treatment compared to control treatment.

In some embodiments, the two-component treatment for an individual experiencing ERT with pompe disease reduces the content of at least one liver glucose accumulation marker after treatment. In some embodiments, the at least one liver glucose accumulation marker comprises urine Hex4. In some embodiments, the individual's urine Hex4 content is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to baseline. In some embodiments, the individual's urine Hex4 content is reduced by at least 25% after 52 weeks of treatment as compared to baseline. In some embodiments, the individual has a significantly reduced urine Hex4 content after treatment as compared to a control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 40% after 52 weeks of treatment as compared to a control treatment.

Drawings

FIG. 1A shows non-phosphorylated higher mannose N-glycans, mono-M6P N-glycans, and di-M6P N-glycans. FIG. 1B shows the chemical structure of the M6P group. Each square represents N-acetylglucosamine (GlcNAc), each circle represents mannose, and each P represents phosphate.

Fig. 2A depicts the production targeting of rhGAA to a target tissue (e.g., muscle tissue of an individual with pompe disease) via M6P-bearing N-glycans. Fig. 2B depicts non-productive drug clearance from non-target tissues (e.g., liver and spleen) or binding to non-target tissues through non-M6P N-glycans.

FIG. 3 is a schematic diagram of an exemplary process for producing, capturing and purifying recombinant lysosomal proteins.

FIG. 4 shows DNA constructs for transforming CHO cells using DNA encoding rhGAA.

Fig. 5 is a graph showing the results of CIMPR affinity chromatography with (embodiment 2) and without (embodiment 1) captured ATB200 rhGAA on an Anion Exchange (AEX) column.

FIGS. 6A-6H show the results of site-specific N-glycosylation analysis of ATB200 rhGAA using two different LC-MS/MS analysis techniques. Fig. 6A shows the site occupancy of seven potential N-glycosylation sites of ATB 200. FIG. 6B shows two analyses of the N-glycosylation pattern of the first potential N-glycosylation site of ATB 200. Fig. 6C shows two analyses of the N-glycosylation pattern of the second potential N-glycosylation site of ATB 200. FIG. 6D shows two analyses of the N-glycosylation pattern of the third potential N-glycosylation site of ATB 200. FIG. 6E shows two analyses of the N-glycosylation pattern of the fourth potential N-glycosylation site of ATB 200. FIG. 6F shows two analyses of the N-glycosylation pattern of the fifth potential N-glycosylation site of ATB 200. FIG. 6G shows two analyses of N-glycosylation patterns of a sixth potential N-glycosylation site of ATB 200. FIG. 6H outlines the relative percentages of mono-and di-phosphorylated species for the first, second, third, fourth, fifth and sixth potential N-glycosylation sites.

FIG. 7 is a display ofGraph of polymax elution profile (thin line, elution to the left) and ATB200 (thick line, elution to the right).

FIG. 8 is a display ofThe summary of N-glycan structures of (a) is compared to the table of three different ATB200rhGAA formulations (identified as BP-rhGAA, ATB200-1 and ATB 200-2).

Fig. 9A and 9B are diagrams showing the respectiveAnd->Is a graph of CIMPR affinity chromatography results.

FIG. 10A is a graph comparing CIMPR binding affinity (left panel) of ATB200rhGAA with that of ATB200rhGAAIs shown in the figure (right panel). FIG. 10B is a graph for comparison->And tables of double M6P content of ATB200 rhGAA.

FIG. 11A is a graph comparing ATB200rhGAA activity inside normal fibroblasts at various GAA concentrations (left panel) withGraph of rhGAA activity (right panel). FIG. 11B is a graph comparing ATB200rhGAA activity (left panel) at various GAA concentrations inside fibroblasts from individuals with pompe diseaseTable of rhGAA activity (right panel). FIG. 11C is a graph showing the comparison of K of fibroblasts from normal individuals and individuals with pompe disease _{Absorption of} Is a table of (2).

Fig. 12 depicts the stability of ATB200 in acidic or neutral pH buffers evaluated in a thermal stability assay using SYPRO orange, as the fluorescence of the dye increases as the protein denatures.

FIG. 13 shows the tissue liver glucose content of WT mice or vector, arabinosidase alpha or ATB200/AT2221 treated Gaa KO mice determined using amyloglucosidase digestion. Bars represent mean ± SEM of 7 mice/group. * p <0.05 compared to the arasidase a in multiple comparisons using the dunnity method under one-way ANOVA analysis.

FIG. 14 depicts LAMP 1-positive vesicles in muscle fibers of Gaa KO mice or WT mice treated with vector, arabinosidase alpha or ATB200/AT 2221. Images were obtained from the lateral femoral muscle and represent 7 mice/group. Magnification = 200x (1,000x in the inset).

FIG. 15A shows LC3 positive aggregates in myofibers of Gaa KO mice or WT mice treated with vector, arabinosidase alpha or ATB200/AT 2221. Images were obtained from the lateral femoral muscle and represent 7 mice/group. Magnification = 400x. Figure 15B shows western blot analysis of LC3 II protein. A total of 30mg protein was loaded in each lane.

FIG. 16 shows plasma membrane repair protein expression in myofibers of vehicle, arabinosidase α or ATB200/AT2221 treated Gaa KO mice or WT mice. Images were obtained from the lateral femoral muscle and represent 7 mice/group. Magnification = 200x.

Fig. 17 depicts co-immunofluorescent staining of LAMP1 (green) (see, e.g., "B") and LC3 (red) (see, e.g., "a") in individual fibers isolated from white gastrocnemius muscle of Gaa KO mice treated with vector, arabinosidase a or ATB 200. "C" depicts the clearance of autophagic debris and the absence of enlarged lysosomes. A minimum of 30 fibers were detected from each animal.

Fig. 18 depicts the stabilization of ATB200 by AT2221 AT 17 μm and 170 μm AT2221, respectively, as compared to ATB200 alone.

FIGS. 19A-19H show the results of a site-specific N-glycosylation analysis of ATB200 rhGAA (including N-glycosylation patterns) at a seventh potential N-glycosylation site using an LC-MS/MS analysis of protease digested ATB 200. Figures 19A-19H provide average data for ten batches of ATBs 200 produced at different scales.

Figure 19A shows the average site occupancy of seven potential N-glycosylation sites of ATB 200. The N-glycosylation site is provided according to SEQ ID NO. 1. Cv=coefficient of variation.

FIGS. 19B-19H show site-specific N-glycosylation assays of all seven potential N-glycosylation sites of ATB200, wherein the site numbering is provided according to SEQ ID NO. 5. Bars represent the maximum and minimum percentages of N-polysaccharide material identified as the particular N-polysaccharide group of the ten batches of ATBs 200 analyzed. FIG. 19B shows the N-glycosylation pattern of the first potential N-glycosylation site of ATB 200. FIG. 19C shows the N-glycosylation pattern of a second potential N-glycosylation site of ATB 200. FIG. 19D shows the N-glycosylation pattern of a third potential N-glycosylation site of ATB 200. FIG. 19E shows the N-glycosylation pattern of the fourth potential N-glycosylation site of ATB 200. FIG. 19F shows the N-glycosylation pattern of a fifth potential N-glycosylation site of ATB 200. FIG. 19G shows the N-glycosylation pattern of a sixth potential N-glycosylation site of ATB 200. FIG. 19H shows the N-glycosylation pattern of the seventh potential N-glycosylation site of ATB 200.

FIGS. 20A-20B further characterize and outline the N-glycosylation pattern of ATB200, as also shown in FIGS. 19A-19H. FIG. 20A shows the localization of 2-anthranilic acid (2-AA) polysaccharide and LC/MS-MS analysis of ATB200 and outlines N-polysaccharide species identified as a percentage of total fluorescence in ATB 200. Data from 2-AA polysaccharide localization and LC-MS/MS analysis are also depicted in table 5. Figure 20B outlines the average site occupancy and average N-glycan profile for all seven potential N-glycosylation sites of ATB200, including total phosphorylation, mono-phosphorylation, di-phosphorylation, and sialylation. Nd=undetected.

FIG. 21 shows a schematic of the ATB200-03 study design.

Figure 22 shows baseline 6-minute walking distance (6 MWD) and sitting Forced Vital Capacity (FVC) characteristics of 122 individuals participating in the ATB200-03 study. AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

Figure 23A depicts 6MWD and FVC data showing baseline of the total population, change from baseline at week 52 ("CFBL"), variance, and p-value (n=122). AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

Fig. 23B depicts 6MWD and FVC data showing changes over time from baseline for the total population (n=122). Sirtuin α/miglutide group: an individual receiving ATB200/AT2221 treatment; the alpha-glucosidase: individuals receiving the arasidase alpha/placebo treatment.

Fig. 24 depicts 6MWD and FVC data showing baseline, CFBL at week 52, differences and p values for the population experiencing ERT (n=95). AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

Fig. 25 depicts 6MWD and FVC changes from baseline at week 12, week 26 and week 38 and week 52 for the population experiencing ERT (n=95).

Fig. 26A depicts 6MWD and FVC data showing baseline, CFBL at week 52, differences, and p values for the population not treated with ERT (n=27). AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

Fig. 26B depicts 6MWD and FVC data showing changes over time from baseline for the population not treated with ERT (n=27). Sirtuin α/miglutide group: an individual receiving ATB200/AT2221 treatment; the alpha-glucosidase: individuals receiving the arasidase alpha/placebo treatment.

Fig. 27 depicts baseline characteristics of key secondary endpoints and biomarkers for the total population and population that experienced ERT. AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

Fig. 28 depicts lower limb freehand muscle force test (MMT) changes at week 12, week 26, week 38, and week 52 relative to baseline for the total population (left side) and the population that underwent ERT (right side).

Fig. 29 depicts gait, stair, golgi, chair (GSGC) changes at week 12, week 26, week 38 and week 52 relative to baseline for the total population (left side) and the population experiencing ERT (right side). Sirtuin α/miglutide group: an individual receiving ATB200/AT2221 treatment; the alpha-glucosidase: individuals receiving the arasidase alpha/placebo treatment.

Fig. 30 depicts the physical function changes of the results measurement information system (proci) from baseline reported by patients at week 12, week 26, week 38 and week 52 for the total population (left) and the population that underwent ERT (right).

Fig. 31 depicts the variation in proci fatigue relative to baseline at week 12, week 26, week 38 and week 52 for the total population (left) and the population experiencing ERT (right).

Fig. 32 depicts Creatine Kinase (CK) biomarker changes relative to baseline at week 12, week 26, week 38, and week 52 for the total population (left) and the population that underwent ERT (right).

Fig. 33 depicts urinary hexose tetra-saccharide (Hex 4) biomarker changes relative to baseline at week 12, week 26, week 38 and week 52 for the total population (left) and the population that underwent ERT (right).

Fig. 34 shows primary, secondary, and biomarker endpoint heatmaps for the total population (left) and the population that underwent ERT (right). AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment.

FIG. 35 summarizes the security data from the ATB200-03 study. AT-GAA group: an individual receiving ATB200/AT2221 treatment; the arabinosidase a group: individuals receiving the arasidase alpha/placebo treatment. TEAE: treating the induced adverse events; IAR: infusion-related reactions.

FIG. 36 summarizes the results from the ATB200-03 study.

FIG. 37 depicts the study goals and statistical methods of the ATB200-03 study.

FIG. 38 depicts the primary and secondary endpoints of the ATB200-03 study.

Figure 39 outlines the patient schedule for the ATB200-03 study.

Figure 40 summarizes baseline demographics for the ATB200-03 study.

Figures 41A-41B show panel analysis of changes in 6MWD and FVC from baseline at baseline in the total population (n=122) (figure 41A) and patients experiencing ERT (n=95) (figure 41B) in an ATB200-03 study.

FIG. 42 shows a list of treatment-induced adverse events (TEAE) for > 10% of patients in any tissue in the ATB200-03 study.

Detailed Description

Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or method steps set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

Provided herein is a method for treating pompe disease comprising administering to an individual a recombinant human alpha-glucosidase (rhGAA) and a pharmacological chaperone. The higher total content of mannose-6-phosphate-bearing N-glycans of rhGAA compared to conventional rhGAA products exhibits excellent uptake into muscle cells and subsequent delivery into lysosomes and has other pharmacokinetic properties that make them particularly effective for enzyme replacement therapy in individuals with pompe disease. Thus, the two-component treatment according to the invention exhibits superior efficacy in treating and reversing disease progression in individuals suffering from pompe disease compared to conventional therapies.

I. Definition of the definition

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

The term "GAA" refers to human acid alpha-Glucosidase (GAA) enzymes, which are enzymes that catalyze the hydrolysis of the alpha-1, 4-glycose bond and the alpha-1, 6-glycose bond of lysosomal liver saccharides; and insertion, related or substituted variants of GAA amino acid sequences and fragments of longer GAA sequences that exert enzymatic activity. Human acid alpha-glucosidase is encoded by the GAA gene (national center for biotechnology information (NCBI) gene ID 2548), which has been mapped to the long arm of chromosome 17 (positions 17q25.2-q 25.3). An exemplary amino acid sequence for GAA is NP 000143.2, which is incorporated by reference. The invention also encompasses DNA sequences encoding the amino acid sequence NP 000143.2. Over 500 mutations have been identified in the human GAA gene, many of which are associated with pompe disease. Mutations that cause misfolding or mishandling of acid alpha-glucosidase include T1064C (Leu 355 Pro) and C2104T (Arg 702 Cys). In addition, GAA mutations that affect enzyme maturation and processing 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 alpha-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to human acid alpha-glucosidase, while the italic abbreviation "GAA" is intended to refer to a human gene encoding human acid alpha-glucosidase. The italic abbreviation "Gaa" is intended to refer to non-human genes encoding non-human acid alpha-glucosidase, including but not limited to rat or mouse genes, and the abbreviation "Gaa" is intended to refer to non-human acid alpha-glucosidase.

The term "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase and is used to distinguish endogenous GAA from synthetically or recombinantly produced GAA (e.g., GAA produced by CHO cells or other host cells transformed with DNA encoding GAA). The term "rhGAA" encompasses a population of individual rhGAA molecules. Characteristics of the population of rhGAA molecules are provided herein. The term "conventional rhGAA product" is intended to mean an product containing an arabinosidase alpha, such asOr->

The term "genetically modified" or "recombinant" refers to a cell, such as a CHO cell, that expresses a particular gene product (such as rhGAA) after introduction of a nucleic acid comprising a coding sequence encoding the gene product and regulatory components 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 term also includes cells that have been engineered, for example, by gene activation techniques, to express or over-express an endogenous gene or gene product that is not normally expressed by the cell.

The term "arabinosidase α" as used herein is intended to mean an enzyme identified as [ 199-arginine, 223-histidine]Prepro-alpha-glucosidase (human); recombinant human acid alpha-glucosidase with chemical abstract accession number 420794-05-0. Alcosidase alpha is approved for use as a product by Genzyme And->Sold in the united states.

As used herein, the term "ATB200" is intended to refer to the recombinant human acid alpha-glucosidase described in U.S.10,961,522, the disclosure of which is incorporated herein by reference. ATB200 is also referred to as "sirtuin α".

As used herein, the term "polysaccharide" is intended to refer to an oligosaccharide that is covalently bound to an amino acid residue on a protein or polypeptide. As used herein, the term "N-polysaccharide" or "N-linked polysaccharide" is intended to refer to a polysaccharide chain linked to an asparagine residue on a protein or a polypeptide covalently bound to a nitrogen atom of an asparagine residue. In some embodiments, the N-glycan unit attached to rhGAA is determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using, for example, thermo Scientific ^TM Orbitrap Velos Pro ^TM Mass spectrometer, thermo Scientific ^TM Orbitrap Fusion ^TM Lumos Tribid ^TM Mass spectrometers or WatersInstrument of G2-XS QTof mass spectrometer.

As used herein, a forced vital capacity or "FVC" is the amount of air that can be forced to exhale from an individual's lungs after the individual has made as deep a breath as possible.

As used herein, a "six minute walk test" (6 MWT) is a test for measuring the distance that a human is given to walk for six minutes in total on a hard flat surface. The test was run so that the individuals walked as far as possible within six minutes.

As used herein, a "ten meter walking test" (10 MWT) is a test for measuring the time required for an individual to walk ten meters on a flat surface while wearing walking shoes.

As used herein, the compound miglutide, also known as n-butyl-1-deoxynojirimycin or NB-DNJ or (2 r,3r,4r,5 s) -1-butyl-2- (hydroxymethyl) piperidine-3, 4, 5-triol, is a compound having the following chemical formula:

one formulation of miglutide is under the trade name monotherapy for Gaucher's disease type 1Commercially available. In some embodiments, miglutt is referred to as AT2221.

Pharmaceutically acceptable salts of meglumine may also be used in the present invention, as discussed below. When a salt of meglumine is used, the dosage of the salt is adjusted so that the patient receives a dose of meglumine equivalent to the amount of the free base of meglumine that it has received to be used.

As used herein, the compound dulcitol, also known as 1-deoxynojirimycin or DNJ or (2 r,3r,4r,5 s) -2- (hydroxymethyl) piperidine-3, 4, 5-triol, is a compound having the following chemical formula:

as used herein, the term "pharmacological chaperone (pharmacological chaperone)" or sometimes just the term "chaperone" is intended to refer to a molecule that specifically binds to an acid alpha-glucosidase and has one or more of the following effects:

Enhancing the formation of stable conformations of proteins;

enhancing proper transport of the protein from the endoplasmic reticulum to another cell site, preferably a primary cell site, in order to prevent endoplasmic reticulum-related degradation of the protein;

preventing aggregation of conformationally unstable or misfolded proteins;

restoring and/or enhancing at least part of the wild-type function, stability and/or activity of the protein; and/or

Improving the phenotype or function of cells carrying an acid alpha-glucosidase.

Thus, pharmacological chaperones for acid alpha-glucosidase are molecules that bind to acid alpha-glucosidase, causing proper folding, transport, non-aggregation and activity of acid alpha-glucosidase. In at least one embodiment, the pharmacological chaperone is meglumine. Another non-limiting example of a pharmacological chaperone for an acid alpha-glucosidase is durum.

As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and do not typically produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia (u.s.pharmacopeia) or other generally recognized pharmacopeia for use in animals, and more particularly in humans. As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or carrier with which the compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in e.w. martin, remington' sPharmaceutical Sciences, 18 th edition or other versions.

The term "pharmaceutically acceptable salt" as used herein means a salt which is, 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 benefit/risk ratio, typically water-or oil-soluble or water-or oil-dispersible, and effective for its intended use. The term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. A list of suitable salts is found in S.M. Berge et al, (J.Pharm. Sci.), 1977,66, pages 1-19, which is incorporated herein by reference. The term "pharmaceutically acceptable acid addition salts" as used herein is intended to mean those salts formed from inorganic acids that retain the biological effectiveness and properties of the free base and are biologically or otherwise undesirable. The term "pharmaceutically acceptable base addition salts" as used herein is intended to mean those salts formed from inorganic bases that retain the biological effectiveness and properties of the free base and are biologically or otherwise undesirable.

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

As used herein, the terms "therapeutically effective dose" and "effective amount" are intended to refer to an amount of acidic alpha-glucosidase and/or miglutide and/or a two-component treatment thereof sufficient to elicit a therapeutic response in an individual.

Therapeutic responses may also include molecular responses such as liver glucose accumulation, lysosomal proliferation, and autophagic region formation. Treatment response can be assessed by comparing physiological and molecular responses of muscle biopsies before and after treatment with rhGAA as described herein. For example, the amount of liver saccharides present in a biopsy sample may be used as a marker to determine the response of a treatment. Another embodiment includes biomarkers, such as LAMP-1, LC3, and plasma membrane repair proteins, which can be used as indicators of lysosomal storage disorders. For example, muscle biopsies collected before and after treatment with rhGAA as described herein can be stained with an antibody that recognizes one of the biomarkers. The therapeutic response may also include a reduction in fatigue or improvement in other patient reported results (e.g., daily life activity, health, etc.).

As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of non-naturally purified enzymes into individuals having defects in such enzymes. The protein administered may be obtained from a natural source or expressed recombinantly. The term also refers to introducing a purified enzyme in an individual that would otherwise be in need of, or benefit from, administration of the purified enzyme. In at least one embodiment, such an individual suffers from enzyme deficiency. The introduced enzyme may be a purified recombinant enzyme produced in vitro, or a protein purified from a tissue or fluid, such as placenta or animal milk, or from a plant.

As used herein, the term "dual component therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In some embodiments, the outcome of the bi-component therapy is enhanced compared to the effect of each therapy when administered alone. Enhancement may include any improvement in the effects of the various therapies, which may yield advantageous results compared to the results achieved by the therapies when performed alone. The enhanced effect or outcome may comprise synergistic enhancement, wherein the enhanced effect exceeds the additive effect of each therapy when administered alone; accumulating the enhancement, wherein the enhancement effect is substantially equal to the additive effect of each therapy when performed by itself; or less than the additive effect, wherein the enhancing effect is less than the additive effect of each therapy when administered by itself, but still better than each therapy when administered alone. The enhanced effect may be measured by any means known in the art that can measure the efficacy or outcome of a treatment.

"Pompe" refers to autosomal recessive LSD characterized by a lack of acid alpha-Glucosidase (GAA) activity, impairing lysosomal hepatic glucose metabolism. Enzyme deficiency leads to accumulation of lysosomal liver glycans and to progressive skeletal muscle weakness, reduced cardiac function, respiratory insufficiency and/or CNS injury in advanced stages of the disease. Gene mutations in the GAA gene lead to lower performance or to the production of mutated forms of the enzyme with altered stability and/or biological activity, ultimately leading to disease (see generally Hirschhorn R,1995, type II liver sugar storage disease: acid glucosidase (acid maltase) deficiency, metabolic and molecular basis of genetic disease, scriver et al, eds., mcGraw-Hill, new York, 7 th edition, pages 2443-2464). Three identified clinical forms of pompe disease (infants, adolescents and adults) are associated with residual α -glucosidase activity levels (user AJ et al, 1995, type ii hepatism (acid maltase deficiency), muscle & Nerve supply 3, S61-S69). Infant pompe disease (type I or a) is the most common and severe and is characterized by failure to thrive, generalized hypotonic, cardiac hypertrophy and heart-lung failure during the second year of life. Adolescent pompe (type II or B) is moderate in severity and is characterized by prominent muscle symptoms without cardiac hypertrophy. Adolescent poincare individuals typically die by the age of 20 years due to respiratory failure. Adult pompe disease (type III or C) is usually manifested as a slowly progressive myopathy in adolescent period or as late as the sixth decade (Felicia K J et al, 1995, clinical changes in adult acid maltase deficiency: report and literature review of affected SIB, pharmaceutical 74, 131-135). In the context of pompe disease, α -glucosidase has been shown to be extensively modified after translation by glycosylation, phosphorylation and proteolytic processing. The conversion of the 110 kilodaltons (kDa) precursor to the 76 and 70kDa mature form by proteolytic cleavage in lysosomes is required for optimal hepatase catalysis. As used herein, the term "pompe disease" refers to all types of pompe disease. The formulations and dosing regimens disclosed in the present application may be used to treat, for example, type I, type II or type III pompe disease.

As used herein, "significant" refers to statistical significance. The term refers to statistical evidence of differences between the two treatment groups. Which is defined as the probability of making a decision to reject a null hypothesis when the null hypothesis is actually true. Decisions are typically derived from a suitable statistical analysis for comparison using p-values < 0.05. See, e.g., example 9.

The "individual" or "patient" is preferably a human, and other mammals and non-human animals suffering from conditions involving accumulation of liver glycans can also be treated. The individual may be a fetus, neonate, child, adolescent or adult suffering from pompe disease or other liver sugar storage or accumulation conditions. One example of a treated individual is an individual (fetal, neonatal, pediatric, adolescent or adult human) with GSD-II (e.g., infant GSD-II, adolescent GSD-II or adult onset GSD-II). The individual may have residual GAA activity or no measurable activity. For example, an individual with GSD-II may have less than about 1% of normal GAA activity (infant GSD-II), about 1-10% of normal GAA activity (adolescent GSD-II), or about 10-40% of normal GAA activity (adult GSD-II). In some embodiments, the individual or patient is a "patient who has undergone ERT" or "ERT shift," referring to a pompe patient who has previously received enzyme replacement therapy. In some embodiments, a patient who has "undergone ERT" or "ERT conversion" is a pompe patient who has received or is currently receiving an arabinosidase a for greater than or equal to 24 months. In some embodiments, the individual or patient is a "not experienced ERT" patient, meaning a pompe patient who has not previously received enzyme replacement therapy. In certain embodiments, the individual or patient is ambulatory (e.g., an ambulatory ERT conversion patient or an ambulatory non-ERT patient). In certain embodiments, the individual or patient is non-ambulatory (e.g., an non-ambulatory ERT conversion patient). The ambulatory or non-ambulatory state may be determined by a six minute walk test (6 MWT). In some embodiments, the ambulatory patient is a pompe patient capable of walking at least 200 meters in a 6 MWT. In some embodiments, the non-ambulatory patient is a pompe patient who is unable to walk or sit on a wheelchair without assistance.

The term "treatment" as used herein refers to ameliorating one or more symptoms associated with a disease, delaying the onset of one or more symptoms of a disease, and/or reducing the severity or frequency of one or more symptoms of a disease. For example, treatment may refer to improving cardiac status (e.g., increasing end diastole and/or end systole volume, or alleviating or ameliorating progressive cardiomyopathy typically found in GSD-II) or pulmonary function (e.g., increasing crying lung capacity beyond baseline capacity and/or oxygen unsaturation normalized during crying); improving neurological development and/or motor skills (e.g., increasing AIMS score); a reduction in hepatic glucose content in the tissues of individuals infected with the disease; or any combination of these effects. In a preferred embodiment, the treatment comprises improving cardiac status, particularly reducing GSD-II related myopathy.

As used herein, the terms "improve," "increase," and "decrease" indicate a value relative to a baseline measurement, such as a measurement in the same individual prior to starting the treatment described herein or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein or a measurement after a control treatment, or a corresponding value from a control treatment. The control subjects are subjects suffering from the same form of GSD-II as the treated subjects (infant, adolescent or adult onset) that are about the same age as the treated subjects (to ensure that the treated subjects are comparable to the disease stage of the control subjects). In some embodiments, the control treatment comprises administering an arabinosidase α and a placebo for pharmacological chaperones (see example 9).

As used herein, the terms "about" and "approximately" shall generally mean an acceptable degree of error in the measured quantity in view of the nature or accuracy of the measurement. For example, the degree of error may be indicated by the number of significant figures provided for the measurement, as understood in the art, and include, but are not limited to, ±1 variations in the most accurate significant figures reported for the measurement. Typical exemplary error levels are within 20 percent (%), preferably within 10%, and more preferably within 5% of the specified value or range of values. Unless otherwise indicated, the numerical quantities specified herein are approximations, by the use of the antecedent "about" or "approximately" when not expressly stated.

All references, papers, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention and reference of any references, articles, publications, patents, patent publications, and patent applications cited herein are not, and should not be taken as, an acknowledgement or any form of suggestion that they form part of the common general knowledge in any country of the world as valid.

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

Recombinant human acid alpha-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.

TABLE 1 nucleotide sequence and protein sequence

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In some embodiments, the rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO:1, as described in U.S. Pat. No. 8,592,362, and has GenBank accession number AHE24104.1 (GI: 568760974). In some embodiments, the rhGAA has the GAA amino acid sequence as encoded in SEQ ID NO. 2 and the mRNA sequence has GenBank accession number Y00839.1. In some embodiments, the rhGAA has the GAA amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the 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.

In some embodiments, the rhGAA initially appears to have 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 the rhGAA undergoes intracellular processing with removal of a portion of the amino acids (e.g., the first 56 amino acids). Thus, the amino acid sequence of the rhGAA secreted by the host cell is shorter than the rhGAA originally expressed in the cell. In some embodiments, the shorter protein has the amino acid sequence set forth in SEQ ID NO. 5, which differs from SEQ ID NO. 1 only in that 56 amino acids before the signal peptide and the precursor peptide have been removed, thus producing a protein having 896 amino acids. In some embodiments, the shorter protein has the amino acid sequence set forth in SEQ ID NO. 6, which differs from SEQ ID NO. 4 only in that 56 amino acids before the signal peptide and the precursor peptide have been removed, thus producing a protein having 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 described by SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO: 6. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid alpha-glucosidase molecules having different amino acid lengths.

In some embodiments, the rhGAA comprises an amino acid sequence that is 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 the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, madison, wis.) and may be used, for example, in preset settings. For example, polypeptides having at least 90%, 95%, 98%, or 99% identity, and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides are contemplated. Unless indicated otherwise, the similarity score will be based on the use of BLOSUM 62. When BLASTP is used, the percent similarity is based on BLASTP positive scores and the percent sequence identity is based on BLASTP identity scores. BLASTP "identity" shows the number and portion of total residues in the same high scoring sequence; and BLASTP "positive numbers" show the number and portions of residues with positive values and similar to each other. Amino acid sequences having these or any moderate degree of identity or similarity to the amino acid sequences disclosed herein are encompassed by the present invention. The polynucleotide sequence of a similar polypeptide is deduced using the genetic code and can be obtained by conventional means, in particular by reverse translation of its amino acid sequence using the genetic code.

In some embodiments, the rhGAA undergoes post-translational and/or chemical modification at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, the N-terminal glutamine in SEQ ID NO. 6 can be further modified to form a pyroglutamate. As another example, an asparagine residue may undergo deamination to aspartic acid. As yet another example, an aspartic acid residue may undergo isomerization to iso-aspartic acid. As another example, unpaired cysteine residues in a protein may form disulfide bonds with free glutathione and/or cysteine. Thus, in some embodiments, the enzyme initially appears to have 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 this disclosure.

N-linked glycosylation of rhGAA

There are seven potential N-linked glycosylation sites on a single rhGAA molecule. These potential glycosylation sites are 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 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. Typically, the Asn-X-Ser or Asn-X-Thr sequence in the protein amino acid sequence indicates a potential glycosylation site, but X may not be His or Pro.

The rhGAA molecules described herein can have an average of 1, 2, 3, or 4 mannose-6-phosphate (M6P) groups on their N-glycans. For example, only one N-glycan on an rhGAA molecule may carry M6P (mono-phosphorylated or mono-M6P), a single N-glycan may have two M6P groups (di-phosphorylated or di-M6P), or two different N-glycans on the same rhGAA molecule may each have a single M6P group. In some embodiments, the rhGAA molecules described herein have on average 3-4 moles of m6p groups on their N-polysaccharide per mole of rhGAA. The recombinant human acid alpha-glucosidase molecule may also have an N-glycan that does not contain an M6P group. In another embodiment, the rhGAA comprises on average greater than 2.5mol m6p per mole of rhGAA and greater than 4mol sialic acid per mole of rhGAA. In some embodiments, the rhGAA comprises on average about 3 to 3.5mol m6p per mole of rhGAA. In some embodiments, the rhGAA comprises about 4 to 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 the total N-glycans on the rhGAA can be in the form of single M6P N-glycans, e.g., about 6.25% of the total N-glycans can carry single M6P groups, and at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total N-glycans on the rhGAA are in the form of double M6P N-glycans on average, and less than 25% of the total rhGAA on average is free of phosphorylated N-glycans that bind to CIMPR. In some embodiments, an average of about 10% to about 14% of the total N-glycans on the rhGAA are monophosphorylated. In some embodiments, an average of about 7% to about 25% of the total N-glycans on the rhGAA are bisphosphorylated. In some embodiments, the rhGAA comprises about 1.3 moles of dual M6P per mole of rhGAA on average.

The rhGAA described herein can average from 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. The rhGAA can be fractionated to provide rhGAA formulations having different average numbers of mono-containing M6P or di-M6P-containing N-glycans, thus permitting further custom targeting of the rhGAA to lysosomes in the target tissue by selection of specific moieties or by selective combination of different moieties.

In some embodiments, up to 60% of the N-glycans on the rhGAA can be fully sialylated, e.g., up to 10%, 20%, 30%, 40%, 50% or 60% of the N-glycans can be fully sialylated. In some embodiments, no more than 50% of the N-glycans on the rhGAA are fully sialylated. In some embodiments, 4% to 20% of the total N-glycans are fully sialylated. In other embodiments, no more than 5%, 10%, 20% or 30% of the N-glycans on the rhGAA carry sialic acid and terminal galactose residues (Gal). This range includes all intermediate values and subranges, e.g., 7% to 30% of the total N-glycans on rhGAA can carry sialic acid and terminal galactose. In 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 do not contain sialic acid. This range includes all intermediate values and subranges, e.g., 8% to 19% of the total N-glycans on the rhGAA in the composition can have only terminal galactose and no sialic acid.

In some embodiments, 40% to 60%, 45% to 60%, 50% to 60%, or 55% to 60% of the total N-glycans on the rhGAA are complex N-glycans; or no more than 1%, 2%, 3%, 4%, 5%, 6%, or 7% of the total N-glycans on the rhGAA are heterozygous N-glycans; no more than 5%, 10%, 15%, 20% or 25% of the high mannose type N-glycans on rhGAA are non-phosphorylated; at least 5% or 10% of the high mannose type N-glycans on rhGAA are monophosphorylated; and/or at least 1% or 2% of the high mannose type N-glycans on the rhGAA are bisphosphorylated. These values include all intermediate values and subranges. The rhGAA may satisfy one or more of the content ranges described above.

In some embodiments, the rhGAA may have an average of 2.0 to 8.0 moles of sialic acid residues per mole of rhGAA. This range includes all intermediate values and subranges thereof, 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 an N-glycan unit bearing a sialic acid residue prevents the inefficient clearance of rhGAA by the asialoglycoprotein receptor.

In one or more embodiments, the rhGAA has a certain N-glycosylation profile at certain potential N-glycosylation sites. In some embodiments, the rhGAA has seven potential N-glycosylation sites. In some embodiments, at least 20% of the 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 the rhgaa may be phosphorylated at the first potential N-glycosylation site. This phosphorylation may be the result of single M6P and/or double 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 carries a single M6P unit 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 carries a double M6P unit at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 1.4 moles of M6P (single M6P and double M6P) per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of at least about 0.5mol of bis M6P per mol of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.25 moles of mono M6P per mole of rhGAA at the first potential N-glycosylation site. In some embodiments, the rhGAA comprises 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 fig. 6A and an N-glycosylation pattern as depicted in fig. 6B. In at least one embodiment the rhGAA comprises a first potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19B or fig. 20B.

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 single M6P and/or double 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 carries a single M6P unit at the second 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 has a double M6P unit at the second N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.5mol M6P (mono-M6P and di-M6P) per mole of rhGAA at the second potential N-glycosylation site. In some embodiments, the rhGAA comprises 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, the rhGAA comprises a second potential N-glycosylation site occupancy as depicted in fig. 6A and an N-glycosylation pattern as depicted in fig. 6C. In at least one embodiment, the rhGAA comprises a second potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19C or fig. 20B.

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, di-, tri-and tetra-antennary complex N-glycans and hybrid N-glycans as the major 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 fig. 6A and an N-glycosylation pattern as depicted in fig. 6D. In at least one embodiment, the rhGAA comprises a third potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19D or fig. 20B.

In some embodiments, at least 20% of the rhGAA is phosphorylated at a fourth potential N-glycosylation site (e.g., N414 of SEQ ID NO:6 and N470 of 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 single M6P and/or double 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 carries a single 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 carries a double 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 comprises an average of about 1.4 moles of M6P (single M6P and double M6P) per mole of rhGAA at the fourth potential N-glycosylation site. In some embodiments, the rhGAA comprises 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 comprises 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, the rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in fig. 6A and an N-glycosylation pattern as depicted in fig. 6E. In at least one embodiment, the rhGAA comprises a fourth potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19E or fig. 20B.

In some embodiments, at least 5% of the rhGAA is phosphorylated at a 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 may have a fucosylated double antenna complex N-glycan as the major 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 fig. 6A and an N-glycosylation pattern as depicted in fig. 6F. In at least one embodiment, the rhGAA comprises a fifth potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19F or fig. 20B.

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 two-antennary, three-antennary, and four-antennary complex N-glycans as the primary 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, the rhGAA comprises 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, the rhGAA comprises 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, the rhGAA comprises an average of at least 0.05 moles of polysaccharide material per mole of rhGAA at the sixth potential N-glycosylation site, which has a poly-N-acetyl-D-lactosamine (poly-LacNAc) residue. In some embodiments, greater than 10% of the rhGAA comprises a polysaccharide carrying a polylactic nac residue at the sixth potential N-glycosylation site. In at least one embodiment, the rhGAA comprises a sixth potential N-glycosylation site occupancy as depicted in fig. 6A and an N-glycosylation pattern as depicted in fig. 6G. In at least one embodiment, the rhGAA comprises a sixth potential N-glycosylation site occupancy as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19G or fig. 20B.

In some embodiments, at least 5% of the rhGAA is phosphorylated at a 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-glycans 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 comprises an average of at least 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of at least 0.8 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of about 0.86 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of at least 0.3 moles of polysaccharide material carrying a polylactic nac residue per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, nearly half of the rhGAA comprises an N-glycan carrying a polylactic nac residue at the seventh potential N-glycosylation site. In at least one embodiment, all of the N-glycans identified at the seventh potential N-glycosylation site are complex N-glycans. In at least one embodiment, the rhGAA comprises a seventh potential N-glycosylation site occupancy as depicted in fig. 6A or as depicted in fig. 19A and an N-glycosylation pattern as depicted in fig. 19H or fig. 20B.

In some embodiments, the rhGAA comprises an average of about 4 to about 7.3 moles of sialic acid per mole of rhGAA 3-4 moles of M6P residues and per mole of rhGAA. In some embodiments, the rhGAA further comprises an average of at least about 0.5 mole of bim 6P per mole of rhGAA at the first potential N-glycosylation site, about 0.4 to about 0.6 mole of monom 6P per mole of rhGAA at the second potential N-glycosylation site, about 0.9 to about 1.2 mole of sialic acid per mole of rhGAA at the third potential N-glycosylation site, about 0.4 to about 0.6 mole of bim 6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.3 to about 0.4 mole of monom 6P per mole of rhGAA at the fourth potential N-glycosylation site, about 0.8 to about 0.9 mole of sialic acid per mole of rhGAA at the fifth potential N-glycosylation site, and about 1.5 to about 2.5 moles of sialic acid per mole of rhGAA at the sixth potential N-glycosylation site. In some embodiments, the rhGAA further comprises an average of at least about 0.5 moles of sialic acid per mole of rhGAA at the seventh potential N-glycosylation site. In some embodiments, the rhGAA comprises an average of 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 an average of 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 site occupancy and N-glycosylation characteristics as depicted in fig. 6A-6H. In at least one embodiment, the rhGAA comprises seven potential N-glycosylation site occupancy and N-glycosylation characteristics as depicted in fig. 19A-19H and 20A-20B.

Methods of preparing rhGAA are disclosed in U.S. provisional patent application No. 62/057,842, filed on 9/30 of 2014, the entire contents of which are incorporated herein by reference.

Once in the lysosome, rhGAA can enzymatically degrade accumulated hepatic glycans. However, conventional rhGAA products have low total content of N-glycans carrying both single and double M6P and thus target myocytes poorly, making rhGAA poorly delivered to lysosomes. Most of the rhGAA molecules in these conventional products do not have phosphorylated N-glycans, and thus lack affinity for CIMPR. Non-phosphorylated high mannose N-glycans can also be cleared by mannose receptors, which results in inefficient clearing of ERT (fig. 2B). In contrast, as depicted in fig. 2A, the rhGAA described herein may contain higher amounts of N-glycans carrying single M6P and double M6P, such that the rhGAA is efficiently absorbed into specific tissues such as muscle.

Production and purification of N-linked glycosylated rhGAA

As described in us 10,961,522, which is incorporated herein by reference in its entirety, cells, such as Chinese Hamster Ovary (CHO) cells, can be used to produce the rhGAA described therein. The expression of high M6PrhGAA in CHO cells is superior to the post-translationally modified rhGAA glycoforms, at least in part because only the former can be converted to the rhGAA form with optimal glycohydrolysis by polysaccharide degradation, thus enhancing therapeutic efficacy.

In some embodiments, the rhGAA is preferably produced from one or more CHO cell lines transformed with a DNA construct encoding the rhGAA described herein. Such CHO cell lines may contain multiple copies of a gene, such as 5, 10, 15, or 20 or more copies of a GAA-encoding polynucleotide. DNA constructs that exhibit acid alpha-glucosidase or other variant acid alpha-glucosidase amino acid sequences, such as those amino acid sequences 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 can select alternative vectors suitable for transforming CHO cells for the production of such DNA constructs.

Methods of making such CHO cell lines are described in us 10,961,522, which is incorporated by reference herein in its entirety. Briefly, these methods involve transforming CHO cells with DNA encoding GAA or GAA variants, selecting CHO cells that stably integrate GAA-encoding DNA into their chromosomes and stably express GAA, and selecting CHO cells that express GAA with high levels of N-glycans carrying mono-M6P or di-M6P, and optionally selecting CHO cells with high sialic acid content of N-glycans and/or with low non-phosphorylated high mannose content of N-glycans. The CHO cell line selected can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell line and recovering the composition from a culture of CHO cells. In some embodiments, rhGAA produced from selected CHO cell lines contains a higher content of N-glycans carrying single M6P or double M6P targeting CIMPR. In some embodiments, the rhGAA produced as described herein has a low content 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 CHO cell line selected encompasses a sub-culture or derivative of such CHO cell culture. In some embodiments, the rhGAA produced from the selected CHO cell line is designated ATB200.

The rhGAA produced as described herein can be purified by the following methods described in U.S.10,227,577 and U.S. provisional application No. 62/506,569, both of which are incorporated herein by reference in their entirety. An exemplary method for producing, capturing and purifying rhGAA produced by CHO cell lines is shown in fig. 3.

Briefly, bioreactor 601 contains a culture of cells, such as CHO cells, that express and secrete rhGAA into the surrounding liquid medium. Bioreactor 601 may be any suitable bioreactor for culturing cells, such as a perfusion, batch or fed-batch bioreactor. After a sufficient period of time for the cells to produce rhGAA, the medium is removed from the bioreactor. Such media removal may be continuous for perfusion bioreactors or batch for batch or fed-batch reactors. The culture medium may be filtered by a filtration system 603 to remove cells. The filtration system 603 may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, tangential Flow Filtration (TFF) system, and/or a centrifugal filtration system. In various embodiments, the filtration system utilizes a filter having a pore size between about 10 nanometers and about 2 microns.

After filtration, the filtrate is loaded onto protein capture system 605. The protein capture system 605 may include one or more chromatography columns. If more than one chromatography column is used, the columns may be placed in series so that the next column may begin loading after loading the first column. Alternatively, the medium removal process may be stopped during the time of switching columns.

In various embodiments, the protein capture system 605 includes one or more Anion Exchange (AEX) columns for directly capturing rhGAA, particularly rhGAA with high M6P content. By varying the pH and/or salt content in the column, the rhGAA captured by the protein capture system 605 is eluted from the column. Exemplary conditions for the AEX column are provided in table 2.

TABLE 2 exemplary conditions for AEX column

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

TABLE 3 exemplary conditions for IMAC columns

After loading the rhGAA onto the second chromatography system 609, the recombinant protein was eluted from the column. The eluted rhGAA may undergo a virus killing step 611. Like virus killing 607, virus killing 611 may include low pH killing, detergent killing, or one or more of other techniques known in the art. In some embodiments, only one of the virus kills 607 or 611 is used, or the virus killing is performed at the same stage in the purification process.

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 to the third chromatography system 613. In various embodiments, the third chromatography system 613 comprises one or more cation exchange Chromatography (CEX) columns and/or Size Exclusion Chromatography (SEC) columns for further removal of impurities. The rhGAA product is then eluted from the third chromatography system 613. Exemplary conditions for the CEX column are provided in table 4 below.

Table 4 exemplary conditions of cex column

The rhGAA product may also undergo further processing. For example, another filtration system 615 may be used to remove viruses. In some embodiments, such filtration may utilize filters with pore sizes between 5 and 50 μm. Other product treatments may include a product conditioning step 617, wherein the recombinant protein product may be sterilized, filtered, concentrated, stored, and/or have additional components for addition for use in a final product formulation.

As used herein, the term "ATB200" refers to rhGAA with higher content of N-glycans carrying single and double M6P, produced from the GA-ATB200 cell line and purified using the methods described herein.

V. pharmaceutical composition

In various embodiments, a pharmaceutical composition is provided that comprises the rhGAA described herein (alone or in combination with other therapeutic agents) and/or a pharmaceutically acceptable carrier.

In one or more embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable salt.

In some embodiments, the pharmaceutically acceptable salt used herein is a pharmaceutically acceptable acid addition salt. Pharmaceutically acceptable acid addition salts may include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, as well as organic acids including, but not limited to, the following: acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphoric acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphoric acid, hemisulfuric acid, caproic acid, formic acid, fumaric acid, 2-hydroxyethanesulfonic acid (hydroxyethanesulfonic acid), lactic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylene sulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, pectic 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, undecanoic acid, and the like.

In some embodiments, the pharmaceutically acceptable salt used herein is a pharmaceutically acceptable base addition salt. Pharmaceutically acceptable base addition salts may include, but are not limited to, ammonia or hydroxides, ammonium or carbonates or bicarbonates of metal cations such as sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of 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, arginine, histidine, caffeine, sea Zhuo An, choline, betaine, ethylenediamine, glucosamine, methylreduced glucosamine, theobromine, purine, piperinePiperidine, N-ethylpiperidine, tetramethylammonium compound, tetraethylammonium compound, pyridine, N-dimethylaniline, N-methylpiperidine, N-methyl +. >In, dicyclohexylamine, benzhydrylamine, N-benzhydrylamine, 1-Anfeamine, N' -benzhydrylamine, polyamineResins and the like.

In some embodiments, the rhGAA or pharmaceutically acceptable salt thereof may be formulated as a pharmaceutical composition suitable for intravenous administration. In some embodiments, the pharmaceutical composition is a sterile isotonic aqueous buffer solution. If desired, the composition may also include a co-solvent and a local anesthetic for reducing pain at the injection site. The components of the pharmaceutical composition may be supplied separately or mixed together in unit dosage form, e.g., in dry lyophilized powder or dry concentrate form in a hermetically sealed container (such as an ampoule or sachet) that indicates the amount of active agent. When the composition is administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. In some embodiments, the infusion may be performed at a hospital or clinic. In some embodiments, the infusion may occur outside of a hospital or clinical setting, such as at a residence of an individual. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

In some embodiments, the rhGAA or pharmaceutically acceptable salt thereof can be formulated for oral administration. Orally administrable compositions may be formulated in the following form: troches, capsules, beads, elixirs, solutions or suspensions, gels, syrups, mouthwashes or dry powders for reconstitution with water or other suitable carrier before use, optionally with flavoring and coloring agents, for immediate release, delayed release, modified release, sustained release, pulsed release or controlled release. Solid compositions such as lozenges, capsules, troches, tablets, pills, boluses, powders, pastes, granules, dragees, or pre-mixed formulations 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. Lozenges 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, dibasic calcium phosphate, magnesium stearate, stearic acid, glyceryl behenate, talc, silica, corn, potato or tapioca starch, sodium starch glycolate, sodium lauryl sulfate, sodium citrate, calcium carbonate, dicalcium phosphate, sodium glycine crosslinked carboxymethylcellulose, and complex silicates. The tablets may be overcoated by methods well known in the art.

In some embodiments, the pharmaceutical compositions described herein may be formulated according to U.S. 10,512,676 and U.S. provisional application No. 62/506,574, both of which are incorporated herein by reference in their entirety. For example, in some embodiments, the pH of the pharmaceutical compositions described herein is from about 5.0 to about 7.0 or from about 5.0 to about 6.0. In some embodiments, the pH is in the range of 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 the target pH by using pH adjusting agents (e.g., alkalizing and acidifying agents), such as sodium hydroxide and/or hydrochloric acid.

The pharmaceutical compositions described herein may comprise a buffer system, such as a citrate system, a phosphate system, and combinations thereof. The citrate and/or phosphate salt may be sodium citrate or sodium phosphate. Other salts include potassium and ammonium salts. In one or more embodiments, the buffer comprises citrate. In other embodiments, the buffer comprises sodium citrate (e.g., a mixture of sodium citrate dehydrate and citric acid monohydrate). In one or more embodiments, the buffer solution comprising citrate may comprise sodium citrate and citric acid. In some embodiments, both citrate and phosphate buffers are present.

In some embodiments, the pharmaceutical compositions described herein comprise at least one excipient. Excipients may act as tonicity agents, bulking agents and/or stabilizers. Tonicity agents are components that help ensure that the formulation has an osmotic pressure similar to or the same as human blood. An bulking agent is an ingredient that adds a substance to a formulation (e.g., lyophilization) and provides the cake with the proper structure. Stabilizers are compounds that prevent or minimize aggregate formation at hydrophobic air-water interface surfaces. An excipient may act as both a tonicity agent and a bulking agent. For example, mannitol may act as a tonicity agent and also provide the benefit of acting as a bulking agent.

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 tonicity agent is in the range of about 10mg/mL to about 50 mg/mL. In other embodiments, the total amount of tonicity agent is in the range of about 10, 11, 12, 13, 14 or 15mg/mL to about 16, 20, 25, 30, 35, 40, 45 or 50 mg/mL.

In some embodiments, the excipient comprises a stabilizer. In some embodiments, the stabilizing agent is a surfactant. In some embodiments, the stabilizer is polysorbate 80. In one or more embodiments, the total amount of stabilizer is in the range of about 0.1mg/mL to about 1.0mg/mL. In other embodiments, the total amount of stabilizer is in the range of about 0.1, 0.2, 0.3, 0.4, or 0.5mg/mL to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0mg/mL. In 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 1.0mg/mL.

In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB 200), (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 acidulant and/or an alkalizing agent.

In some embodiments, the pharmaceutical composition comprises (a) rhGAA (such as ATB 200) at a concentration of about 5-50mg/mL, about 5-30mg/mL, or about 15mg/mL, (b) sodium citrate buffer at a concentration of about 10-100mM or about 25mM, (c) mannitol at a concentration of about 10-50mg/mL or about 20mg/mL, (d) polysorbate 80 present at a concentration of about 0.1-1mg/mL, about 0.2-0.5mg/mL, or about 0.5mg/mL, and (e) water, and has a pH of about 6.0. In at least one embodiment the pharmaceutical composition comprises (a) 15mg/mL rhGAA (such as ATB 200), (b) 25mM sodium citrate buffer, (c) 20mg/mL mannitol, (d) 0.5mg/mL polysorbate 80, and (e) water, and has a pH of about 6.0. In some embodiments, the composition may further comprise an acidulant and/or an alkalizing agent.

In some embodiments, the pharmaceutical composition comprising rhGAA is diluted prior to administration to an individual in need thereof.

In some embodiments, the pharmaceutical compositions described herein comprise a chaperone. In some embodiments, the chaperone is meglumine or a pharmaceutically acceptable salt thereof. In another embodiment, the partner is durum or a pharmaceutically acceptable salt thereof.

In some embodiments, the rhGAA described herein is formulated in one pharmaceutical composition, while a partner such as meglumine is formulated in another pharmaceutical composition. In some embodiments, the pharmaceutical composition comprising meglumine is based on asThe formulation of (Actelion Pharmaceuticals) is commercially available.

In some embodiments, the pharmaceutical compositions described herein may be subjected to a lyophilization (freeze drying) process to provide a cake or powder. Thus, in some embodiments, the pharmaceutical compositions described herein relate to rhGAA compositions after lyophilization. The lyophilized mixture can comprise the rhGAA (e.g., ATB 200) described herein, 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 ingredients (e.g., other excipients) may be added to the lyophilized mixture. Vials of pharmaceutical compositions comprising the lyophilized formulations may be provided which may then be stored, transported, reconstituted and/or administered to a patient.

VI therapeutic methods

A. Treatment of disease

Another aspect of the invention relates to a method of treating a disease or disorder associated with a glycostorage disorder by administering the rhGAA or pharmaceutical composition described herein. In some embodiments, the disease is pompe disease (also known as Acid Maltase Deficiency (AMD) and type II liver sugar storage disease (GSD II)). In some embodiments, the rhGAA is ATB200. In some embodiments, the pharmaceutical composition comprises ATB200. Also provided herein is the use of rhGAA or ATB200 for the treatment of pompe disease.

In some embodiments, the individual treated by the methods disclosed herein is a patient who has experienced ERT. In some embodiments, the individual treated by the methods disclosed herein is an ERT untreated patient.

The rhGAA or pharmaceutical compositions described herein are administered by a suitable route. In one embodiment, the rhGAA or pharmaceutical composition is administered intravenously. 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., intramuscular) or the nervous system (e.g., direct injection into the brain; within the brain; intrathecal). In some embodiments, the rhGAA or pharmaceutical composition is administered orally. If desired, more than one route may be used simultaneously.

In some embodiments, the therapeutic effect of the rhGAA or pharmaceutical compositions described herein can be assessed based on one or more of the following criteria: (1) Cardiac status (e.g., increasing end diastole and/or end systole volume, or reducing, ameliorating, or preventing progressive cardiomyopathy typically found in GSD-II), (2) pulmonary function (e.g., increasing crying vital capacity beyond baseline capacity, and/or oxygen unsaturation normalized during crying); (3) Neural development and/or motor skills (e.g., increasing AIMS score), and (4) reduced liver glucose levels in tissues of individuals infected with the disease.

In some embodiments, after administration of one or more doses of the rhGAA or pharmaceutical composition described herein, the myocardial condition of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) compared to the myocardial condition of the subject treated with the vehicle or the subject prior to treatment. The individual's myocardial state may be assessed by measuring end diastole and/or end systole volumes and/or by clinically assessing cardiomyopathy. In some embodiments, the lung function of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200 as compared to the lung function of the subject treated with the carrier or prior to treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period in between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves lung function in an individual after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period therebetween) of administration.

In some embodiments, after administration of one or more doses of the rhGAA or pharmaceutical composition described herein, the pulmonary function of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) compared to the pulmonary function of the subject treated with the vector or the subject prior to treatment. The lung function of an individual can be assessed by normalization of crying lung capacity relative to a baseline capacity and/or oxygen saturation during crying. In some embodiments, the lung function of the subject is improved by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200 as compared to the lung function of the subject treated with the carrier or prior to treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period in between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves lung function in an individual after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period therebetween) of administration.

In some embodiments, the subject's neurological and/or motor skills are improved by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more doses of the rhGAA or pharmaceutical composition described herein, as compared to the subject treated with the vector or the subject's neurological and/or motor skills prior to treatment. The neurological development and/or motor skills of an individual may be assessed by determining an AIMS score. AIMS is a 12-item anchoring scale for clinical administration and scoring (see Rush JA jr., handbook of Psychiatric Measures, american society of psychosis, 2000, 166-168). Items 1-10 were rated on a 5 point anchor scale. Items 1 to 4 assess orofacial movement. Items 5 to 7 deal with limb and torso movement difficulties. Items 8-10 deal with overall severity as determined by the censor, as well as patient motor awareness and pain associated therewith. Items 11 to 12 are what are/are non-problems with respect to teeth and/or dentures (these problems may cause misidentification diagnostics of movement difficulties). In some embodiments, the subject's neurological and/or motor skills are improved by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200, as compared to the subject treated with the carrier or prior to treatment. In certain embodiments, the improvement is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period in between) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 improves neurogenesis and/or motor skills in an individual after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period in between) of administration.

In some embodiments, the liver sugar content of a tissue of an individual is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage in between) after administration of one or more doses of the rhGAA or pharmaceutical composition described herein, as compared to the liver sugar content of the individual treated with the carrier or of the individual prior to treatment. In some embodiments, the tissue is a muscle, such as quadriceps, triceps, and gastrocnemius. The liver sugar content of the tissue may be analyzed using methods known in the art. Determination of liver saccharide content is well known based on amyloglucosidase digestion and is described in the disclosure, such as: amalfitano et al (1999), "systemic correction of muscle disorder type II liver sugar storage disease following liver targeting of modified adenovirus vectors encoding human acid alpha-glucosidase", "Proc. Natl. Acad. Sci. USA, 96:8861-8866. In some embodiments, the muscle liver glucose content of the subject is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200, as compared to the muscle liver glucose content of the subject treated with the carrier or of the subject prior to treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period therebetween) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces liver saccharide content in a muscle of an individual after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period therebetween) of administration.

B. Biomarkers and their use

Biomarkers of liver glucose accumulation in individuals, such as urine hexose tetraose (Hex 4), can be used to assess and compare the therapeutic effect of enzyme replacement therapies in individuals with pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on liver saccharide accumulation is assessed by measuring the urine Hex4 content of the individual.

Biomarkers of muscle injury or damage, such as Creatine Kinase (CK), alanine Aminotransferase (ALT), and aspartate Aminotransferase (AST), can be used to evaluate and compare the therapeutic effect of enzyme replacement therapies in individuals with pompe disease. In some embodiments, the therapeutic effect of rhGAA or a pharmaceutical composition comprising rhGAA on muscle injury is assessed by measuring CK, ALT, and/or AST levels in the individual. In at least one embodiment, the effect of rhGAA or a pharmaceutical composition comprising rhGAA on the treatment of muscle injury is assessed by measuring CK content in the individual.

Biomarkers such as LAMP-1, LC3 and plasma membrane repair proteins can also be used to evaluate and compare the therapeutic effects of rhGAA or pharmaceutical compositions described herein. In pompe disease, GAA's inability to hydrolyze lysosomal liver sugar causes abnormal accumulation of large lysosomes filled with liver sugar in some tissues. Studies in a mouse model of (Raben et al, JBC 273:19086-19092,1998.) pompe disease have shown that amplified lysosomes in skeletal muscle do not adequately account for the decrease in mechanical potency and that the presence of large inclusions containing degraded myofibrils (i.e. autophagic accumulation) contributes to the impairment of muscle function. (Raben et al, human molecular Gene 17:3897-3908,2008). The report also shows that impaired autophagic flux is associated with poor outcome in treatment of pompe patients. (Nascimbini et al, neuropathology and applied neurobiology doi:10.1111/nan.12214,2015; fukuda et al, molecular therapy (Mol Ther) 14:831-839,2006). In addition, delayed pompe disease commonly occurs in unclassified distant limb muscular dystrophy (LGMD) (Preisler et al, mol Genet meta 110:287-289,2013), which is a group of genetic heterogeneous neuromuscular diseases of more than 30 genetically defined subtypes of varying severity. IHC assay discloses the presence of plasma membrane repair proteins in skeletal muscle fibers of Gaa KO mice.

Various known methods can be used to measure the gene expression and/or protein content of such biomarkers. For example, a sample, such as a biopsy of tissue (particularly muscle), from an individual treated with the rhGAA or pharmaceutical composition described herein may be obtained. In some embodiments, the sample is a biopsy of an individual. In some embodiments, the muscle is selected from the group consisting of quadriceps, triceps, and gastrocnemius. Samples obtained from an individual may be stained with one or more antibodies or other detection agents that detect such biomarkers or are identified and quantified by mass spectrometry. The sample may also or alternatively be treated to detect the presence of a nucleic acid (such as mRNA) encoding a biomarker via, for example, RT-qPCR methods.

In some embodiments, the gene expression and/or protein content of one or more biomarkers is measured in muscle biopsies obtained from an individual before and after treatment with the rhGAA or pharmaceutical compositions described herein. In some embodiments, the gene expression and/or protein content of one or more biomarkers is measured in a muscle biopsy obtained from a vector treated individual. In some embodiments, the gene expression level and/or protein content of one or more biomarkers is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more doses of the rhGAA or pharmaceutical composition described herein, as compared to the gene expression level and/or protein content of the subject treated with the vector or of the subject prior to treatment. In some embodiments, the gene expression level and/or protein content of one or more biomarkers is reduced by 10%, 20%, 30%, 40% or 50% (or any percentage therebetween) after administration of one or more doses of ATB200 or a pharmaceutical composition comprising ATB200, as compared to the gene expression level and/or protein content of one or more biomarkers of an individual treated with the vector or prior to treatment. In certain embodiments, the reduction is achieved after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period therebetween) of administration. In certain embodiments, ATB200 or a pharmaceutical composition comprising ATB200 reduces gene expression and/or protein content of one or more biomarkers after 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or longer (or any period of time therebetween) of administration.

Dosage of rhGAA

The pharmaceutical formulation or reconstituted composition is administered in a therapeutically effective amount (e.g., a dose sufficient to treat the disease when administered at regular intervals, 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 for treating the disease can depend on the nature and extent of the effect of the disease and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may be employed as appropriate to assist in identifying optimal dosage ranges. In at least one embodiment, the rhGAA or pharmaceutical composition comprising rhGAA described herein is administered at a dose of about 1mg/kg to about 100mg/kg, such as about 5mg/kg to about 30mg/kg, typically about 5mg/kg to about 20 mg/kg. In at least one embodiment, the rhGAA or pharmaceutical composition described herein is administered at a dose of about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg, or about 100 mg/kg. In some embodiments, rhGAA is administered at a dose of 5mg/kg, 10mg/kg, 20mg/kg, 50mg/kg, 75mg/kg, or 100 mg/kg. In at least one embodiment, the rhGAA or pharmaceutical composition is administered at a dose of about 20 mg/kg. In some embodiments, the rhGAA or pharmaceutical composition is administered simultaneously or sequentially with the pharmacological chaperone. In some embodiments, the pharmacological chaperone is miglutide. In at least one embodiment, the meglumine is administered at an oral dose of about 260 mg. In at least one embodiment, the meglumine is administered at an oral dose of about 195 mg. The effective dosage for a particular individual may vary (e.g., increase or decrease) over time depending on the individual's needs. For example, the amount of rhGAA and/or meglumine may be adjusted at the time of a physical disease or stress, or when an acid-fast α -glucosidase antibody becomes present or increased, or at the time of worsening of the symptoms of the disease.

In some embodiments, a therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is lower than conventional rhGAA products. For example, if the therapeutically effective dose of a conventional rhGAA product is 20mg/kg, the dose of rhGAA or pharmaceutical composition described herein required to produce the same or better therapeutic effect as the conventional rhGAA product may be less than 20mg/kg. The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., myocardial status, liver glucose content, or biomarker performance). In some embodiments, a therapeutically effective dose of the rhGAA or pharmaceutical composition described herein is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower than a therapeutically effective dose of a conventional rhGAA product.

In some embodiments, the therapeutic effect of the rhGAA or pharmaceutical compositions described herein comprises improved motor function, improved muscle strength (upper, lower, or systemic), improved lung function, reduced fatigue, reduced levels of at least one muscle damage biomarker, reduced levels of at least one liver glucose accumulation biomarker, or a combination thereof. In some embodiments, the therapeutic effect of the rhGAA or pharmaceutical composition described herein comprises reversal of lysosomal pathology in muscle fibers, faster and/or more effective reduction of liver sugar content in muscle fibers, increase in six-minute walk test distance, decrease in standing-walk timing test time, decrease in four-layer climb test time, decrease in ten-meter walk test time, decrease in gait-stair-golgi-chair, increase in upper limb strength, improvement in shoulder adduction, improvement in shoulder abduction, improvement in elbow flexion, improvement in elbow extension, improvement in upper body strength, improvement in lower body strength, improvement in systemic strength, improvement in erectile (sitting) effort lung capacity, improvement in maximum expiratory pressure, improvement in maximum inspiratory pressure, decrease in fatigue severity scale score, decrease in urine hexose tetraose content, decrease in creatine kinase content, decrease in alanine aminotransferase content, decrease in aspartate aminotransferase content, or any combination thereof.

In some embodiments, the rhGAA or pharmaceutical compositions described herein achieve the desired therapeutic effect faster than conventional rhGAA products when administered at the same dose. The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., myocardial status, liver glucose content, or biomarker performance). For example, if a single dose of a conventional rhGAA product reduces the amount of liver saccharide in the tissue of the treated individual by 10% in one week, the same degree of reduction can be achieved in less than one week when the same dose of the rhGAA or pharmaceutical composition described herein is administered. In some embodiments, the rhGAA or pharmaceutical compositions described herein achieve the desired therapeutic effect at least about 1.25, 1.5, 1.75, 2.0, 3.0 or faster than conventional rhGAA products when administered at the same dose.

In some embodiments, a therapeutically effective amount of rhGAA (or a composition or medicament comprising rhGAA) is administered more than once. In some embodiments, the rhGAA or pharmaceutical compositions described herein are administered at regular intervals, and continue, depending on the nature and extent of the effect of the disease. Administration at "regular time intervals" as used herein indicates periodic administration of a therapeutically effective amount (as distinguished from a single dose). The time interval may be determined by standard clinical techniques. In certain embodiments, the rhGAA is administered monthly, weekly, twice weekly, or daily. In some embodiments, the rhGAA is administered intravenously twice weekly, once weekly, or every other week. The individual administration intervals need not be fixed intervals, but may vary over time depending on the individual needs. For example, the time interval between doses may decrease when a physical disorder or stress, or when an anti-rhGAA antibody becomes present or increasing, or when a symptom of the disorder worsens.

In some embodiments, the rhGAA or pharmaceutical composition as described herein is administered less frequently than a conventional rhGAA product when used at the same dose and still is capable of producing the same or better therapeutic effect as the conventional rhGAA product. For example, if a conventional rhGAA product is administered at 20mg/kg weekly, the rhGAA or pharmaceutical composition as described herein may produce the same or better therapeutic effect than a conventional rhGAA product when administered at 20mg/kg, although the rhGAA or pharmaceutical composition is administered less frequently (e.g., once every two weeks or once a month). The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., myocardial status, liver glucose content, or biomarker performance). In some embodiments, the time interval between two doses of the rhGAA or pharmaceutical composition described herein is longer than the time interval between conventional rhGAA products. In some embodiments, the time interval between two doses of rhGAA or pharmaceutical composition is at least about 1.25, 1.5, 1.75, 2.0, 3.0 or more longer than the time interval of a conventional rhGAA product.

In some embodiments, the rhGAA or pharmaceutical compositions described herein provide therapeutic effects to a degree that is superior to the degree provided by conventional rhGAA products under the same treatment conditions (e.g., the same dose administered at the same time interval). The therapeutic effect may be assessed based on one or more criteria discussed above (e.g., myocardial status, liver glucose content, or biomarker performance). For example, administration of rhGAA or pharmaceutical composition at 20mg/kg per week may reduce the amount of liver saccharide in the tissue of the treated individual to a higher extent when compared to conventional rhGAA product administered at 20mg/kg per week. In some embodiments, the rhGAA or pharmaceutical compositions described herein provide a therapeutic effect of at least about 1.25, 1.5, 1.75, 2.0, 3.0 or greater than the therapeutic effect of a conventional rhGAA product when administered under the same treatment conditions.

D. Two-component therapy

In one or more embodiments, the rhGAA or pharmaceutical composition comprising the rhGAA described herein is administered simultaneously or sequentially with a pharmacological chaperone. In some embodiments, the rhGAA or pharmaceutical composition is administered via 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.

In various embodiments, the pharmacological chaperone is miglutide. Without wishing to be bound by any theory, it is believed that when co-administered, miglutide denatures and stabilizes the ATB200 in the systemic circulation, which enhances delivery of the active ingredient ATB200 to the lysosome.

In some embodiments, the meglumine is administered at an oral dose of about 50mg to about 600 mg. In at least one embodiment, the meglumine is administered at an oral dosage of about 200mg to about 600mg, or at an oral dosage of about 200mg, about 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, or about 600 mg. In at least one embodiment, the meglumine is administered at an oral dose of about 233mg to about 500 mg. In at least one embodiment, the meglumine is administered at an oral dosage of about 250 to about 270mg, or at an oral dosage of about 250mg, about 255mg, about 260mg, about 265mg, or about 270 mg. In at least one embodiment, the meglumine is administered at an oral dose of about 260 mg.

It will be appreciated by those skilled in the art that oral doses of miglutide in the range of about 200mg to 600mg or any smaller range therewith may be suitable for adult patients depending on his/her body weight. For example, for patients weighing significantly less than about 70kg, including (but not limited to) infants, children, or underweighing adults, smaller doses may be considered suitable by a physician. Thus, in at least one embodiment, the meglumine is administered at an oral dosage of about 50mg to about 200mg, or at an oral dosage of about 50mg, about 75mg, about 100mg, about 125mg, about 130mg, about 150mg, about 175mg, about 195mg, about 200mg, or about 260 mg. In at least one embodiment, the meglumine is administered at an oral dosage of about 65mg to about 195mg, or at an oral dosage of about 65mg, about 130mg, or about 195 mg.

In some embodiments, rhGAA is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and meglumine is administered orally at a dose of about 50mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and meglumine is administered orally at a dose of about 50mg to about 200 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and meglumine is administered orally at a dose of about 200mg to about 600 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and meglumine is administered orally at a dose of about 200mg to about 500 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20mg/kg and meglumine is administered orally at a dose of about 260 mg. In some embodiments, rhGAA is administered intravenously at a dose of about 5mg/kg to about 20mg/kg and meglumine is administered orally at a dose of about 130mg to about 200 mg. In one embodiment, rhGAA is administered intravenously at a dose of about 20mg/kg and meglumine is administered orally at a dose of about 195 mg.

In some embodiments, the meglumine and rhGAA are administered simultaneously. For example, meglumine 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, the meglumine is administered within 5, 4, 3, 2, or 1 minutes before or after administration of the rhGAA.

In some embodiments, the meglumine and rhGAA are administered sequentially. In at least one embodiment, the meglumine is administered prior to the administration of rhGAA. In at least one embodiment, the meglumine is administered less than three hours prior to administration of the rhGAA. In at least one embodiment, the meglumine is administered about two hours prior to administration of the rhGAA. For example, meglumine is administered about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes prior to administration of rhGAA. In at least one embodiment, the meglumine is administered about one hour prior to administration of the rhGAA.

In some embodiments, the meglumine is administered after the administration of rhGAA. In at least one embodiment, the meglumine is administered within three hours after administration of the rhGAA. In at least one embodiment, the meglumine is administered within two hours after administration of the rhGAA. For example, meglumine may be administered within about 1.5 hours, about 1 hour, about 50 minutes, about 30 minutes, or about 20 minutes after administration of rhGAA.

In some embodiments, the subject is fasted at least two hours before and at least two hours after administration of the meglumine.

In some embodiments, a two-component treatment according to the invention ameliorates one or more disease symptoms in an individual with pompe disease compared to (1) baseline, or (2) a control treatment comprising administration of an arabinosidase a and a placebo for a pharmacological chaperone. In such control treatments, a placebo, but not a pharmacological chaperone, is administered. In some embodiments, the individual treated by the bi-component therapy is a patient who has experienced ERT. In some embodiments, the individual treated by the bi-component therapy is an ERT untreated patient.

In some embodiments, the two-component treatment according to the invention improves the motor function of the individual as measured by gait, stair, golgi, chair (GSGC) tests. In some embodiments, the GSGC score of the individual is improved after 12, 26, 38, or 52 weeks of treatment as compared to baseline, as indicated by at least a 0.1, 0.3, 0.5, 0.7, 1.0, 1.5, or 2.5 point decrease. In some embodiments, the GSGC score of the individual is improved after 52 weeks of treatment as compared to baseline, as indicated by at least a 0.5 point decrease. In some embodiments, the GSGC score of the individual is significantly improved after treatment compared to control treatment. In some embodiments, the GSGC score of the individual is significantly improved compared to a control treatment, 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. In some embodiments, the GSGC score of the individual is significantly improved compared to the control treatment, as indicated by a decrease of at least 1.0 point after 52 weeks of treatment.

In some embodiments, a two-component treatment according to the invention reduces the amount of at least one liver glucose accumulation marker after treatment. In some embodiments, the at least one liver sugar accumulation marker comprises urine hexose tetraose (Hex 4). In some embodiments, the individual's urine Hex4 content is reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to baseline. In some embodiments, the individual's urine Hex4 content is reduced by at least 30% after 52 weeks of treatment as compared to baseline. In some embodiments, the individual has a significantly reduced urine Hex4 content after treatment as compared to a control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% after 12, 26, 38 or 52 weeks of treatment as compared to control treatment. In some embodiments, the individual's urine Hex4 content is significantly reduced by at least 40% after 52 weeks of treatment as compared to a control treatment.

In some embodiments, the two-component treatment of an individual experiencing ERT with pompe disease improves the motor function of the individual as measured by 6 MWT. In some embodiments, the individual'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 compared to baseline. In some embodiments, the individual's 6MWD increases by at least 15 meters or at least 5% after 52 weeks of treatment. In some embodiments, the individual's 6MWD is significantly improved after treatment compared to control treatment. In some embodiments, the individual'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 compared to control treatment. In some embodiments, the subject's 6MWD is improved by at least 15 meters after 52 weeks of treatment compared to control treatment. In some embodiments, the individual has a baseline 6MWD of less than 300 meters. In some embodiments, the individual has a baseline 6MWD of greater than or equal to 300 meters.

E. Set of parts

Another aspect of the invention relates to a kit suitable for use in performing the rhGAA therapies described herein. In one or more embodiments, the kit comprises a container (e.g., vial, tube, bag, etc.) comprising the rhGAA or pharmaceutical composition (either before or after lyophilization) and instructions for reconstitution, dilution, and administration. In one or more embodiments, the kit comprises a container (e.g., vial, tube, bag, etc.) comprising a pharmacological chaperone (e.g., meglumine) and a pharmaceutical composition comprising rhGAA (either before or after lyophilization) and instructions for reconstitution, dilution, and administration of the rhGAA with the pharmacological chaperone.

Examples

Example 1: rhGAA-producing CHO cells were prepared with higher levels of mono-or di-M6P-carrying N-glycans.

DG44 CHO (DHFR-) cells were transfected with a DNA construct expressing rhGAA. The DNA construct is shown in fig. 4. Following transfection, CHO cells containing stably integrated GAA gene were selected by medium lacking hypoxanthine/thymidine (-HT). GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM).

Cell pools exhibiting high amounts of GAA were identified by GAA enzymatic activity analysis and used to establish individual clones producing rhGAA. Individual clones were generated on semisolid flasks, extracted by the ClonePix system, and transferred to 24 deep-well plates. The GAA enzyme activity of these individual clones was analyzed to identify clones exhibiting high GAA levels. An improved medium for determining GAA activity uses a 4-MU-alpha-glucosidase matrix. Further evaluation of viability, growth ability, GAA productivity, N-glycan structure and stable protein performance of higher GAA producing clones as measured by GAA enzyme analysis. CHO cell lines including CHO cell line GA-ATB200 exhibiting rhGAA with increased mono-M6P or di-M6P N-glycans were isolated using this procedure.

Example 2: purification of rhGAA

Multiple batches of rhGAA of the invention were produced in shake flasks and perfusion bioreactors using CHO cell line GA-ATB200, the product of which was designated "ATB200". ATB200 rhGAA was fractionated according to terminal phosphate and sialic acid using weak anion exchange ("WAX") liquid chromatography. The dissolution profile results from the use of increased amounts of salt to dissolve ERT. The profile was monitored by UV (A280 nm). A similar CIMPR receptor binding (at least-70%) profile was observed for purified ATB200 rhGAA from different production batches (fig. 5), indicating that ATB200 rhGAA was consistently produced.

Example 3: oligosaccharide characterization of ATB200 rhGAA

The site-specific N-glycan profile of ATB200 rhGAA was analyzed using different LC-MS/MS analysis techniques. The results of the first two LC-MS/MS methods are shown in FIGS. 6A-6H. The results of the third LC-MS/MS method using 2-AA polysaccharide localization are shown in fig. 19A-19H, fig. 20A-20B and table 5.

In the first LC-MS/MS analysis, proteins were denatured, reduced, alkylated and digested prior to LC-MS/MS analysis. In protein changesDuring sex and reduction, 200. Mu.g of protein sample, 5. Mu.L of 1mol/L tris-HCl (final concentration 50 mM), 75. Mu.L of 8mol/L guanidine HCl (final concentration 6M), 1. Mu.L of 0.5mol/L EDTA (final concentration 5 mM), 2. Mu.L of 1mol/L DTT (final concentration 20 mM) and Water was added to the 1.5mL tube to provide a total volume of 100. Mu.L. The samples were mixed and incubated in a dry bath at 56℃for 30 minutes. During alkylation, denatured and reduced protein samples were mixed with 5. Mu.L of 1mol/L iodoacetamide (IAM, final concentration 50 mM) followed by incubation in the dark at 10-30℃for 30 min. After alkylation, 400 μl of pre-cooled acetone was added to the sample and the mixture was frozen at-80 ℃ for 4 hours under refrigeration. The sample was then centrifuged at 13000rpm at 4 ℃ for 5min and the supernatant removed. 400 μl of pre-cooled acetone was added to the pellet, which was then centrifuged at 13000rpm at 4 ℃ for 5min, and the supernatant removed. The sample was then air dried on ice in the dark to remove acetone residues. Forty microliters of 8M urea and 160 μl of 100mM NH ₄ HCO ₃ Added to the sample to solubilize the protein. During trypsin digestion, 50. Mu.g of protein was then added to a final volume of 100. Mu.L with trypsin digestion buffer, and 5. Mu.L of 0.5mg/mL trypsin (protein to enzyme ratio 20/1 w/w) was added. The solution was mixed thoroughly and incubated overnight (16.+ -. 2 hours) at 37 ℃. Two and five microliters of 20% TFA (final concentration 0.5%) was added to quench the reaction. Subsequent use of Thermo Scientific ^TM Orbitrap Velos Pro ^TM The mass spectrometer analyzes the sample.

In a second LC-MS/MS analysis, ATB200 samples were prepared according to similar denaturation, reduction, alkylation and digestion procedures, except that iodoacetic acid (IAA) was used as the alkylating agent instead of IAM, and Thermo Scientific was subsequently used ^TM Orbitrap Fusion ^TM Lumos Tribid ^TM The mass spectrometer performs the analysis.

The results of the first and second analyses are shown in fig. 6A-6H. In fig. 6A-6H, the results of the first analysis are represented by the left bar (dark gray) and the results from the second analysis are represented by the right bar (light gray). The symbol nomenclature for polysaccharides is according to Varki, a., cummings, r.d., esko j.d., et al, glycobiology basis, 2 nd edition (2009).

As can be seen from fig. 6A-6H, both analyses provided similar results, but there was some variation between the results. This variation can be attributed to a variety of factors including the instrument used and the integrity of the N-polysaccharide analysis. For example, if some phosphorylated N-polysaccharide material is not identified and/or quantified, the total number of phosphorylated N-glycans at that site may be underestimated and the percentage of rhGAA carrying phosphorylated N-glycans may be underestimated. As another example, if some non-phosphorylated N-glycans are not identified and/or are not quantified, the total number of non-phosphorylated N-glycans at the site may be underestimated and the percentage of rhGAA carrying phosphorylated N-glycans may be underestimated.

FIG. 6A shows the N-glycosylation site occupancy of ATB 200. As can be seen from fig. 6A, the first, second, third, fourth, fifth and sixth N-glycosylation sites are largely occupied, with about 90% or more and up to about 100% of the ATB200 enzyme detected by both assays, and N-glycans detected at each potential N-glycosylation site. However, the seventh potential N-glycosylation site is N-glycosylated about half the time.

FIG. 6B shows the N-glycosylation pattern of the first potential N-glycosylation site N84. As can be seen from fig. 6B, the main N-polysaccharide material is bis M6P N-polysaccharide. Both the first and second assays detected more than 75% of ATB200 with dual M6P at the first site, corresponding to an average of about 0.8 moles of dual M6P per mole of ATB200 at the first site.

FIG. 6C shows the N-glycosylation pattern of a second potential N-glycosylation site N177. As can be seen from fig. 6C, the main N-polysaccharide species are mono M6P N-polysaccharide and non-phosphorylated higher mannose N-polysaccharide. Both the first analysis and the second analysis detected more than 40% of ATB200 with single M6P at the second site, corresponding to an average of about 0.4 to about 0.6 moles of single M6P per mole of ATB200 at the second site.

FIG. 6D shows the N-glycosylation pattern of a third potential N-glycosylation site N334. As can be seen from fig. 6D, the main N-polysaccharide species are non-phosphorylated high mannose N-glycans, di-, tri-and tetra-antennary complex N-glycans and hybrid N-glycans. Both the first and second assays detected more than 20% of ATB200 with sialic acid residues at the third site, corresponding to an average of about 0.9 to about 1.2 moles of sialic acid per mole of ATB200 at the third site.

FIG. 6E shows the N-glycosylation pattern of the fourth potential N-glycosylation site N414. As can be seen from fig. 6E, the main N-polysaccharide species are double M6P and single M6P N-polysaccharide. Both the first analysis and the second analysis detected more than 40% of ATB200 with dual M6P at the fourth site, corresponding to an average of about 0.4 to about 0.6 moles of dual M6P per mole of ATB200 at the fourth site. Both the first analysis and the second analysis detected more than 25% of ATB200 with single M6P at the fourth site, corresponding to an average of about 0.3 to about 0.4 moles of single M6P per mole of ATB200 at the fourth site.

FIG. 6F shows the N-glycosylation pattern of the fifth potential N-glycosylation site N596. As can be seen from fig. 6F, the main N-polysaccharide material is a fucosylated biantennary complex N-polysaccharide. Both the first and second assays detected more than 70% of the ATBs 200 with sialic acid residues at the fifth site, corresponding to an average of about 0.8 to about 0.9 moles of sialic acid per mole of ATB200 at the fifth site.

FIG. 6G shows the N-glycosylation pattern of the sixth potential N-glycosylation site N826. As can be seen from fig. 6G, the main N-polysaccharide materials are two-antenna, three-antenna and four-antenna complex N-polysaccharide. Both the first and second assays detected more than 80% of ATB200 with sialic acid residues at the sixth site, corresponding to an average of about 1.5 to about 1.8 moles of sialic acid per mole of ATB200 at the sixth site.

Analysis of N-glycosylation at the seventh site N869 showed about 40% N-glycosylation, with the most common N-glycans being A4S 3GF (12%), A5S3G2F (10%), A4S2G2F (8%) and A6S3G3F (8%).

Fig. 6H shows an overview of phosphorylation at each of seven potential N-glycosylation sites. As can be seen from fig. 6H, the first and second assays detect higher phosphorylation levels at the first, second, and fourth potential N-glycosylation sites. Analysis at the first locus detected more than 80% of ATB200 as mono-or di-phosphorylated, at the second locus more than 40% of ATB200 as mono-or di-phosphorylated, and at the fourth locus more than 80% of ATB200 as mono-or di-phosphorylated.

Another N-glycosylation analysis of ATB200 was performed according to the LC-MS/MS method as follows. This analysis resulted in an average N-glycosylation pattern on ten batches of ATB200 (FIGS. 19A-19H, 20A-20B).

N-linked glycans from ATB200 are enzymatically released with PNGase-F and are 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. Purified 2-AA N-polysaccharide was dissolved in acetonitrile/water (20/80; v/v) and 10. Mu.g was loaded on an aminopolymer analytical column (apHera ^TM Supelco) for high performance liquid chromatography (HPLC-FLD) and High Resolution Mass Spectrometry (HRMS) analysis using fluorescence detection.

Liquid Chromatography (LC) separation was performed in gradient elution mode under normal phase conditions with mobile phase a (2% acetic acid/acetonitrile) and mobile phase B (5% acetic acid; 20 mmol ammonium acetate/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-20 Axs, shimadzu) are excitation (Ex): 320nm; emission (Em): 420nm. HRMS analysis was performed using a quadrupole time-of-flight mass spectrometer (Sciex X500B QTOF) operating in Independent Data Acquisition (IDA) mode. The data file was converted to mzML file using MSConverter from Proteowizard, and GRITS Toolbox 1.2 morning mixing software (UGA) was then used for the polysaccharide database search and subsequent labeling of the identified N-glycans. N-glycans were identified using precursor monoisotopic mass (m/z) and product ion m/z. The experimental product ion and fragmentation patterns were confirmed by computer simulation using a GlycoWorkbench2 application.

To determine the relative quantification of N-linked glycans from ATB200, data obtained from HPLC-FLD-QTOF MS/MS experiments were processed as follows. All N-glycan peaks in the FLD chromatogram were integrated and each peak was designated as a percentage of the total area of all peaks in the FLD chromatogram. The fluorescent signal, expressed as peak area, is a quantitative measure of the amount of each N-polysaccharide in the sample. However, in most cases, the same FLD peak contains multiple N-glycans. Thus, a mass spectrometer is also required to obtain relative quantification of each N-polysaccharide substance (table 5). The ionic strength signal of each N-polysaccharide is "acquired" from the data to create a chromatographic peak called the extracted ion chromatograph (XIC). XIC aligns with FLD chromatographic peaks and is specific for only one N-polysaccharide substance. XIC peaks generated from the ionic strength signals are then integrated and this peak area is a relative quantitative measure of the amount of polysaccharide present. Both the FLD peak area and the mass spectrometer XIC peak area were used to achieve the relative quantification of all N-linked polysaccharide species of ATB200 reported herein.

The results of this LC-MS/MS analysis are provided in table 5 below. The symbol nomenclature for polysaccharide is according to Wepeis W et al 2006. Hypersialylated O-glycans in siallia patients are aberrantly glycosylated Biochimica et Biophysica acta.1762:598-607; gornik O, et al 2007. Serum polysaccharide changes during sepsis and acute pancreatitis. Sugar biology 17:1321-1332; kattla JJ, et al 2011, murray Moo-Young (ed.), incorporated by reference, second edition, 3:467-486; themamalingam-Jaikaran T, et al, N-polysaccharide analysis of bovine follicular fluid under the key major follicular development stage 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. The single chain antibody-fragment M6P-1 has a mannose 6-phosphate-specific binding pocket that discriminates between N-glycan phosphorylation in a branching-specific manner. Sugar biology 26-2:181-192.

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Based on this 2-AA and LC-MS/MS analysis, and as further summarized, the ATB200 tested had an average M6P content of 3-5 moles per mole of ATB200 (considering single M6P and double M6P) and a sialic acid content of 4-7 moles per mole of ATB 200.

As shown in fig. 19A-19H and summarized in fig. 20B, the average M6P content of the first potential N-glycosylation site of ATB200 is about 1.4 moles of M6P per mole of ATB200, the average single M6P content is about 0.25 moles of single-M6P per mole of ATB200, and the average double M6P content is about 0.56 moles of double M6P per mole of ATB200; the second potential N-glycosylation site of ATB200 has an average M6P content of about 0.5 moles of M6P per mole of ATB200, wherein the predominantly phosphorylated N-polysaccharide material is mono M6P N-polysaccharide; the third potential N-glycosylation site of ATB200 has an average sialic acid content of about 1 mole sialic acid per mole of ATB200; the fourth potential N-glycosylation site of ATB200 has an average M6P content of about 1.4 moles of M6P per mole of ATB200, an average single M6P content of about 0.35 moles of single M6P per mole of ATB200, and an average double M6P content of about 0.52 moles of double M6P per mole of ATB200; the fifth potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mole sialic acid/ATB 200; the sixth potential N-glycosylation site of ATB200 has an average sialic acid content of about 4.2 moles sialic acid per mole of ATB200; and the seventh potential N-glycosylation site of ATB200 has an average sialic acid content of about 0.86 mole sialic acid per mole of ATB200.

Also in accordance with this 2-AA and LC-MS/MS analysis technique, the first potential N-glycosylation site of ATB200 averages about 65% of the N-glycans at the high mannose N-glycans, the second potential N-glycosylation site of ATB200 is about 89% of the N-glycans at the high mannose N-glycans, the third potential N-glycosylation site of ATB200 is more than half of the N-glycans sialylated (wherein almost 20% is fully sialylated), and the third potential N-glycosylation site of ATB200 is about 85% of the N-glycans at the complex N-glycans, the fourth potential N-glycosylation site of ATB200 is about 84% of the N-glycans at the high mannose N-glycans, the fifth potential N-glycosylation site of ATB200 is about 70% of the N-glycans at the high mannose N-glycans, and the fifth potential N-glycans at the about 26% of the saliva N-glycans at the third potential N-glycosylation site is about 100% of the complex N-glycans at the about 10% of the five N-glycosylation site of ATB200, and the seventh potential N-glycans at the about 100% of the complex N-glycans at the potential N-glycosylation site of ATB200 is about 80% of the complex N-glycans at the N-glycosylation site of about 100%.

Example 4: ATB200Analytical comparison of (a)

Purified ATB200 andn-glycans were evaluated by MALDI-TOF to determine the individual N-glycan structures found on each ERT. />Are obtained from commercial sources. As depicted in fig. 7, the ATB200 isThe right side shows four main peak elution. This demonstrates that ATB200 has a higher degree of phosphorylation thanThe evaluation is therefore performed by terminal charge rather than CIMPR affinity. As summarized in FIG. 8, ATB200 samples were found to contain less than +.>Non-phosphorylated high mannose type N-glycans in the amount of (a).

To evaluateAnd->Two conventional rhGAA formulations were injected onto a CIMPR affinity column (which binds to rhGAA with M6P groups) and the flow-through was collected. Bound material was eluted with a free M6 gradient. Fractions were collected in 96-well plates and passed through 4MU- α -glucosylPlasma analysis of GAA activity. The relative amounts of unbound (flowed through) and bound (dissociated M6P) rhGAA were determined based on GAA activity and reported as fractions of total enzyme. Fig. 9A and 9B show at +.>And->Binding profile of rhGAA in (a):73% rhGAA and +.in (FIG. 9B)>The 78% rhgaa in (fig. 9A) did not bind to CIMPR. In fact, the _on>Only 27% of rhGAA and +. >The 22% rhgaa of (c) contains M6P, which is effective in targeting it to CIMPR on muscle cells. In contrast, as depicted in fig. 5, more than 70% of rhgaa in ATB200 was found to bind to CIMPR under the same conditions.

In addition to having a large percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of this interaction.Binding to ATB200 receptor was determined using CIMPR disc binding assay. Briefly, CIMPR coated discs were used to capture GAA. Different concentrations of rhGAA were applied to the immobilized receptor and unbound rhGAA was washed away. The amount of rhGAA remaining was determined by GAA activity. As shown in FIG. 10A, ATB200 and +.>Significantly better binding to CIMPR than to CIMPR. FIG. 10B shows->(conventional rhGAA product) and the relative content of bis M6P N-polysaccharide in ATB200 according to the invention. For->On average only 10% of the molecules have bisphosphorylated N-glycans. In contrast, the average of at least one biphosphorylated N-glycan per rhGAA molecule in ATB 200.

In general, withIn contrast, higher levels of M6P N-polysaccharide in ATB200 indicated that higher portions of the rhGAA molecule in ATB200 could target muscle cells. As shown above, the high percentage of mono-and di-phosphorylated structures determined by MALDI is consistent with the CIMPR profile, which demonstrates significantly greater binding of ATB200 to CIMPR receptors. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that each ATB200 molecule contained at least one native bis M6P N-glycan structure on average. This higher content of bis-M6P N-polysaccharide of ATB200 was associated with high affinity (K) for binding to CIMPR in the M6P receptor disc binding assay _D About 2-4 nM) is directly related.

Comparison of ATB200 with Pompe fibroblast cell linesrelative cellular uptake of rhGAA. Comparison of ATB200 of 5-100nM related to and of the invention with 10-500nM conventional rhGAA product +.>After 16hr incubation, external rhGAA was inactivated with TRIS base and the cells were washed 3 times with PBS prior to harvest. Internalized GAA as measured by 4 MU-a-glucoside hydrolysis and plotted against total cellular protein, and the results are presented in fig. 11A-11C.

ATB200 is also shown to be efficiently internalized into cells. As depicted in fig. 11A-11B, ATB200 internalizes into both normal and poincare fibroblasts and is produced more than conventional rhGAAArticle (B)The degree of internalization is higher. ATB200 saturates the cellular receptor at about 20nM, while requiring about 250nM +.>To saturate the cellular receptors. As shown in FIG. 11C, the absorption efficiency constant (K _{Absorption of} ) Is ATB200:2-3nm and->56nM. These results indicate that ATB200 is a very good targeted treatment for pompe disease.

Example 5: ATB200 and pharmacological accompanying proteins

The stability of ATB200 in acidic or neutral pH buffers was evaluated in a thermal stability assay using SYPRO orange, as the fluorescence of the dye increased as the protein denatured. As depicted in fig. 12, addition of AT2221 stabilized the ATB200 AT pH 7.4 in a concentration-dependent manner, similar to the stability of ATB200 AT pH 5.2, mimicking the acidic environmental conditions of lysosomes. As summarized in table 6, addition of AT2221 caused the melting temperature (T _m ) The increase was almost 10 ℃.

TABLE 6 stability of ATB200 with AT2221

Test conditions	Temperature (. Degree. C.)
		pH 7.4	56.2
pH 7.4+10μM AT2221	61.6
		pH 7.4+30μM AT2221	62.9
pH 7.4+100μM AT2221	66.0
		pH 5.2	67.3

Example 6: co-administration of ATB200 and AT2221 in Gaa KO mice

The therapeutic effects of ATB200 and AT2221 were evaluated and compared against the therapeutic effects of arabinoxylan alpha in Gaa KO mice. For the study, male Gaa KO (3 to 4 months old) and age-matched wild-type (WT) mice were used. The arosidase a was administered via bolus injection tail vein Intravenous (IV) injection. In the co-administration regimen, AT2221 was administered via oral gavage (PO) 30 minutes prior to IV injection of ATB 200. Treatment was given once every two weeks. Treated mice were sacrificed 14 days after the last administration and various tissues were collected for further analysis. Table 7 summarizes the study design:

TABLE 7 Co-administration study design

As discussed above, the tissue liver saccharide content in the tissue sample is determined using amyloglucosidase digestion. As depicted in fig. 13, the combination of 20mg/kg ATB200 with 10mg/kg AT2221 significantly reduced hepatic glucose content in four different tissues (quadriceps, triceps, gastrocnemius and heart) compared to the same dose of araboxylase α.

The tissue samples were also analyzed for biomarker changes according to the methods discussed in the following: khanna R, et al (2012), "pharmacological chaperone protein AT2220 increases recombinant human acid alpha glucosidase uptake and hepatic glucose reduction in a pompe mouse model", plos One 7 (7): e40776; and Khanna, R et al (2014), "pharmacological concomitant protein AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha glucosidase and promotes hepatic glucose reduction in pompe gene transgenic mouse models," PLoS ONE 9 (7): e102092. As depicted in fig. 14, a significant increase and expansion of LAMP 1-positive vesicles was observed in muscle fibers in Gaa KO animals compared to WT, indicating lysosomal proliferation. Co-administration of ATB200/AT2221 resulted in more fibers AT standardized LAMP1 content, while the size of the remaining LAMP 1-positive vesicles was also reduced (inset).

Similarly, severe LC3 positive aggregates in muscle fibers of untreated Gaa KO mice indicate the presence of autophagic regions and autophagy accumulation. LC3 positive aggregates (red) were preferably reduced in mice treated with ATB200/AT2221 co-administration compared to mice treated with arasidase a (fig. 15A). Similar observations were made when assessing LC3 performance using the western blot method. As depicted in fig. 15B, most animals treated with ATB200/AT2221 exhibited a significant decrease in LC3 II (lipidated form associated with autophagosomes), indicating an improvement in autophagy flux. In contrast, the effect of the arabinosidase α on autophagy was modest.

Plasma membrane repair proteins, a protein involved in and membrane repair and defective/mistrafficking associated with multiple muscle malnutrition, were also evaluated. As depicted in fig. 16, plasma membrane repair proteins (brown) accumulated in large amounts in the sarcoplasmic slurry of Gaa KO mice. Compared to the arabinosidase a, ATB200/AT2221 is able to restore plasma membrane repair proteins to the myofiber membrane in a greater number of myofibers.

These data are consistent with the improvement at cellular levels exhibited in human pompe patients treated with ATB200 and mighty (e.g., patients exhibiting reduced levels of biomarkers of liver saccharide accumulation and muscle damage), leading not only to effective treatment of pompe but also to reversal of disease progression. Clinical data in human pompe patients are summarized in examples 8 and 9 below.

Example 7: single fiber analysis

As depicted in fig. 17, most of the vector-treated mice exhibitedLysosomes are shown to be substantially enlarged (green) (see e.g. "B") and to exhibit massive autophagy accumulation (red) (see e.g. "a"). Warp yarnThe treated mice did not exhibit any significant differences compared to the vehicle treated mice. In contrast, most of the fibers isolated from ATB200 treated mice exhibited significantly reduced lysosomal size (see, e.g., "C"). In addition, the area of autophagy accumulation is also reduced to a different extent (see, e.g., "C"). Thus, most of the muscle fibers (36-60%) analyzed from ATB200 treated mice appeared normal or near normal. Table 8 below summarizes the individual fiber analysis shown in fig. 17.

TABLE 8 Single fiber analysis

* This includes fibers with a different degree of reduced autophagy accumulation. Overall, the degree of accumulation was less in the ATB200 treated group compared to the vehicle or the arasidase a treated group.

Overall, the data demonstrate that ATB200 with higher M6P content is further stabilized by the pharmacological accompanying protein AT2221 alone and AT neutral pH of blood, more efficient in tissue targeting and lysosomal transport when administered to Gaa KO mice than arasidase α, consistent with the stability of AT2221 to ATB200 as depicted in fig. 18. Thus, administration of ATB200 and co-administration of ATB200/AT2221 are more effective than arabinosidase a in correcting some disease-related pathologies such as liver sugar accumulation, lysosomal proliferation and autophagy region formation. Thanks to these positive therapeutic effects, it was shown that administration of ATB200 and ATB200/AT2221 co-administration improved the probability of myofiber recovery due to disruption, and even improved reversal of damage caused by clearance of hepatic saccharides accumulated in cells due to lack of optimal GAA activity. As in example 6, these data are also consistent with the improvement in cell content demonstrated in human pompe patients that caused both effective treatment of pompe disease and reversal of disease progression following administration of ATB200 and meglumine. Clinical data in human pompe patients are summarized in examples 8 and 9 below.

Example 8: ATB200-02 test

Phase 1/2 (ATB 200-02, NCT-02675465) open label, fixed order, dose escalation clinical studies were performed to assess the safety, tolerability, pharmacokinetics and temporary efficacy of intravenous infusion of ATB200 and AT2221 in adult individuals with pompe disease. The data are reported in International publication No. WO 2020/163480, the disclosure of which is incorporated herein by reference.

Example 9: ATB200-03 test: study in phase 3 humans of ATB200/AT2221 in patients with Pompe disease

The ATB200-03 trial was a phase 3 double blind, randomized, multicenter, international study of ATB200/AT2221 compared to the arasidase alpha/placebo in adult individuals with delayed poincare disease (LOPD) who had received enzyme replacement therapy with arasidase alpha (i.e., experienced ERT) or had never received ERT (i.e., not been ERT treated).

Study design

As depicted in fig. 21, the trial consisted of a screening period of up to 30 days, a 12 month treatment period, and a 30 day safety follow-up period. Eligible individuals were randomly allocated AT a 2:1 ratio to receive either ATB200/AT2221 or arabinosidase alpha/placebo and graded by ERT status (ERT experienced), no ERT treatment and baseline 6 minute walking distance (6 MWD) (75 to <150 meters, 150 to <400 meters, > 400 meters).

The effectiveness assessment (i.e., functional assessment) included evaluating walking function (6 MWT), motor function tests (gait, stair, golgi, chair maneuver (GSGC) test and upright-walking Timing (TUG) test), muscle strength (freehand muscle test and quantitative muscle test), and lung function tests (FVC, SVC, MIP, MEP and SNIP). Recording patient reported results (Rasch created Pompe specific Activity (R Pact) scale, euroQol 5-dimensional 5 level (EQ-5D-5L), results measurement information System reported for patients with physical function, fatigue, dyspnea and upper extremitiesInstrument, and overall impression of changes by individual). The physician's overall impression of the change is also made. />

The pharmacodynamic assessment includes measurement of biomarkers of muscle injury (creatine kinase (CK)) and disease substrate (hexose tetraose (Hex 4) in urine). Rare blood samples were collected for determining total GAA protein content and AT2221 concentration of plasma from population PK analysis in individuals who underwent ERT. Continuous blood sampling for characterizing PK profiles of total GAA protein and AT2221 was performed in individuals not treated with ERT.

Safety assessments include monitoring Adverse Events (AEs) including infusion-related reactions (IARs), clinical laboratory tests (chemical, hematology and urine sample analysis), vital signs, physical examinations including body weight, electrocardiogram (ECG) and immunogenicity. Concomitant medication and non-medication is also noted.

Individual selection

The individuals who participated in the study met all of the following inclusion criteria and no exclusion criteria. In total, 122 individuals participated in the ATB200-03 test. Of these, 85 individuals (treated with ERT:65; untreated with ERT: 20) received ATB200/AT2221 treatment, and 37 individuals (treated with ERT:30; untreated with ERT: 7) received the arabinoxylate alpha/placebo treatment. As shown in fig. 22, baseline 6MWD and FVC data represent this population and are generally similar in both treatment groups.

Inclusion criteria:

1. prior to conducting any study related procedures, individuals provided signed informed consent.

2. Male and female individuals are not less than 18 years old and weigh not less than 40kg at the time of screening.

3. Female and male individuals with fertility potential agree to use medically accepted methods of contraception during the study and last for 90 days after the last dose of study drug.

4. Individuals were diagnosed as LOPD based on literature of one of the following:

GAA enzyme deficiency

GAA genotyping

5. Individuals are categorized in terms of ERT status as one of the following:

a. ERT was experienced, defined as receiving standard care ERT (arabinoxylate) for > 24 months at recommended doses and regimens (i.e., 20mg/kg dose every 2 weeks).

Experienced ERT, defined as standard care ERT (Alsidase. Alpha.) at a dose of 20mg/kg at the recommended dose and regimen, based on lean or ideal body weight, per 2 weeks, specifically in Australia

b. Without ERT treatment, defined as never receiving research or commercially available ERT

6. The individual's sitting FVC at screening was > 30% predictive value for healthy adults (national health and nutrition examination survey III)

7. The individuals were subjected to two 6 MWTs at the time of screening, as determined to be effective by the clinical evaluator, and met all of the following criteria:

two screening values of a.6MWD are more than or equal to 75 m

b.6MWD predictive value for healthy adults with two screenings less than or equal to 90%

The lower limit of the c.6MWD is within 20% of the upper limit of the 6MWD

Exclusion criteria:

1. the individual has received any investigational therapy or pharmacological treatment for pompe disease (except for the arabinosidase a) within 30 days or 5 times half-life of treatment or treatment (whichever is longer), prior to day 1 or is expected to do so during the study.

2. Individuals have received gene therapy for pompe disease.

3. The subject took any of the following prohibited agents 30 days prior to day 1:

miglitol

Meigulute

Acarbose (acarbose)

Voglibose (voglibose)

4. Individuals need to support for >6 hours with invasive or non-invasive ventilation every day while awake.

5. The individual is hypersensitive to any of the excipients ATB200, albomase a or AT 2221.

6. The situation where an individual has a medical condition or any other predisposition, as far as the researcher or medical monitor is concerned, poses undue safety risks to the individual or compromises his/her ability to comply with or adversely affect the protocol requirements. This includes clinical depression with uncontrollable or poorly controlled symptoms (as diagnosed by psychiatric or other mental health professionals).

7. At the time of screening, the individual (in the case of females) is pregnant or lactating.

8. Individuals (whether male or female) plan pregnant children during the study.

9. Individuals refused to conduct genetic testing.

Study product, dose and mode of administration

Individuals were randomized AT a randomization rate of AT least 2:1 to receive either ATB200/AT2221 or arabinosidase alpha/placebo. Table 9 below summarizes the treatment of the enrolled individuals.

TABLE 9 treatment assignments and protocols

Abbreviations: IV = intravenous

Note that: the subject is required to fasted AT least 2 hours prior to administration of AT2221 or placebo and 2 hours after administration of AT2221 or placebo.

Data evaluation and statistical considerations

The primary efficacy endpoint was a change from baseline at week 52 from 6 MWD. The primary endpoint was tested for superiority of ATB200/AT2221 versus arasidase alpha/placebo using a mixed action model of repeated measures (MMRM) in the event of a violation of normalization and pre-specified non-parametric tests.

Important secondary efficacy endpoints in a pre-specified layering order are as follows. These secondary endpoints were analyzed using the covariate analysis (ANCOVA) model and last observed value transfer method (ITT LOCF).

Change from baseline by week 52 of sitting FVC (predictive%)

Freehand muscle strength test score of lower limb changes from baseline by week 52

Changes from baseline in the total score of PROMIS-entity function up to week 52

PROMIS-fatigue Total score changes from baseline by week 52

Change from baseline in GSGC Total score by week 52

Other secondary efficacy endpoints were as follows:

changes from baseline by week 52 in the following variables related to motor function:

time to complete 10 meter walking (i.e., gait assessment) of GSGC test

Stage 4 stair climbing time to complete GSGC testing

Golgi time to complete GSGC test

Time since chair up as part of GSGC test

Time to complete TUG test

Changes from baseline by week 52 in the following variables related to muscle strength:

freehand muscle strength test scoring of upper limbs

Total score for freehand muscle strength test

Quantitative muscle strength test value (Kg) of upper limb

Quantitative muscle strength test value (Kg) of lower limb

Quantitative myodynamia test total (Kg)

The following variables measured from the results reported by the patient were changed from baseline by week 52:

PROMIS-general score of dyspnea

PROMIS-total score of upper limbs

-total score of R-PAct scale

-EQ-5D-5L Normal State

At week 52, the actual value of the individual's physical state (improvement, stabilization or decline) with respect to the effect of the study drug in the following life areas, as measured by the individual's overall impression of change

-overall physical health

-respiratory operation

Muscle strength

-muscle function

Free-running ability

Activities of daily living

-energy level

-muscle pain level

At week 52, the actual value of the individual's physical state (improvement, stabilization or decline), as measured by the physician's overall impression of the change

Changes from baseline by week 52 in the following pulmonary function measurements, as follows:

Sitting FVC (predictive%)

-MIP(cmH ₂ O)

MIP (predictive%)

-MEP(cmH ₂ O)

MEP (predictive%)

SNIP(cmH ₂ O)

The pharmacokinetic endpoints were as follows:

changes in serum CK content from baseline up to week 52

Changes from baseline in urine Hex4 content by week 52

For individuals experiencing RTT, pharmacokinetic endpoints from population PK analysis of total GAA protein levels and AT2221 concentrations were collected. Plasma total GAA protein concentration and PK parameters of AT2221 were calculated for individuals not treated with ERT.

The safety profile of ATB200/AT2221 uses treatment-induced adverse events (TEAEs), severe Adverse Events (SAE) and any abnormal characterization mentioned in the incidence of AEs causing disruption of study medication, frequency and severity of immediate and late IAR, and other safety assessments. The effect of immunogenicity on ATB200 and alfa on safety and efficacy was also assessed.

Statistical methods include the following considerations regarding sample randomization, sample size calculation, efficacy analysis, and safety analysis.

The following two factors are determined as design layering variables: 1. baseline 6MWD (75 to <150 meters, 150 to <400 meters, > 400 meters); ERT status (ERT-treated, not ERT-treated). These two factors form six factor combinations (i.e., horizontal, layer). Centralized block randomization procedure is used to balance the risk factors described above, 1) reduce bias and increase accuracy of statistical analysis, and 2) allow for various plans and unscheduled subset analysis. Each of the 6 layers is block randomized. Randomization ratio was 2:1ATB200/AT2221 vs alpha-glucosidase/placebo, fixed.

In the superiority test, the 2-group t-test, which determines that the 2-sided significant content is 0.05 and the 2:1 randomization regimen (66 individuals in the ATB200/AT2221 group and 33 individuals in the arabinosidase alpha/placebo group, total sample size 99 individuals), has a normalized effect size of 0.7 between about 90% of the ability to detect 2 groups. Using NqueryThis calculation is performed. Assuming a 10% discard rate, the sample size will be about 110 individuals.

The primary efficacy endpoint (i.e., the change from baseline by week 52 of 6 MWD) was analyzed using parametric analysis of the co-variation (ANCOVA) model to compare the new treatment to the control. This model will typically adjust the baseline 6MWD (as a continuous co-variable), and a factor of 2 is used to stratify randomization: ERT status (no ERT treatment versus ERT experienced) and baseline 6MWD (75 to <150 meters, 150 to <400 meters, > 400 meters). However, baseline 6MWD is not available for modeling twice (both continuous and categorical variables) due to the expected high-point bicontinuous correlation between them. Thus, the 6MWD continuous variable is maintained in the model, but the classification 6MWD is removed. The ANCOVA model then has terms for treatment, baseline 6MWD (continuous) and ERT status (category).

In addition, potential therapeutic interactions with covariates were detected (i.e., treatment and treatment continued with baseline 6MWD via ERT status). If the interaction term is statistically significant (e.g., p <0.10,2 edge) and there is a logical biological interpretation, the interaction term can potentially be added to the final ANCOVA model to be used for primary endpoint analysis. The data was then analyzed based on the ANCOVA model and all relevant evaluations (e.g., LS mean difference, 95% Confidence Interval (CI) of LS mean difference for each treatment group, and p-values compared between 2 treatment groups) were provided.

To support interpretation of clinical benefit, complex individual-content responses are defined based on the ensemble of processing result data. Based on the treatment results, individuals were classified by sequential response variables consisting of significant improvement, moderate improvement, or little/no improvement.

The critical secondary endpoints were analyzed according to the hierarchical order using a step-wise closed test program to control type I error rates. The key secondary endpoint and other secondary endpoints were analyzed using similar methods for primary endpoint analysis, respectively.

The security data is summarized using the counts and percentages of the classified data and descriptive statistics (mean, standard deviation, median, minimum, maximum) of the continuous data.

Efficacy results from the ATB200-03 test

In the total population, the ATB200/AT2221 treatment demonstrated a 6MWD and stability improvement relative to baseline AT week 52 (fig. 23A) and predicted FVC percentage over time (fig. 23B). The ATB200/AT2221 treatment exhibited a greater improvement in 6MWD in the total population AT week 52 compared to the arabinosidase α/placebo (fig. 23A). Furthermore, as depicted in fig. 23A, ATB200/AT2221 treatment exhibited a clinically significant improvement in the predicted FVC percentage in the total population AT week 52 compared to the arabinosidase α/placebo.

In the population that underwent ERT, ATB200/AT2221 treatment exhibited an improvement in 6MWD and stability of the predicted FVC percentage AT week 52 relative to baseline (figure 24). Compared to the arabinosidase α/placebo, ATB200/AT2221 exhibited an improvement in 6MWD over time and predicted improvement in FVC percentage over time in the population that experienced ERT (fig. 25). Furthermore, as depicted in fig. 24, ATB200/AT2221 treatment exhibited clinically significant improvement in both 6MWD and predicted FVC percentage in the population experiencing ERT AT week 52 compared to the arabinosidase α/placebo.

As shown in fig. 26A and 26B, ATB200/AT2221 treatment exhibited improved and predicted stability of FVC percentage relative to baseline and 6MWD over time (fig. 26B) AT week 52 (fig. 26A) in the smaller ERT untreated population (n=27). The difference between the two treatment groups was greater and no clinically significant improvement was observed in the 6MWD or predicted FVC percentages (fig. 26A).

As shown in fig. 28, lower limb MMT was numerically favored over the arabinosidase α/placebo in the population where the total population underwent ERT 200/AT2221 treatment.

As shown in fig. 29, ATB200/AT2221 treatment exhibited clinically significant improvement in GSGC AT week 52 compared to the arabinosidase α/placebo in the population as a whole and in the population that experienced ERT.

As shown in fig. 30, proci body function was numerically favorable to ATB200/AT2221 treatment compared to the arabinosidase alpha/placebo in the total and ERT-experienced populations.

As depicted in fig. 31, proci fatigue similarly improved between the two treatment groups in the total population and the population that underwent ERT.

Biomarker results from the ATB200-03 assay

In the total population and the population that underwent ERT, ATB200/AT2221 treatment exhibited improvement over time of biomarkers of muscle injury (CK) and disease substrate (Hex 4) (fig. 32 and 33). Furthermore, as depicted in fig. 32 and 33, the decrease in CK and urine Hex4 AT week 52 was significantly greater under ATB200/AT2221 treatment compared to the arabinosidase α/placebo in the total and ERT-experienced populations.

As outlined in fig. 34, endpoints between motor function, pulmonary function, muscle strength, patient reported results (PRO) and biomarkers consistently favor ATB200/AT2221 over the albedo alpha/placebo in the total population and the population that experienced ERT. Furthermore, of the 17 efficacy and biomarker endpoints assessed, 16 favored ATB200/AT2221 treatment over the arabinoxylase alpha/placebo.

Safety results from the ATB200-03 test

As depicted in fig. 35, the overall safety profile of the ATB200/AT2221 treated group was similar to that of the arabinosidase α/placebo group.

Fig. 36-40 describe additional aspects of the ATB200-03 test.

Example 10: results of PROPEL 3 phase clinical trial

AT-GAA shows clinically significant and significant improvement in musculoskeletal and respiratory measurements in terms of delayed pompe disease compared to standard care in critical phase 3 propeller studies. PROPEL is also known as "ATB200-03", see example 9.

Patients were far walked an average of 17 meters (p=0.046) from the approved standard care ERT (arabinosidase a) to AT-GAA.

Patients who turned to AT-GAA also showed a percent predicted improvement in Forced Vital Capacity (FVC), which is the most important measure of respiratory function for pompe disease, compared to the decline in patients treated with arabinosidase a (FVC difference 4.1%; p=0.006).

AT-GAA exhibited nominally statistically significant and clinically significant differences in advantage AT the first key secondary endpoint compared to the decline in patients treated with arabinosidase a (3.0% difference in FVC; p=0.023).

In the combined study population of patients turning to ERT and non-ERT treatment, AT-GAA outperformed the arabinosidase a 14 meters (21 m compared to 7 m) AT the primary endpoint and was not statistically significant for superiority (p=0.072).

The improvement of two important biomarkers of pompe disease (Hex-4 and CK) in the combined study population clearly favors AT-GAA (compared to the arasidase a (p < 0.001)).

PROPEL is a 52 week double blind randomized whole study designed to evaluate the effectiveness, safety, and tolerability of AT-GAA compared to current standard care (arabinosidase alpha, an Enzyme Replacement Therapy (ERT)). The study recruited 123 adult pompe patients, who were still able to walk and breathe without mechanical ventilation and were conducted in 62 clinical centers in 24 countries in 5 continents. It is the largest controlled clinical study that has been conducted in lysosomal disorders.

Patients participating in PROPEL were randomized to a 2:1 group such that every two patients were randomized to AT-GAA treatment and one randomized to Jing A glycosidase alpha treatment. In pompe patients who participated in propeller, 77% were treated with arabinosidase α (n=95) immediately prior to participation, and 23% were never treated with any ERT (n=28). 117 patients completed the propeller study, and all 117 had voluntarily participated in the long-term extension study, and were now treated with AT-GAA alone for their pompe disease.

6 min walking distance (6 MWD) and percentage in combined diverted ERT and non-ERT treated study population Pre-specified analysis of predicted Forced Vital Capacity (FVC):

The primary endpoint of the study was the average change in 6-minute walking distance compared to baseline measurements at 52 weeks in the entire combination-diverted ERT and non-ERT treated patient population. In this combined population, patients using AT-GAA (n=85) were on average 21 meters away AT 52 weeks (table 10) compared to 7 meters (n=37) for those treated with arabinosidase a. The superiority of this primary endpoint in the combined population was assessed and although numerically greater, no statistical significance of the superiority of this combined population was achieved for the AT-GAA arm compared to the arabinosidase a arm (p=0.072).

The first key secondary endpoint of the study was the average change in the predicted FVC percentage at 52 weeks throughout the combined population, according to the hierarchy of the statistical analysis program. In this combined population, patients using AT-GAA demonstrated nominally statistically significant and clinically significant differences in advantage over those treated with arabinosidase a. AT-GAA significantly slowed the patient's hypopnea rate after 52 weeks. Patients treated with AT-GAA showed a 0.9% absolute decrease in the percentage of FVC as predicted compared to a 4.0% absolute decrease in the alpha arm of the arabinosidase (p=0.023) (table 11). The percentage of FVC is predicted to be the most important measure of airway muscle function for pompe disease and is the basis for approval of the enzyme alpha.

Table 10. 6MWD (m) in the population of study with and without ERT treatment

TABLE 11 overall shift to ERT and FVC (predictive%) in study population without ERT treatment

Pre-specified analysis of 6 min walking distance (6 MWD) and percent predictive Forced Vital Capacity (FVC) in study population turning to ERT (n=95):

patients turning to PROPEL have been treated with arabinoxylan alpha for a minimum of two years when they entered the study. Those patients who exceeded three minutes of two (67% +) had been on ERT for more than five years (average 7.4 years) prior to entering the propeller study.

Pre-specified analysis of patients switched from araboxylase alpha over 6 minutes walking distance showed that AT-GAA treated patients (n=65) were 16.9 meters (p=0.046) more than their baseline after 52 weeks self-switching compared to 0.0 meters of those patients with random groupings resting on araboxylase alpha (n=30) (table 12).

Pre-specified analysis of patients who converted from ara-glycosidase a based on the predicted percentage of FVC showed that AT-GAA treated patients stabilized and slightly improved their respiratory function based on this important measure, while those remaining in ara-glycosidase a continued to significantly decrease in respiratory muscle function. AT-GAA patients showed an absolute increase in the percentage of FVC predicted of 0.1%, whereas the arasidase alpha patients showed an absolute decrease of 4.0% over the course of one year (p=0.006) (table 13).

Table 12. Turning to 6MWD (m) in ERT study population

TABLE 13 FVC (predicted%) in the steering ERT study population

Pre-specified analysis of 6 min walking distance (6 MWD) and percent predictive Forced Vital Capacity (FVC) in the non ERT-treated population (n=28):

pre-specified analysis of patients who had never previously been treated with any ERT AT 6 minutes walking distance showed that AT-GAA treated patients (n=20) were walking 33 meters away from their baseline after 52 weeks. The arabinosidase a-treated patient (n=7) was 38 meters away from its baseline. The difference between the two groups was statistically insignificant (p=0.60) (table 14).

Pre-specified analysis of patients pre-treated with any ERT showed similar percentages of predicted Forced Vital Capacity (FVC) from patients not pre-treated with any ERT AT week 52, -4.1% for AT-GAA treated patients and-3.6% for arasidase alpha treated patients (table 15). The difference between the two groups was statistically insignificant (p=0.57).

TABLE 14 6MWD (m) for population not treated with ERT

TABLE 15 FVC (predicted%) in the population without ERT treatment

Note that: one patient in the alpha arm of the arabinosidase was excluded from the study analysis due to the use of a study anabolic steroid affecting its baseline efficacy.

Pre-specified analysis of other key secondary endpoints and biomarker endpoints between the overall diversion to ERT and non-ERT treated study population:

musculoskeletal and other key secondary endpoints:

GSGC (gait, stairs, golgi, chair): GSGC is an important and general endpoint in pompe disease for capturing strength, coordination and mobility. Patients treated with AT-GAA demonstrated statistically significant improvement in scores (p < 0.05) compared to the exacerbation of patients treated with arasidase a in the total population in this important assessment.

MMT (freehand muscle strength test), proci body function: of these validated measurements of both muscle strength and patient reported results, AT-GAA treated patients improved numerically more than the arabinosidase a treated patients, but the results were not statistically significant.

Procis fatigue: fatigue as measured by this scale is slightly beneficial to AT-GAA treated patients, rather than to the arasidase alpha treated patients.

Biomarkers for treating effects on disease:

urine Hex-4: for the combined study population of patients turning to ERT and non-ERT treatment, patients receiving AT-GAA showed substantial improvement in this biomarker, with an average decrease in Hex-4 of-31.5% after 52 weeks, whereas Hex-4 increased by +11.0% (i.e., worsened) in the arabinosidase a-treated patients (p= < 0.001). Urine Hex-4 is a common biomarker in pompe disease and is used as an indirect measure of bone hepatics clearance in pompe patients receiving ERT. The liver sugar accumulates as a substrate in the lysosomes of the muscles of patients with pompe disease.

CK (creatine kinase): after 52 weeks, AT-GAA treated patients exhibited substantial improvement of this biomarker and an average decrease in CK of-22.4% compared to +15.6% increase (i.e., exacerbation) in the arasidase alpha treated patients. (p < 0.001). CK is an enzyme that leaks out of damaged muscle cells and is elevated in pompe patients.

AT-GAA shows a similar safety profile as that of the arabinosidase alpha. Two patients receiving AT-GAA (2.4%) discontinued treatment due to adverse events, while one (2.6%) for arasidase a was discontinued independent of treatment. Injection-related responses (IAR) were reported in 25% of AT-GAA participants and 26% of the arosidase alpha patients.

Post hoc analysis panel analysis:

baseline 6MWD and FVC categories: population not treated with ERT (n=27): baseline 6MWD for three patients <300m and baseline for FVC three patients <55%; CFBL analysis was not performed in these subgroups due to the smaller number of patients. Baseline 6MWD ∈10 m: the sirtuin α/miglutide (AT-GAA) (n=18) and the arabinosidase α/placebo (n=6) groups had similar improvements over time (average [ SE ] CFBL to week 52: +34.4[12.1] m and +30.8[9.6] m, respectively). Baseline FVC is greater than or equal to 55%: the sirtuin α/miglutide (n=19) and the arabinosidase α/placebo (n=5) groups decreased with time (average [ SE ] CFBL to week 52: 3.7[1.5] and-3.3 [2.6] percent, respectively). In patients with baseline 6MWD <300m and ≡300m, and FVC <55% and ≡55%, the results consistently favor patients with sirtuin alpha/miglutide in the total and ERT-experienced populations, as depicted in fig. 41A and 41B.

In the general study population including patients not treated and experienced ERT, sirtuin α/miglutide exhibited positive trends or clinically significant improvements in motor and respiratory function compared to approved ERT, was independent of baseline 6MWD and FVC% assessment, and in both the pre-and post-hoc analyses.

The sirtuin alpha/miglutide exhibited a similar safety profile as for the aratuin alpha/placebo (fig. 42).

With respect to AT-GAA

AT-GAA is a research two-component therapy consisting of sirtuin alpha (ATB 200), a unique recombinant human acid alpha-glucosidase (rhGAA) with optimized carbohydrate structure, especially a bisphosphorylated mannose-6 phosphate (bis M6P) polysaccharide, administered in combination with a stabilizer for sirtuin alpha (AT 2221), to enhance absorption into cells. In preclinical studies, increased amounts of mature lysosomal forms of AT-GAA and GAA are associated with reduced hepatic saccharide content in muscle, relief of autophagy defects, and improvement of muscle strength.

For pompe disease

Pompe disease is an inherited lysosomal disorder caused by a deficiency of the enzyme acid alpha-Glucosidase (GAA). The reduced or absent GAA content causes accumulation of liver glycans in cells, which are believed to cause clinical manifestations of pompe disease. The disease may be debilitating and characterized by severe muscle weakness that worsens over time. Pompe disease ranges from a rapidly fatal form of infants with a significant impact on cardiac function to a more slowly progressive form that affects mainly skeletal muscle. Pompe disease is estimated to affect about 5,000 to 10,000 people worldwide.

Number embodiments

The present disclosure sets forth the following numbered embodiments, in spite of the existence of the appended claims:

1. a method of treating pompe disease in an individual in need thereof, comprising: administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules to the individual simultaneously or sequentially with a pharmacological chaperone;

wherein the rhGAA molecule comprises seven potential N-glycosylation sites;

wherein 40% to 60% of the N-glycans on the rhGAA molecule are complex N-glycans;

wherein the rhGAA molecule comprises at least 0.5 moles of bimannose-6-phosphate (bim 6P) per mole of rhGAA at the first potential N-glycosylation site, as determined by using liquid chromatography tandem mass spectrometry (LC-MS/MS); and is also provided with

Wherein the method improves one or more disease outcome in the individual compared to (1) baseline or (2) a control treatment comprising administration of an arabinosidase alpha and a placebo for the pharmacological chaperone.

2. The method of embodiment 1, wherein the method improves athletic function of the individual as measured by a 6 minute walk test.

3. The method of embodiment 2, wherein the 6 minute walk distance (6-minute walk distance;6 MWD) varies by at least 20 meters from baseline.

4. The method of embodiment 3, wherein after 52 weeks of treatment, the 6MWD varies by at least 20 meters from baseline.

5. The method of embodiment 2, wherein the 6MWD of the individual is increased by at least 10 compared to the control treatment.

6. The method of embodiment 5, wherein the individual's 6MWD is improved by at least 13 meters after 52 weeks of treatment compared to the control treatment.

7. The method of any one of embodiments 2-6, wherein the individual has a baseline 6MWD of less than 300 meters.

8. The method of any one of embodiments 2-6, wherein the individual has a baseline 6MWD of greater than or equal to 300 meters.

9. The method of embodiment 1, wherein the method improves lung function of the individual as measured by a forced vital capacity (forced vital capacity; FVC) test.

10. The method of embodiment 9, wherein after treatment, the individual's predicted FVC percentage increases from baseline or decreases by less than 3% from baseline.

11. The method of embodiment 10, wherein after treatment, the individual's predicted FVC percentage is reduced by less than 1% from baseline.

12. The method of embodiment 10 or embodiment 11, wherein the predicted FVC percentage of the individual is reduced by less than 1% from baseline after 52 weeks of treatment.

13. The method of embodiment 9, wherein the predicted FVC percentage of the individual is significantly improved or stabilized after treatment compared to the control treatment.

14. The method of embodiment 13, wherein the predicted FVC percentage of the individual is improved by at least 3% after treatment as compared to the control treatment.

15. The method of embodiment 12 or embodiment 13, wherein the predicted FVC percentage of the individual is improved by at least 3% after 52 weeks of treatment compared to the control treatment.

16. The method of any one of embodiments 9 to 15, wherein the individual has a baseline FVC of less than 55%.

17. The method of any one of embodiments 9 to 15, wherein the individual has a baseline FVC of greater than or equal to 55%.

18. The method of embodiment 1, wherein the method improves the motor function of the individual as measured by a gait, stair, golgi, chair (GSGC) test.

19. The method of embodiment 18, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 0.5 score as compared to baseline.

20. The method of embodiment 19, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 0.5 score as compared to baseline.

21. The method of embodiment 18, wherein the GSGC score of the individual is significantly improved after treatment compared to the control treatment.

22. The method of embodiment 21, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

23. The method of embodiment 21 or embodiment 22, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

24. The method of embodiment 1, wherein the method reduces the level of at least one muscle damage marker and/or at least one liver sugar accumulation marker.

25. The method of embodiment 24, wherein the at least one muscle damage marker comprises Creatine Kinase (CK) and/or the at least one liver glucose accumulation marker comprises urinary hexose tetrasaccharide (hexose tetrasaccharide; hex 4).

26. The method of embodiment 25, wherein the CK content of the subject is reduced by at least 20% after treatment and/or the urine Hex4 content of the subject is reduced by at least 30% after treatment as compared to baseline.

27. The method of embodiment 26, wherein the CK content of the individual is reduced by at least 20% after 52 weeks of treatment and/or the urine Hex4 content of the individual is reduced by at least 30% after 52 weeks of treatment, as compared to baseline.

28. The method of embodiment 25, wherein the CK and/or urine Hex4 content of the subject is significantly reduced after treatment compared to the control treatment.

29. The method of embodiment 28, wherein the CK content of the subject is reduced by at least 30% after treatment and/or the urine Hex4 content of the subject is reduced by at least 40% after treatment as compared to the control treatment.

30. The method of embodiment 28 or embodiment 29, wherein the CK content of the individual is reduced by at least 30% after 52 weeks of treatment and/or the urine Hex4 content of the individual is reduced by at least 40% after 52 weeks of treatment as compared to the control treatment.

31. The method of any one of embodiments 1-30, wherein the individual is a patient who has undergone ERT.

32. The method of any one of embodiments 1-30, wherein the individual is an ERT untreated patient.

33. A method of treating pompe disease in an individual in need thereof, comprising:

Administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules to the individual simultaneously or sequentially with a pharmacological chaperone;

wherein the rhGAA molecule comprises seven potential N-glycosylation sites;

wherein the rhGAA molecule comprises at least 0.5 moles of bimannose-6-phosphate (bim 6P) per mole of rhGAA at the first potential N-glycosylation site, as determined by using liquid chromatography tandem mass spectrometry (LC-MS/MS);

wherein the method ameliorates one or more disease symptoms of the individual as compared to (1) baseline, or (2) a control treatment comprising administration of an arabinosidase alpha and a placebo for the pharmacological chaperone, and

wherein the individual is a patient who has experienced ERT.

34. The method of embodiment 33, wherein the method improves athletic performance of the individual as measured by a 6 minute walk test.

35. The method of embodiment 34, wherein the subject's 6-minute walking distance (6-minute walk distance;6 MWD) is increased by at least 15 meters or at least 5% after treatment, as compared to baseline.

36. The method of embodiment 35, wherein the subject's 6-minute walking distance (6 MWD) is increased by at least 15 meters or at least 4% after 52 weeks of treatment, as compared to baseline.

37. The method of embodiment 33, wherein the individual's 6MWD is significantly improved after treatment compared to the control treatment.

38. The method of embodiment 37, wherein the individual's 6MWD is improved by at least 15 meters after treatment compared to the control treatment.

39. The method of embodiment 37 or embodiment 38, wherein the individual's 6MWD is improved by at least 15 meters after 52 weeks of treatment compared to the control treatment.

40. The method of any one of embodiments 34-39, wherein the individual has a baseline 6MWD of less than 300 meters.

41. The method of any one of embodiments 34-39, wherein the individual has a baseline 6MWD of greater than or equal to 300 meters.

42. The method of embodiment 33, wherein the method improves lung function of the individual as measured by a forced vital capacity (forced vital capacity; FVC) test.

43. The method of embodiment 42, wherein the predicted FVC percentage of the individual increases by at least 0.1% from baseline after treatment.

44. The method of embodiment 43, wherein the predicted FVC percentage of the individual increases by at least 0.1% from baseline after 52 weeks of treatment.

45. The method of embodiment 42, wherein the predicted FVC percentage of the individual is significantly improved after treatment compared to the control treatment.

46. The method of embodiment 45, wherein the predicted FVC percentage of the individual is improved by at least 4% after treatment as compared to the control treatment.

47. The method of embodiment 45 or embodiment 46, wherein the predicted FVC percentage of the individual is improved by at least 4% after 52 weeks of treatment as compared to the control treatment.

48. The method of any one of embodiments 42 to 47, wherein the individual has a baseline FVC of less than 55%.

49. The method of any one of embodiments 42 to 47, wherein the individual has a baseline FVC of greater than or equal to 55%.

50. The method of embodiment 33, wherein the method improves the locomotor function of the individual as measured by a gait, stair, golgi, chair (GSGC) test.

51. The method of embodiment 50, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 0.5 score as compared to baseline.

52. The method of embodiment 51, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 0.5 score as compared to baseline.

53. The method of embodiment 50, wherein the GSGC score of the individual is significantly improved after treatment compared to the control treatment.

54. The method of embodiment 53, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

55. The method of embodiment 53 or embodiment 54, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

56. The method of embodiment 33, wherein the method reduces the level of at least one muscle damage marker and/or at least one liver glucose accumulation marker.

57. The method of embodiment 56, wherein said at least one muscle injury marker comprises Creatine Kinase (CK) and/or said at least one liver glucose accumulation marker comprises urinary hexose tetrasaccharide (hexose tetrasaccharide; hex 4).

58. The method of embodiment 57, wherein the CK content of the subject is reduced by at least 15% after treatment and/or the urine Hex4 content of the subject is reduced by at least 25% after treatment as compared to baseline.

59. The method of embodiment 58, wherein the CK content of the subject is reduced by at least 15% after treatment and/or the urine Hex4 content of the subject is reduced by at least 25% after 52 weeks of treatment, as compared to baseline.

60. The method of embodiment 57, wherein the CK and/or urine Hex4 content of the individual is significantly reduced after treatment compared to the control treatment.

61. The method of embodiment 60, wherein the CK content of the subject is reduced by at least 30% after treatment and/or the urine Hex4 content of the subject is reduced by at least 40% after treatment as compared to the control treatment.

62. The method of embodiment 60 or embodiment 61, wherein the CK content of the individual is reduced by at least 30% after treatment and/or the urine Hex4 content of the individual is reduced by at least 40% after 52 weeks of treatment as compared to the control treatment.

63. The method of any one of embodiments 1 to 62, wherein the population of rhGAA molecules is administered at a dose of 5mg/kg to 20mg/kg, optionally 20 mg/kg.

64. The method of any one of embodiments 1 to 63, wherein the population of rhGAA molecules is administered every two weeks.

65. The method of any one of embodiments 1 to 64, wherein the population of rhGAA molecules is administered intravenously.

66. The method of any one of embodiments 1 to 65, wherein the pharmacological chaperone is miglutide (miglutstat) or a pharmaceutically acceptable salt thereof, wherein further optionally the miglutide or a pharmaceutically acceptable salt thereof is administered orally.

67. The method of embodiment 66, wherein the meglumine or a pharmaceutically acceptable salt thereof is administered at a dose of 195mg or 260 mg.

68. The method of embodiment 66 or embodiment 67, wherein the meglumine or a pharmaceutically acceptable salt thereof is administered prior to administering the population of rhGAA molecules, optionally one hour prior to administering the population of rhGAA molecules.

69. The method of embodiment 68, wherein the subject is fasted for at least two hours prior to administration of the meglumine or a pharmaceutically acceptable salt thereof and is fasted for at least two hours after administration of the meglumine or a pharmaceutically acceptable salt thereof.

70. The method of any of embodiments 1 to 69 wherein the rhGAA molecule comprises an amino acid sequence having at least 95% identity to SEQ ID No. 4 or SEQ ID No. 6.

71. The method of any of embodiments 1 to 70, wherein the rhGAA molecule comprises the amino acid sequence of SEQ ID No. 4 or SEQ ID No. 6.

72. The method of any one of embodiments 1 to 71, wherein at least 30% of the rhGAA molecules comprise one or more N-glycan units carrying one mannose-6-phosphate residue (single M6P) or double M6P, as determined by using LC-MS/MS.

73. The method of any of embodiments 1 to 72, wherein the rhGAA molecule comprises an average of 0.5 to 7.0 moles of single M6P or double M6P per mole of rhGAA as determined by using LC-MS/MS.

74. The method of any one of embodiments 1 to 73, wherein the rhGAA molecule comprises an average of 2.0 to 8.0 moles of sialic acid per mole of rhGAA as determined by using LC-MS/MS.

75. The method of any one of embodiments 1 to 73, wherein the rhGAA molecule comprises an average of at least 2.5mol per mole of rhGAA m6p and at least 4 mol of sialic acid per mole of rhGAA, as determined by using LC-MS/MS.

76. The method of any one of embodiments 1 to 75, wherein the rhGAA molecule comprises an average per mole of rhGAA:

(a) 0.4 to 0.6 moles of mono-M6P at the second potential N-glycosylation site;

(b) 0.4 to 0.6 moles of bis-M6P at the fourth potential N-glycosylation site; or alternatively

(c) 0.3 to 0.4 moles of mono-M6P at the fourth potential N-glycosylation site;

wherein (a) - (c) are determined using LC-MS/MS.

77. The method of embodiment 76 wherein the rhGAA molecule further comprises 4 to 7.3 moles of sialic acid per mole of rhGAA; and is also provided with

Wherein the rhGAA molecule comprises an average per mole of rhGAA:

(a) 0.9 to 1.2 moles of sialic acid at the third potential N-glycosylation site;

(b) 0.8 to 0.9 moles of sialic acid at the fifth potential N-glycosylation site; or alternatively

(c) 1.5 to 4.2 moles of sialic acid at the sixth potential N-glycosylation site;

wherein (a) - (c) are determined using LC-MS/MS.

78. The method of any one of embodiments 1 to 77, wherein the population of rhGAA molecules is formulated into a pharmaceutical composition.

79. The method of embodiment 78, wherein the pharmaceutical composition further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof; wherein the pharmaceutical composition has a pH of 5.0 to 7.0.

80. The method of embodiment 79, wherein the pharmaceutical composition has a pH of 5.0 to 6.0.

81. The method of embodiment 78 or embodiment 79, wherein the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

82. The method of embodiment 81 wherein in the pharmaceutical composition the population of rhGAA molecules is present at a concentration of 5-50mg/mL, the at least one buffer is sodium citrate buffer present at a concentration of 10-100mM, the at least one excipient is mannitol present at a concentration of 10-50mg/mL and polysorbate 80 present at a concentration of 0.1-1mg/mL, and the pharmaceutical composition further comprises water, and optionally an acidifying and/or alkalizing agent; wherein the pharmaceutical composition has a pH of 6.0.

83. The method of embodiment 82, wherein in the pharmaceutical composition the population of rhGAA molecules is present at a concentration of 15mg/mL, the sodium citrate buffer is present at a concentration of 25mM, the mannitol is present at a concentration of 20mg/mL, and the polysorbate 80 is present at a concentration of 0.5 mg/mL.

84. The method of any one of embodiments 1 to 83, wherein the rhGAA is produced by chinese hamster ovary cells.

Sequence listing

<110> amikus therapeutics company (Amicus Therapeutics, inc.)

<120> recombinant human acid alpha-glucosidase and uses thereof

<130> AMCS-013/02WO 337351-2416

<150> US 63/162,683

<151> 2021-03-18

<150> US 63/148,596

<151> 2021-02-11

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 952

<212> PRT

<213> artificial sequence

<220>

<223> recombinant human acid alpha-glucosidase

<400> 1

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His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

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Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

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Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

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Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

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Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

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Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

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Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

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Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

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Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu

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Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

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cagttgggaa agctgaggtt gtcgccgggg ccgcgggtgg aggtcgggga tgaggcagca 60

ggtaggacag tgacctcggt gacgcgaagg accccggcca cctctaggtt ctcctcgtcc 120

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tgctgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 600

ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatgggc 660

tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 720

cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 780

aacaggcgct acgaggtgcc cttggagacc ccgcgtgtcc acagccgggc accgtcccca 840

ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcaccg gcagctggac 900

ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 960

ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 1020

atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 1080

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cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 1200

cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctggg cccagagccc 1260

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ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1380

gtggagaaca tgaccagggc ccacttcccc ctggacgtcc aatggaacga cctggactac 1440

atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1500

gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1560

agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1620

atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1680

cccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1740

gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcaga 1800

ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1860

gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1920

cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1980

gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 2040

cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 2100

gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 2160

ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 2220

taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 2280

agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2340

cacctctaca cactgttcca ccaggcccac gtcgcggggg agaccgtggc ccggcccctc 2400

ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2460

gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2520

cccttgggca catggtacga cctgcagacg gtgccaatag aggcccttgg cagcctccca 2580

cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2640

ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2700

ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2760

accaagggtg gagaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2820

ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2880

gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2940

ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 3000

tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 3060

ctcgtcagct ggtgttagcc gggcggagtg tgttagtctc tccagaggga ggctggttcc 3120

ccagggaagc agagcctgtg tgcgggcagc agctgtgtgc gggcctgggg gttgcatgtg 3180

tcacctggag ctgggcacta accattccaa gccgccgcat cgcttgtttc cacctcctgg 3240

gccggggctc tggcccccaa cgtgtctagg agagctttct ccctagatcg cactgtgggc 3300

cggggcctgg agggctgctc tgtgttaata agattgtaag gtttgccctc ctcacctgtt 3360

gccggcatgc gggtagtatt agccaccccc ctccatctgt tcccagcacc ggagaagggg 3420

gtgctcaggt ggaggtgtgg ggtatgcacc tgagctcctg cttcgcgcct gctgctctgc 3480

cccaacgcga ccgcttcccg gctgcccaga gggctggatg cctgccggtc cccgagcaag 3540

cctgggaact caggaaaatt cacaggactt gggagattct aaatcttaag tgcaattatt 3600

ttaataaaag gggcatttgg aatc 3624

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<213> artificial sequence

<220>

<223> recombinant human acid alpha-glucosidase

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Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

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Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

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His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

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Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

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Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

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Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

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Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

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Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

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Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

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Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

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Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

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Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

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Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

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Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

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Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

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Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

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Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

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Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

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Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

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Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

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Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

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Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

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Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 4

<211> 952

<212> PRT

<213> artificial sequence

<220>

<223> recombinant human acid alpha-glucosidase

<400> 4

Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys

1 5 10 15

Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu

20 25 30

His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val

35 40 45

Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly

50 55 60

Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr

65 70 75 80

Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys

85 90 95

Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe

115 120 125

Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser

130 135 140

Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe

145 150 155 160

Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu

165 170 175

Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu

180 185 190

Val Pro Leu Glu Thr Pro His Val His Ser Arg Ala Pro Ser Pro Leu

195 200 205

Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val Arg Arg

210 215 220

Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe

225 230 235 240

Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser

260 265 270

Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly

275 280 285

Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly

290 295 300

Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val

305 310 315 320

Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile

325 330 335

Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln

340 345 350

Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly

355 360 365

Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr

370 375 380

Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val

385 390 395 400

Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe

405 410 415

Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His

420 425 430

Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser

435 440 445

Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg

450 455 460

Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val

465 470 475 480

Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu

485 490 495

Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe

500 505 510

Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly

515 520 525

Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val

530 535 540

Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser

545 550 555 560

Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly

565 570 575

Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly

580 585 590

Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg

595 600 605

Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu

610 615 620

Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro

625 630 635 640

Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu

645 650 655

Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg

660 665 670

Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser

675 680 685

Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala

690 695 700

Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly

705 710 715 720

Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser

725 730 735

Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile

740 745 750

Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro

755 760 765

Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Val Glu Ala Leu Gly

770 775 780

Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser

785 790 795 800

Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val

805 810 815

His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr

820 825 830

Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr

835 840 845

Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser

850 855 860

Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala

865 870 875 880

Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly

885 890 895

Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala

900 905 910

Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr

915 920 925

Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 5

<211> 896

<212> PRT

<213> artificial sequence

<220>

<223> recombinant human acid alpha-glucosidase

<400> 5

Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro

1 5 10 15

Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser

20 25 30

Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu

35 40 45

Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala

50 55 60

Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr

65 70 75 80

Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu

85 90 95

Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg

100 105 110

Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys

115 120 125

Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro Arg Val

130 135 140

His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu

145 150 155 160

Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg Val Leu Leu

165 170 175

Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu

180 185 190

Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu

195 200 205

Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn

210 215 220

Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro

225 230 235 240

Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu

245 250 255

Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu

260 265 270

Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly

275 280 285

Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr

290 295 300

Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp

305 310 315 320

Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr

325 330 335

Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met

340 345 350

Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe

355 360 365

Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met

370 375 380

Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg

385 390 395 400

Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr

405 410 415

Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro

420 425 430

Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala

435 440 445

Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn

450 455 460

Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn

465 470 475 480

Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu

485 490 495

Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His

500 505 510

Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His

515 520 525

Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg

530 535 540

Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp

545 550 555 560

Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu

565 570 575

Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly

580 585 590

Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu

595 600 605

Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu

610 615 620

Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg

625 630 635 640

Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu

645 650 655

Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe

660 665 670

Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu

675 680 685

Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys

690 695 700

Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln

705 710 715 720

Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala

725 730 735

Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro

740 745 750

Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile

755 760 765

Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro

770 775 780

Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu

785 790 795 800

Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala

805 810 815

Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu

820 825 830

Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val

835 840 845

Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly

850 855 860

Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp

865 870 875 880

Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

885 890 895

<210> 6

<211> 896

<212> PRT

<213> artificial sequence

<220>

<223> recombinant human acid alpha-glucosidase

<400> 6

Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln Ala His Pro

1 5 10 15

Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro Pro Asn Ser

20 25 30

Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu Gln Cys Glu

35 40 45

Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu Gln Gly Ala

50 55 60

Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr Pro Ser Tyr

65 70 75 80

Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr Ala Thr Leu

85 90 95

Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu Thr Leu Arg

100 105 110

Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe Thr Ile Lys

115 120 125

Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr Pro His Val

130 135 140

His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe Ser Glu Glu

145 150 155 160

Pro Phe Gly Val Ile Val Arg Arg Gln Leu Asp Gly Arg Val Leu Leu

165 170 175

Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe Leu Gln Leu

180 185 190

Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala Glu His Leu

195 200 205

Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr Leu Trp Asn

210 215 220

Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly Ser His Pro

225 230 235 240

Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly Val Phe Leu

245 250 255

Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser Pro Ala Leu

260 265 270

Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile Phe Leu Gly

275 280 285

Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val Val Gly Tyr

290 295 300

Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu Cys Arg Trp

305 310 315 320

Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu Asn Met Thr

325 330 335

Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu Asp Tyr Met

340 345 350

Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe Arg Asp Phe

355 360 365

Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg Tyr Met Met

370 375 380

Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly Ser Tyr Arg

385 390 395 400

Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr Asn Glu Thr

405 410 415

Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr Ala Phe Pro

420 425 430

Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp Met Val Ala

435 440 445

Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile Asp Met Asn

450 455 460

Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys Pro Asn Asn

465 470 475 480

Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly Gly Thr Leu

485 490 495

Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu Ser Thr His

500 505 510

Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile Ala Ser His

515 520 525

Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val Ile Ser Arg

530 535 540

Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp Thr Gly Asp

545 550 555 560

Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro Glu Ile Leu

565 570 575

Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp Val Cys Gly

580 585 590

Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp Thr Gln Leu

595 600 605

Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu Leu Ser Leu

610 615 620

Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln Ala Met Arg

625 630 635 640

Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu Tyr Thr Leu

645 650 655

Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg Pro Leu Phe

660 665 670

Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp His Gln Leu

675 680 685

Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln Ala Gly Lys

690 695 700

Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr Asp Leu Gln

705 710 715 720

Thr Val Pro Val Glu Ala Leu Gly Ser Leu Pro Pro Pro Pro Ala Ala

725 730 735

Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val Thr Leu Pro

740 745 750

Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly Tyr Ile Ile

755 760 765

Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg Gln Gln Pro

770 775 780

Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala Arg Gly Glu

785 790 795 800

Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu Arg Gly Ala

805 810 815

Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile Val Asn Glu

820 825 830

Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu Gln Lys Val

835 840 845

Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu Ser Asn Gly

850 855 860

Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys Val Leu Asp

865 870 875 880

Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val Ser Trp Cys

885 890 895

<210> 7

<211> 6

<212> PRT

<213> unknown

<220>

<223> conservation hexapeptide

<400> 7

Trp Ile Asp Met Asn Glu

1 5

Claims

wherein the rhGAA molecule comprises seven potential N-glycosylation sites;

2. The method of claim 1, wherein the method improves athletic function of the individual as measured by a 6 minute walk test.

3. The method of claim 2, wherein the 6 minute walk distance (6-minute walk distance;6 MWD) varies by at least 20 meters from baseline.

4. The method of claim 3, wherein the 6MWD after 52 weeks of treatment varies from baseline by at least 20 meters.

5. The method of claim 2, wherein the individual has an increase in 6MWD of at least 10 compared to the control treatment.

6. The method of claim 5, wherein the individual's 6MWD is improved by at least 13 meters after 52 weeks of treatment compared to the control treatment.

7. The method of any one of claims 2-6, wherein the individual has a baseline 6MWD of less than 300 meters.

8. The method of any one of claims 2-6, wherein the individual has a baseline 6MWD of greater than or equal to 300 meters.

9. The method of claim 1, wherein the method improves lung function of the individual as measured by a forced vital capacity (forced vitalcapacity; FVC) test.

10. The method of claim 9, wherein the individual's predicted FVC percentage increases from baseline or decreases by less than 3% from baseline after treatment.

11. The method of claim 10, wherein the individual's predicted FVC percentage is reduced by less than 1% from baseline after treatment.

12. The method of claim 10 or claim 11, wherein the predicted FVC percentage of the individual is reduced by less than 1% from baseline after 52 weeks of treatment.

13. The method of claim 9, wherein the individual's predicted FVC percentage is significantly improved or stabilized after treatment compared to the control treatment.

14. The method of claim 13, wherein the predicted FVC percentage of the individual is improved by at least 3% after treatment compared to the control treatment.

15. The method of claim 12 or claim 13, wherein the predicted FVC percentage of the individual is improved by at least 3% after 52 weeks of treatment compared to the control treatment.

16. The method of any one of claims 9 to 15, wherein the individual has a baseline FVC of less than 55%.

17. The method of any one of claims 9 to 15, wherein the individual has a baseline FVC of greater than or equal to 55%.

18. The method of claim 1, wherein the method improves motor function of the individual as measured by a gait, stair, golgi, chair (GSGC) test.

19. The method of claim 18, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 0.5 points compared to baseline.

20. The method of claim 19, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 0.5 points compared to baseline.

21. The method of claim 18, wherein the GSGC score of the individual is significantly improved after treatment compared to the control treatment.

22. The method of claim 21, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

23. The method of claim 21 or claim 22, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

24. The method of claim 1, wherein the method reduces the level of at least one muscle damage marker and/or at least one liver sugar accumulation marker.

25. The method of claim 24, wherein the at least one muscle damage marker comprises Creatine Kinase (CK) and/or the at least one liver glucose accumulation marker comprises urinary hexose tetrasaccharide (hexose tetrasaccharide; hex 4).

26. The method of claim 25, wherein the CK content of the individual is reduced by at least 20% after treatment and/or the urine Hex4 content of the individual is reduced by at least 30% after treatment as compared to baseline.

27. The method of claim 26, wherein the CK content of the individual is reduced by at least 20% after 52 weeks of treatment and/or the urine Hex4 content of the individual is reduced by at least 30% after 52 weeks of treatment, as compared to baseline.

28. The method of claim 25, wherein the CK and/or urine Hex4 content of the individual is significantly reduced after treatment compared to the control treatment.

29. The method of claim 28, wherein the CK content of the individual is reduced by at least 30% after treatment and/or the urine Hex4 content of the individual is reduced by at least 40% after treatment as compared to the control treatment.

30. The method of claim 28 or claim 29, wherein the CK content of the individual is reduced by at least 30% after 52 weeks of treatment and/or the urine Hex4 content of the individual is reduced by at least 40% after 52 weeks of treatment as compared to the control treatment.

31. The method of any one of claims 1-30, wherein the individual is a patient who has experienced ERT.

32. The method of any one of claims 1-30, wherein the individual is an ERT untreated patient.

33. A method of treating pompe disease in an individual in need thereof, comprising: administering a population of recombinant human acid alpha-glucosidase (rhGAA) molecules to the individual simultaneously or sequentially with a pharmacological chaperone;

wherein the rhGAA molecule comprises seven potential N-glycosylation sites;

wherein the individual is a patient who has experienced ERT.

34. The method of claim 33, wherein the method improves athletic function of the individual as measured by a 6 minute walk test.

35. The method of claim 34, wherein the subject's 6-minute walking distance (6-minutewalk distance;6 MWD) is increased by at least 15 meters or at least 5% after treatment, as compared to baseline.

36. The method of claim 35, wherein the subject's 6-minute walking distance (6 MWD) is increased by at least 15 meters or at least 4% after 52 weeks of treatment, as compared to baseline.

37. The method of claim 33, wherein the individual's 6MWD is significantly improved after treatment compared to the control treatment.

38. The method of claim 37, wherein the individual's 6MWD is improved by at least 15 meters after treatment compared to the control treatment.

39. The method of claim 37 or claim 38, wherein the individual's 6MWD is improved by at least 15 meters after 52 weeks of treatment compared to the control treatment.

40. The method of any one of claims 34-39, wherein the individual has a baseline 6MWD of less than 300 meters.

41. The method of any one of claims 34-39, wherein the individual has a baseline 6MWD of greater than or equal to 300 meters.

42. The method of claim 33, wherein the method improves lung function of the individual as measured by a forced vital capacity (forced vital capacity; FVC) test.

43. The method of claim 42, wherein the individual's predicted FVC percentage is increased by at least 0.1% from baseline after treatment.

44. The method of claim 43, wherein the predicted FVC percentage of the individual increases by at least 0.1% from baseline after 52 weeks of treatment.

45. The method of claim 42, wherein the predicted FVC percentage of the individual is significantly improved after treatment compared to the control treatment.

46. The method of claim 45, wherein the predicted FVC percentage of the individual is improved by at least 4% after treatment as compared to the control treatment.

47. The method of claim 45 or claim 46, wherein the predicted FVC percentage of the individual is improved by at least 4% after 52 weeks of treatment compared to the control treatment.

48. The method of any one of claims 42 to 47, wherein the individual has a baseline FVC of less than 55%.

49. The method of any one of claims 42 to 47, wherein the individual has a baseline FVC of greater than or equal to 55%.

50. The method of claim 33, wherein the method improves motor function of the individual as measured by a gait, stair, golgi, chair (GSGC) test.

51. The method of claim 50, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 0.5 points compared to baseline.

52. The method of claim 51, wherein the GSGC score of the individual is improved after 52 weeks of treatment as indicated by a decrease of at least 0.5 points as compared to baseline.

53. The method of claim 50, wherein the GSGC score of the individual is significantly improved after treatment compared to the control treatment.

54. The method of claim 53, wherein the GSGC score of the individual is improved after treatment as indicated by a decrease of at least 1 score as compared to the control treatment.

55. The method of claim 53 or claim 54, wherein the GSGC score of said subject is improved after 52 weeks of treatment as indicated by a decrease of at least 1 score as compared to said control treatment.

56. The method of claim 33, wherein the method reduces the level of at least one muscle damage marker and/or at least one liver sugar accumulation marker.

57. The method of claim 56, wherein said at least one muscle injury marker comprises Creatine Kinase (CK) and/or said at least one liver glucose accumulation marker comprises urine hexose tetraose (hexose tetrasaccharide; hex 4).

58. The method of claim 57, wherein the CK content of the subject is reduced by at least 15% after treatment and/or the urine Hex4 content of the subject is reduced by at least 25% after treatment as compared to baseline.

59. The method of claim 58, wherein the CK content of the subject is reduced by at least 15% after treatment and/or the urine Hex4 content of the subject is reduced by at least 25% after 52 weeks of treatment, as compared to baseline.

60. The method of claim 57, wherein the CK and/or urine Hex4 content of the subject is significantly reduced after treatment compared to the control treatment.

61. The method of claim 60, wherein the CK content of the subject is reduced by at least 30% after treatment and/or the urine Hex4 content of the subject is reduced by at least 40% after treatment as compared to the control treatment.

62. The method of claim 60 or claim 61, wherein the CK content of the individual is reduced by at least 30% after treatment and/or the urine Hex4 content of the individual is reduced by at least 40% after 52 weeks of treatment as compared to the control treatment.

63. The method of any one of claims 1 to 62 wherein the population of rhGAA molecules is administered at a dose of 5mg/kg to 20mg/kg, optionally 20 mg/kg.

64. The method of any one of claims 1 to 63, wherein the population of rhGAA molecules is administered every two weeks.

65. The method of any one of claims 1 to 64, wherein the population of rhGAA molecules is administered intravenously.

66. The method of any one of claims 1 to 65, wherein the pharmacological chaperone is miglutide (miglutstat) or a pharmaceutically acceptable salt thereof, wherein further optionally the miglutide or a pharmaceutically acceptable salt thereof is administered orally.

67. The method of claim 66, wherein the meglumine or pharmaceutically acceptable salt thereof is administered at a dose of 195mg or 260 mg.

68. The method of claim 66 or claim 67 wherein the meglumine or a pharmaceutically acceptable salt thereof is administered prior to the administration of the population of rhGAA molecules, optionally one hour prior to the administration of the population of rhGAA molecules.

69. The method of claim 68, wherein the subject is fasted for at least two hours prior to administration of the meglumine or a pharmaceutically acceptable salt thereof and is fasted for at least two hours after administration of the meglumine or a pharmaceutically acceptable salt thereof.

70. The method of any one of claims 1 to 69 wherein the rhGAA molecule comprises an amino acid sequence having at least 95% identity to SEQ ID No. 4 or SEQ ID No. 6.

71. The method of any one of claims 1 to 70, wherein the rhGAA molecule comprises the amino acid sequence of SEQ ID No. 4 or SEQ ID No. 6.

72. The method of any one of claims 1 to 71, wherein at least 30% of the rhGAA molecules comprise one or more N-glycan units bearing one mannose-6-phosphate residue (single M6P) or double M6P, as determined by using LC-MS/MS.

73. The method of any one of claims 1 to 72, wherein the rhGAA molecule comprises an average of 0.5 to 7.0 moles of single M6P or double M6P per mole of rhGAA as determined by using LC-MS/MS.

74. The method of any one of claims 1 to 73, wherein the rhGAA molecule comprises an average of 2.0 to 8.0 moles of sialic acid per mole of rhGAA, as determined by using LC-MS/MS.

75. The method of any one of claims 1 to 73 wherein the rhGAA molecule comprises an average of at least 2.5mol m6p per mole of rhGAA and at least 4 mol sialic acid per mole of rhGAA, as determined by using LC-MS/MS.

76. The method of any one of claims 1 to 75, wherein the rhGAA molecule comprises an average per mole of rhGAA:

(a) 0.4 to 0.6 moles of mono-M6P at the second potential N-glycosylation site;

(c) 0.3 to 0.4 moles of mono-M6P at the fourth potential N-glycosylation site;

wherein (a) - (c) are determined using LC-MS/MS.

77. The method of claim 76 wherein the rhGAA molecule further comprises between 4 and 7.3 moles of sialic acid per mole of rhGAA; and is also provided with

Wherein the rhGAA molecule comprises an average per mole of rhGAA:

wherein (a) - (c) are determined using LC-MS/MS.

78. The method of any one of claims 1 to 77 wherein the population of rhGAA molecules is formulated into a pharmaceutical composition.

79. The method of claim 78, wherein the pharmaceutical composition further comprises at least one buffer selected from the group consisting of citrate, phosphate, and combinations thereof, and at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof; wherein the pharmaceutical composition has a pH of 5.0 to 7.0.

80. The method of claim 79, wherein the pharmaceutical composition has a pH of 5.0 to 6.0.

81. The method of claim 78 or claim 79, wherein the pharmaceutical composition further comprises water, an acidifying agent, an alkalizing agent, or a combination thereof.

82. The method of claim 81, wherein in the pharmaceutical composition the population of rhGAA molecules is present at a concentration of 5-50mg/mL, the at least one buffer is sodium citrate buffer present at a concentration of 10-100mM, the at least one excipient is mannitol present at a concentration of 10-50mg/mL and polysorbate 80 present at a concentration of 0.1-1mg/mL, and the pharmaceutical composition further comprises water, and optionally an acidifying and/or basifying agent; wherein the pharmaceutical composition has a pH of 6.0.

83. The method of claim 82, wherein in the pharmaceutical composition the population of rhGAA molecules is present at a concentration of 15mg/mL, the sodium citrate buffer is present at a concentration of 25mM, the mannitol is present at a concentration of 20mg/mL, and the polysorbate 80 is present at a concentration of 0.5 mg/mL.

84. The method of any one of claims 1 to 83, wherein the rhGAA is produced by chinese hamster ovary cells.