Highly potent acid alpha-glucosidase with enhanced carbohydrates

Abstract

Recombinant human alpha glucosidase (rhGAA) compositions derived from CHO cells are disclosed, comprising a more optimal glycan composition consisting of rhGAA with higher amounts of N-glycans bearing mannose-6-phosphate (M6P) or bis-M6P, along with low amounts of non-phosphorylated high mannose glycans, than conventional rhGAA, along with low amounts of terminal galactose on complex oligosaccharides. Compositions comprising the rhGAA are described, as are methods of use.

Description

[0001] Cross Reference to Related Applications

[0002] This application is a divisional application of the patent application with the application number 201580052512.5, filed on September 30, 2015, and the title "High-strength acid alpha-glucosidase with enhanced carbohydrates". BACKGROUND

[0004] TECHNICAL FIELD

[0005] The present invention relates to the fields of medicine, genetics, and recombinant glycoprotein biochemistry, and in particular to recombinant human alpha glucosidase (rhGAA) compositions having a higher total content of mannose 6-phosphate-bearing glycans that are effective in targeting CIMPR on muscle cells and subsequently delivering rhGAA to lysosomes, where it can break down abnormally high levels of accumulated glycogen. The rhGAA of the present invention exhibits superior targeting to muscle cells and subsequent delivery to lysosomes compared to conventional rhGAA products, and exhibits other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy in subjects with Pompe disease.

[0006] TECHNICAL DESCRIPTION

[0007] Existing enzyme replacement therapy for Pompe disease uses conventional rhGAA products that have a low total content of M6P- and bi-M6P-bearing glycans. It is known that and the conventional product under the name of Alphalucosidase alfa. "Lumizyme" and "Myozyme" are conventional forms of rhGAA produced or sold as a biologic by Genzyme and approved by the U.S. Food and Drug Administration, and are described by reference to the Physician's Desk Reference (2014), which is incorporated herein by reference, or the product approved by the FDA for use in the United States on October 1, 2014, under the name or Alphalucosidase alfa is identified as the chemical name [199-arginine, 223-histidine] prepro- alpha-glucosidase (human); molecular formula: C 4758 H 7262 N 1274 O 1369 S 35; CAS Number 420794-05-0. These products are administered to subjects with Pompe disease (also known as glycogen storage disease type II (GSD-II) or acid maltase deficiency). Enzyme replacement therapy seeks to treat Pompe disease by administering rhGAA to replace the missing GAA in lysosomes, thereby restoring the ability of the cells to break down lysosomal glycogen.

[0008] Pompe disease is an inherited lysosomal storage disorder due to deficiency in the activity of acid alpha-glucosidase (GAA). People with Pompe disease have little or reduced levels of acid alpha-glucosidase (GAA), an enzyme that breaks down glycogen and a substance the body uses for energy. This deficiency in the enzyme leads to an accumulation of excess glycogen in lysosomes, which are intracellular organelles that contain enzymes that normally break down glycogen and other cellular debris or waste. The accumulation of glycogen in certain tissues of subjects with Pompe disease, especially muscle, impairs the ability of the cells to function normally. In Pompe disease, glycogen is not properly metabolized and gradually accumulates in lysosomes, particularly in skeletal muscle cells, and in the infantile onset form of the disease, in cardiac muscle cells. The accumulation of glycogen impairs muscle and nerve cells and those muscle and nerve cells in other affected tissues.

[0009] Traditionally, Pompe disease is clinically identified as either an early infantile form or a late onset form, depending on the age of onset. The age of onset tends to correspond to the severity of the genetic mutation that leads to Pompe disease. The most severe genetic mutations result in complete loss of GAA activity, manifesting as early onset disease in infancy. Genetic mutations that reduce GAA activity but do not completely eliminate GAA activity are associated with forms of Pompe disease that have a delayed onset and progression. Infantile onset Pompe disease manifests soon after birth and is characterized by muscle weakness, respiratory insufficiency, and heart failure. Untreated, it is usually fatal within two years. Childhood and adult onset Pompe disease manifests later in life and generally progresses more slowly than infantile onset. This form of the disease, while it generally does not affect the heart, can also lead to death due to weakening of skeletal muscle and those muscles involved in respiration.

[0010] Current non-palliative treatment of Pompe disease involves enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA) such as Myozyme® or Lumizyme®. rhGAA is administered in an attempt to replace or supplement the missing or deficient GAA in subjects with Pompe disease. However, because the majority of rhGAA in conventional rhGAA products is not targeted to muscle tissue, it is non-productively eliminated after administration.

[0011] This occurs because conventional rhGAA lacks high total levels of M6P- and bis-M6P-bearing glycans that target the rhGAA molecule to the CIMPR on target muscle cells, followed by transport of the rhGAA into the lysosomes of these target muscle cells. Cellular uptake of rhGAA for enzyme replacement therapy is facilitated by specialized carbohydrates (mannose-6-phosphate (M6P)) that bind to the cation-independent mannose 6-phosphate receptor (CIMPR) present on the cell surface for subsequent delivery of the exogenous enzyme to the lysosome.

[0012] There are seven potential N-linked glycosylation sites on rhGAA. Because each glycosylation site is heterogeneous in the type of N-linked oligosaccharide (N-glycan) present, rhGAA consists of a complex mixture of proteins with N-glycans that have different binding affinities for the M6P receptor and other carbohydrate receptors. rhGAA containing high mannose N-glycans with one M6P group (mono-M6P) binds to the CIMPR with low (about 6,000 nM) affinity, while rhGAA containing two M6P groups on the same N-glycan (bis-M6P) binds with high (about 2 nM) affinity. Representative structures of the non-phosphorylated, mono-M6P, and bis-M6P glycans are shown below. FIG. 1A FIG. 1B Once inside the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA has low total levels of M6P- and bis-M6P-bearing glycans and therefore poorly targets muscle cells, resulting in poor delivery of rhGAA to the lysosome. Most rhGAA molecules in these conventional products do not have phosphorylated N-glycans, and thus lack affinity for the CIMPR. Non-phosphorylated high mannose glycans can also be cleared by mannose receptors, which results in non-productive clearance of ERT (Figure 2).

[0013] Other types of N-glycans, complex carbohydrates, containing galactose and sialic acid are also present on rhGAA. Because the complex N-glycans are not phosphorylated, they have no affinity for the CIMPR. However, complex-type N-glycans with exposed galactose residues have moderate to high affinity for the asialoglycoprotein receptor on liver hepatocytes, which results in rapid non-productive clearance of rhGAA (Figure 2).

[0014] ​Glycosylation of GAA or rhGAA can be enzymatically modified in vitro by phosphotransferase and uncovering enzyme as described in Canfield et al., U.S. Patent No. 6,534,300 to produce M6P groups. Enzymatic glycosylation cannot be adequately controlled and results in rhGAA with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA can contain only high-mannose N-glycans, which can all potentially be enzymatically phosphorylated in vitro by phosphotransferase / uncovering enzyme, and can contain an average of 5-6 M6P groups per GAA. The glycosylation pattern produced by in vitro enzyme treatment of GAA is problematic because the additional terminal mannose residues, particularly non-phosphorylated terminal mannose residues, negatively impact the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannose groups increase non-productive clearance of GAA, increase uptake of the enzymatically modified GAA by immune cells, and decrease rhGAA therapeutic efficacy due to less GAA reaching target tissues, such as cardiac or skeletal muscle cells. For example, terminal non-phosphorylated mannose residues are known ligands for mannose receptors in the liver and spleen, which result in rapid clearance of the enzymatically modified rhGAA and decreased targeting of rhGAA to target tissues. In addition, the glycosylation pattern of the enzymatically modified GAA with high-mannose N-glycans containing terminal non-phosphorylated mannose residues is similar to the glycosylation pattern on glycoproteins produced in yeast, molds, and functions, which increases the risk of eliciting an immune or allergic reaction, such as a life-threatening severe allergic (anaphylactic) or hypersensitivity reaction, to the enzymatically modified rhGAA.

[0015] As explained above, conventional rhGAA products such as have low levels of mono-phosphorylated glycans and even lower levels of di-phosphorylated glycans. To be effective in Pompe disease therapy, rhGAA must be delivered to lysosomes in muscle cells. The low total amount of mono- and di-M6P targeting groups on conventional rhGAA limits cellular uptake via CIMPR and lysosomal delivery, thus making conventional enzyme replacement therapy inefficient. For example, while 20 mg / kg or higher doses of conventional rhGAA products do improve some aspects of Pompe disease, they do not sufficiently reduce glycogen accumulation in many target tissues, particularly skeletal muscle, to reverse disease progression.

[0016] Due to the inefficiency of delivering conventional enzyme replacement therapy to lysosomes, these therapies are often associated with other problems, including the generation of an immune response to GAA. Most of the GAA in conventional rhGAA does not contain glycans with mono- or bi-M6P, which target rhGAA to muscle cells. The immune system of the subject is exposed to this excess of non-phosphorylated GAA and can generate a harmful immune response that recognizes GAA. Inducing an immune response to non-phosphorylated GAA that does not enter the target tissue and is delivered to the lysosome increases the risk of treatment failure due to immunoinactivation of the administered rhGAA and increases the risk of the patient experiencing a harmful autoimmune or allergic reaction to rhGAA therapy. The rhGAA according to the present invention contains significantly less of this non-targeted, non-phosphorylated rhGAA, thereby reducing the exposure of the immune system of the patient to it.

[0017] Logically, larger doses place additional burdens on the subject as well as the medical professionals treating the subject, such as the infusion time required to administer rhGAA intravenously. This is because conventional rhGAA contains a higher content of non-phosphorylated rhGAA that does not target CIMPR on muscle cells. rhGAA that does not bind to CIMPR on muscle cells and then enters the lysosome does not enzymatically degrade glycogen there. When an equivalent dose of conventional rhGAA and rhGAA according to the present invention is administered, more of the rhGAA in the composition according to the present invention binds to CIMPR on muscle cells and then is delivered to the lysosome. The rhGAA of the present invention provides the physician with the option of administering a lower amount of rhGAA while delivering the same or more rhGAA to the lysosome.

[0018] The existing manufacturing process for making conventional rhGAA (such as Lumizyme® or alglucosidase alfa) does not significantly increase the content of M6P or bi-M6P because cellular carbohydrate processing is naturally complex and extremely difficult to manipulate. In view of these deficiencies of conventional rhGAA products, the inventors have endeavored to seek and identify methods to effectively target rhGAA to muscle cells and deliver it to the lysosome, minimize non-productive clearance of rhGAA once administered, and thereby more productively target rhGAA to muscle tissue. SUMMARY

[0020] In response to the problems associated with targeting and administering rhGAA in its conventional form and in response to the difficulties associated with producing such a well-targeted form of rhGAA, the inventors have investigated and developed a procedure for making rhGAA that is more effectively targeted to CIMPR and delivered to lysosomes in muscle tissue because it has a higher content of M6P-glycans and bi-M6P glycans than conventional rhGAA compositions. Moreover, the rhGAA of the present invention has well-processed complex N-glycans that minimize unproductive clearance of rhGAA by non-target tissues.

[0021] In view of the problems associated with current enzyme replacement therapy using conventional rhGAA products (such as ) the inventors have, through diligent research and investigation, developed a method for producing rhGAA in CHO cells that have a significantly higher total content of mono- and bi-M6P glycans that target CIMPR on muscle cells and then deliver rhGAA to lysosomes.

[0022] The rhGAA produced by this method also has advantageous pharmacokinetic properties by virtue of its overall glycosylation pattern that increases target tissue uptake and decreases unproductive clearance after administration to subjects with Pompe disease. The inventors have shown that the rhGAA of the present invention, as exemplified by the rhGAA designated ATB-200, is more potent and more effective at targeting skeletal muscle tissue than conventional rhGAA (such as ). As illustrated by Figure 2, the rhGAA according to the present invention has the superior ability to productively target muscle tissue in patients with Pompe disease and decrease unproductive clearance of rhGAA.

[0023] The superior rhGAA according to the present invention can be further finished or conjugated with a chaperone or other moiety that targets CIMPR in muscle tissue (e.g., the portion of IGF2 that binds this receptor). The following examples show that the rhGAA of the present invention (as exemplified by ATB-200 rhGAA) outperforms the existing standard of care for enzyme replacement therapy by providing significantly better glycogen clearance in skeletal muscle compared to the existing regimen using conventional rhGAA products

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] The present application document contains at least one drawing in color.

[0026] Figure 1 FIG. 1A shows non-phosphorylated high mannose glycans, mono-M6P glycans, and bi-M6P glycans. FIG. 1B shows the chemical structure of a M6P group.​

[0027] Figure 2 FIG. 2A Productive targeting of rhGAA to target tissues (e.g., muscle tissue of a subject with Pompe disease) via M6P-bearing glycans is described. FIG. 2B Non-productive drug clearance from non-target tissues (e.g., liver and spleen) or binding by non-M6P glycans to non-target tissues is described.

[0028] Figure 3 FIG. 3A CIMPR receptor (also known as IGF2 receptor) and domains of the receptor are illustrated. FIG. 3B is a table showing binding affinity (nanomolar) of glycans with bi- and mono-M6P to CIMPR, binding affinity of high mannose type glycans to mannose receptor, and binding affinity of asialo complex glycans to asialoglycoprotein receptor. RhGAA with M6P- and bi-M6P-bearing glycans can productively bind to CIMPR on muscle target cells. RhGAA with high mannose glycans and asialo glycans can non-productively bind to non-target cells with the corresponding receptors.

[0029] Figure 4 FIG. 4A and 4B Results of CIMPR affinity chromatography of and are shown, respectively. The dashed line indicates the M6P elution gradient. Elution with M6P replaces binding of GAA molecules to CIMPR via M6P-containing glycans. As shown in FIG. 4A , 78% of GAA activity was eluted prior to M6P addition in FIG. 4B 73% of GAA activity was eluted prior to M6P addition. In or Myozyme, only 22% or 27% of rhGAA, respectively, was eluted with M6P. These graphs show that the majority of rhGAA in these two conventional rhGAA products lack the M6P-bearing glycans required for targeting CIMPR in target muscle tissue.

[0030] FIG. 5 DNA construct used to transform CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA (SEQ ID NO: 4).

[0031] Figure 6 FIG. 6A and 6B show results of CIMPR affinity chromatography of Myozyme and ATB-200 rhGAA. As shown in FIG. 6B about 70% of rhGAA in ATB-200 rhGAA contains M6P.

[0032] FIG. 7A TB-200 rhGAA purification, Examples 1 and 2.

[0033] FIG. 8 Polywax elution profiles of ATB-200 rhGAA and ATB-200.rhGAA.

[0034] FIG. 9 Comparison of three different preparations of ATB200 rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2, Overview of N-glycan structures.

[0035] Figure 10 FIG. 10A CIMPR binding affinity of ATB-200 rhGAA (left trace) and rhGAA (right trace) were compared. FIG. 10B The and ATB-200 rhGAA were compared.

[0036] Figure 11 FIG. 11A ATB-200 rhGAA activity in normal fibroblasts (left trace) and rhGAA activity (right trace) were compared at different GAA concentrations. FIG. 11B ATB-200 rhGAA activity in fibroblasts from subjects with Pompe disease (left trace) and rhGAA activity (right trace) were compared at different GAA concentrations. FIG. 11C ATB-200 rhGAA activity in fibroblasts from normal subjects and subjects with Pompe disease (K 摄取 ).

[0037] Figure 12 FIG. 12A shows the amount of glycogen relative to protein in the myocardium after contact with vehicle (negative control), with 20 mg / ml FIG. 12B or 5 mg / kg, 10 mg / kg or 20 mg / kg ATB-200 rhGAA. shows the amount of glycogen relative to protein in the quadriceps after contact with vehicle (negative control), with 20 mg / ml FIG. 12C or 5 mg / kg, 10 mg / kg or 20 mg / kg ATB-200 rhGAA. After exposure to ATB-200 rhGAA at doses of 5 mg / kg, 10 mg / kg, or 20 mg / kg, the amount of glycogen relative to protein in the triceps muscle was compared with the negative control and with... In comparison, ATB-200 rhGAA produced a significant reduction in glycogen in the quadriceps and triceps muscles.

[0038] Figure 13 shows that the stability of ATB-200 rhGAA is improved in the presence of molecular chaperone AT2221. FIG. 13A The first left trajectory line shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 7.4 (blood pH). The last right trajectory line shows the percentage of unfolded ATB-200 rhGAA protein at various temperatures at pH 5.2 (lysosomal pH). The three intermediate trajectories show the effect of 10 μg, 30 μg, or 100 μg of the AT2221 molecular chaperone on protein folding. These data show that, compared to the control sample, AT2221 prevents the unfolding of ATB-200 rhGAA at blood pH. The improvement in Tm of AT2221 at neutral pH is summarized in... FIG. 13B middle.

[0039] FIG. 14 The table shows that the combination of ATB-200 rhGAA and the molecular chaperone AT2221 is more effective than other methods in GAA knockout mice. With AT2221 or without AT2221 molecular chaperone The treatment compared to the ATB200 rhGAA control provided significantly better glycogen clearance.

[0040] FIG. 15 Residual glycogen in quadriceps muscles after treatment with Lumizyme, ATB-200 rhGAA, or ATB-200 rhGAA and various concentrations of AT2221 molecular chaperone.

[0041] Figure 16 shows the improvement in skeletal muscle pathology in mice treated with ATB200 + Miglustat (AT2221) compared to those treated with ERT alone. PAS glycogen staining of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200 rhGAA and Miglustat (AT-2221) FIG. 16A ) and EM ( FIG. 16B ). FIG. 16C Assessment of lysosomal proliferation using LAMP-1 markers. FIG. 16D Identification of type I and type II muscle fibers.

[0042] Figure 17. Improvement in skeletal muscle pathology in mice treated with ATB-200 + magistat (AT2221) over those treated with ERT alone. PAS glycogen staining of muscle tissue from GAA KO mice treated with either conventional rhGAA or ATB-200 rhGAA and magistat (AT-2221) FIG. 17A FIG. 17B Evaluation of lysosomal proliferation by LAMP-1 marker. DETAILED DESCRIPTION

[0044] DEFINITIONS : In the context of the present invention and in the particular context of each term's use, the terms used in this specification generally have their ordinary meaning in the art. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the compositions and methods of the present invention and how to make and use them.

[0045] The term "GAA" refers to human acid alpha-glucosidase (GAA) (an enzyme that catalyzes the hydrolysis of alpha-1,4-glucosidic bonds and alpha-1,6-glucosidic bonds of lysosomal glycogen), as well as to insertional, related or substitution variants of the GAA amino acid sequence and fragments of longer GAA sequences that exhibit enzymatic activity. The term "rhGAA" is used to distinguish endogenous GAA from GAA produced synthetically or recombinantly, such as GAA produced by transforming CHO cells with DNA encoding GAA. An exemplary DNA sequence encoding GAA is NP_000143.2 (SEQ ID NO: 4), which is incorporated by reference. GAA and rhGAA can be present in a composition that comprises a mixture of GAA molecules having different glycosylation patterns, such as a mixture of rhGAA molecules bearing mono-M6P or bi-M6P groups on their glycans and GAA molecules that do not bear M6P or bi-M6P. GAA and rhGAA can also be accomplished with other compounds (such as chaperones) or can be conjugated to other moieties in the GAA or rhGAA conjugate, such as to an IGF2 moiety that targets the conjugate to CIMPR and subsequently delivers it to the lysosome.

[0046] ​A "subject" or "patient" is preferably a human, although other mammals and non-human animals suffering from a disorder involving glycogen accumulation can also be treated. The subject can be a fetus, neonate, infant, child, or adult suffering from Pompe's disease or another glycogen storage or accumulation disorder. One example of an individual being treated is an individual (fetus, neonate, infant, child, adolescent, or adult) suffering from GSD-II (e.g., infantile GSD-II, childhood GSD-II, or adult-onset GSD-II). The individual can have residual GAA activity, or no measurable activity. For example, an individual suffering from GSD-II can have GAA activity of less than about 1% of normal GAA activity (infantile GSD-II), GAA activity of about 1% to 10% of normal GAA activity (childhood GSD-II), or GAA activity of about 10% to 40% of normal GAA activity (adult GSD-II).

[0047] The term "treat" or "treatment" as used herein refers to the amelioration of one or more symptoms associated with a disease, the prevention or delay of onset of one or more symptoms of a disease, and / or the lessening of severity or frequency of one or more symptoms of a disease. For example, treatment can refer to improvement in cardiac status (e.g., an increase in end diastolic and / or end systolic volume, or a reduction, amelioration, or prevention of the progressive cardiomyopathy typically found in GSD-II) or improvement in pulmonary function (e.g., an increase in crying lung capacity over baseline capacity, and / or normalization of oxygen desaturation during crying); improvement in neurodevelopment and / or motor skills (e.g., an increase in AIMS score); a reduction in glycogen levels in tissues of an individual affected by the disease; or any combination of these effects. In a preferred embodiment, treatment includes improvement in cardiac status, specifically a reduction or prevention of GSD-II-associated cardiomyopathy.

[0048] The terms "improve," "increase," or "reduce" as used herein mean relative to a baseline measurement, either a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or individuals) in the absence of the treatment described herein. The control individual is an individual suffering from the same form of GSD-II (infantile, childhood, or adult-onset) as the individual being treated, and is approximately the same age as the individual being treated (to ensure that the stage of the disease in the treated individual and the control individual is comparable).

[0049] The term "purified" as used herein refers to a material that is isolated under conditions that reduce or eliminate the presence of unrelated material, i.e., contaminants, including the natural material from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell, and a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules that can be found in a cell with it. As used herein, the term "substantially free of" is used operationally in the context of analytical testing of a material. Preferably, purified material that is substantially free of contaminants is at least 95% pure; more preferably at least 97% pure, more preferably still at least 99% pure. Purity can be assessed by chromatography, gel electrophoresis, immunoassay, compositional analysis, bioassay, enzymatic assay, and other methods known in the art. In particular embodiments, purified means that the level of contaminants is below an acceptable level for safe administration to a human or non-human animal by a regulatory agency. Recombinant proteins can be isolated or purified from CHO cells using methods known in the art, including by chromatographic size separation, affinity chromatography, or anion exchange chromatography.

[0050] The term "genetically modified" or "recombinant" refers to a cell (e.g., a CHO cell) that expresses a particular gene product (e.g., rhGAA or ATB-200 rhGAA) after introduction of a nucleic acid comprising a coding sequence encoding the gene product, along with regulatory elements that control expression of the coding sequence. Introduction of the nucleic acid can be accomplished by any method known in the art, including gene targeting and homologous recombination. As used herein, the term also includes a cell that has been engineered (e.g., by gene activation technology) to express or overexpress an endogenous gene or gene product that is normally not expressed by such a cell.

[0051] Pompe disease is an autosomal recessive latent leukemia (LSD) characterized by a deficiency of acid alpha-glucosidase (GAA) activity, which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation and results in progressive skeletal muscle weakness, decreased cardiac function, respiratory failure, and / or CNS damage in the later stages of the disease. Genetic mutations in the GAA gene result in lower expression or the production of mutant forms of the enzyme with altered stability and / or ultimately disease-causing biological activity. (See, generally, Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, edited by Scriver et al., McGraw-Hill, New York, 7th ed., pp. 2443-2464). The three recognized clinical forms of Pompe disease (infants, children, and adults) are associated with residual α-glucosidase activity levels (Reuser AJ et al., 1995, Glycogen Storage Disease Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement, 3, pp. 61-69). Infantile Pompe disease (type I or A) is the most common and severe, characterized by failure to thrive in the second year of life, systemic hypoosmolarity, cardiac hypertrophy, and cardiopulmonary failure. Childhood Pompe disease (type II or B) is of moderate severity and is characterized by the absence of muscular symptoms associated with cardiac hypertrophy. Individuals with childhood Pompe disease typically die before the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) usually presents as a slowly progressive myopathy in adolescence or as late as sixty years of age (Felicia KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135). In Pompeii, it has been shown that α-glucosidase is significantly modified post-translationally through glycosylation, phosphorylation, and proteolytic processing.The optimal glycogen catalysis requires the conversion of the 110 kDalton (child) precursor into 76 and 70 child mature forms via proteolysis in lysosomes. As used herein, the term "Pompe disease" refers to all types of Pompe disease. The formulations and dosing regimens disclosed in this application may be used to treat, for example, type I, II, or III Pompe disease.

[0052] NON-RESTRICTIVE EMBODIMENTS OF THE INVENTION

[0053] A rhGAA composition derived from CHO cells, the rhGAA composition comprising more than through (Aglucosidase α; CAS 420794-05-0) exemplifies a higher amount of rhGAA than conventional rhGAA, which comprises an N-glycan carrying mono-mannose-6-phosphate (M6P) or bis-M6P. An exemplary rhGAA composition according to the invention is ATB-200 (sometimes referred to as ATB-200, ATB-200, or CBP-rhGAA) described in the examples. The rhGAA (ATB-200) of the invention has been shown to have high affinity (K... D Approximately 2-4 nM) binds to CIMPR and is induced by Pompe fibroblasts and skeletal myoblasts (K). 摄取 It can be effectively internalized (approximately 7-14 nM). ATB-200 was characterized in vivo and showed to have better performance than current rhGAA ERT(t). 1 / 2 Shorter apparent plasma half-life (t0) (approximately 60 min) 1 / 2 (Approximately 45 minutes).

[0054] The amino acid sequence of the rhGAA can be at least 70%, 75%, 80%, 85%, 95%, or 99% identical to, or comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more deletions, substitutions, or additions from, the amino acid sequence described by SEQ ID NO: 1, 3, or 4. In some embodiments of the GAA or rhGAA of the application, as in the ATB-200 rhGAA, the GAA or rhGAA will comprise a wild-type GAA amino acid sequence, such as the amino acid sequence of SEQ ID NO: 1 or 3. In other non-limiting embodiments, the rhGAA comprises a subset of the amino acid residues present in wild-type GAA, wherein the subset comprises the amino acid residues of wild-type GAA that form the active site for substrate binding and / or substrate reduction. In one embodiment, the rhGAA is a glucosidase alpha, which is the human enzyme acid alpha-glucosidase (GAA), encoded by the most prevalent nine observed haplotypes of the gene. The rhGAA of the application, including the ATB-200 rhGAA, can comprise an amino acid sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of human alpha glucosidase, such as the amino acid sequence given by accession number AHE24104.1 (GI:568760974) (SEQ ID NO: 1) and incorporated by reference to U.S. Patent No. 8,592,362 or to the amino acid sequence of NP_000143.2 (SEQ ID NO: 4). The nucleotide and amino acid sequences of GAA are also given by SEQ ID NOs: 2 and 3, respectively. Variants of the amino acid sequence also include those having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acid deletions, insertions, or substitutions from the following GAA amino acid sequence. Polynucleotide sequences encoding GAA and such variant human GAA are also contemplated and can be used to recombinantly express the rhGAA according to the application.

[0055] Different alignment algorithms and / or programs can be used to calculate identity between two sequences, including FASTA or BLAST available as part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used with, for example, default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein, and preferably exhibiting essentially the same function, are contemplated, as are polynucleotides encoding such polypeptides. Unless otherwise specified, similarity scores will be based on the use of BLOSUM62. When using BLASTP, the percent similarity is based on the BLASTP positive score, and the percent sequence identity is based on the BLASTP consistency score. BLASTP "consistency" shows the number and fraction of total residues in high-scoring sequence pairs that are identical; and BLASTP "positives" show the number and fraction of residues that have a positive alignment score and are similar to each other. The present disclosure contemplates and encompasses amino acid sequences having these degrees of identity or similarity, or any intermediate degree of identity or similarity, to the amino acid sequences disclosed herein. Polynucleotide sequences of similar polypeptides deduced using the genetic code, and can be obtained by routine means, particularly by reverse-transcribing their amino acid sequences using the genetic code.

[0056] Preferably, no more than 70%, 65%, 60%, 55%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total rhGAA in the composition according to the present application lacks N-glycans bearing M6P or bis-M6P or lacks the ability to bind the cation-independent mannose-6-phosphate receptor (CIMPR). Alternatively, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, <100%, or more of the rhGAA in the composition comprises at least one N-glycan bearing M6P and / or bis-M6P or has the ability to bind CIMPR.

[0057] The rhGAA molecules in the rhGAA compositions of the present application can have 1, 2, 3, or 4 M6P groups on their glycans. For example, the only N-glycan on an rhGAA molecule can bear M6P (mono-phosphorylated), a single N-glycan can bear two M6P groups (di-phosphorylated), or two different N-glycans on the same rhGAA molecule can bear a single M6P group. The rhGAA molecules in the rhGAA compositions can also have N-glycans that do not bear M6P groups. In another embodiment, the N-glycans, on average, contain greater than 3 mol / mol of M6P and greater than 4 mol / mol of sialic acid. On average, at least about 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total glycans on the rhGAA can be in the mono-M6P glycan form, for example, about 6.25% of the total glycans can carry a single M6P group, and on average, at least about 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0% of the total glycans on the rhGAA are in the di-M6P glycan form, and on average, less than 25% of the total rhGAA of the present application does not contain phosphorylated glycans that bind to CIMPR.

[0058] The rhGAA compositions according to the present application can have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0 mol / mol rhGAA, or any intermediate value of the subranges 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 mol / mol rhGAA. As shown in the examples, the rhGAA of the present application can be fractionated to provide rhGAA compositions having different average numbers of M6P-bearing or di-M6P-bearing glycans on the rhGAA, allowing further customization of the rhGAA to target lysosomes in target tissues by selecting particular fractions or by selectively combining different fractions.

[0059] Up to 60% of the N-glycans on the rhGAA can be fully sialylated, for example, up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans can be fully sialylated. In some embodiments, from 4% to 20% of the total N-glycans in the rhGAA composition are fully sialylated.

[0060] In other embodiments, no more than 5%, 10%, 20%, or 30% of the N-glycans on the rhGAA carry sialic acid and terminal Gal. This range includes all intermediate values and subranges, for example, 7% to 30% of the total N-glycans on the rhGAA in the composition can carry sialic acid and terminal Gal.

[0061] In yet other embodiments, no more than 5%, 10%, 15%, 16%, 17%, 18%, 19%, or 20% of the N-glycans on the rhGAA in the composition have only terminal Gal and do not include sialic acid. This range includes all intermediate values and subranges, for example, from 8% to 19% of the total N-glycans on the rhGAA in the composition can have only terminal Gal and do not include sialic acid.

[0062] In other embodiments of the application, 40%, 45%, 50%, 55% to 60% of the total N-glycans on the rhGAA in the composition are complex type N-glycans; or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7% of the total N-glycans on the rhGAA in the composition are hybrid type N-glycans; no more than 5%, 10%, or 15% of the high mannose type N-glycans on the rhGAA in the composition are unphosphorylated; at least 5% or 10% of the high mannose type N-glycans on the rhGAA in the composition are mono-M6P phosphorylated; and / or at least 1% or 2% of the high mannose type N-glycans on the rhGAA in the composition are di-M6P phosphorylated. These values include all intermediate values and subranges. The rhGAA compositions according to the application can meet one or more of the above content ranges.

[0063] In some embodiments, the rhGAA compositions of the application will carry an average of 2.0 to 8.0 sialic acid residues per mol of rhGAA. This range includes all intermediate values and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 residues per mol of rhGAA. The sialic acid residues can prevent non-productive clearance by asialoglycoprotein receptors.

[0064] The rhGAA compositions of the application are preferably produced by CHO cells, such as the CHO cell line GA-ATB-200, or by subcultures or derivatives of such CHO cell cultures. DNA constructs expressing allelic variants of GAA or other variant GAA amino acid sequences, such as those having at least 90%, 95%, or 99% identity to SEQ ID NO: 1, can be constructed and expressed in CHO cells. One of ordinary skill in the art can select alternative vectors suitable for transforming CHO cells for producing such DNA constructs.

[0065] The inventors have discovered that a glycosylation pattern can be used to produce rhGAA with superior ability to target CIMPR and lysosomes, as well as reduce non-productive clearance in vivo, using Chinese hamster ovary (CHO) cells. These cells can be induced to express rhGAA with significantly higher levels of total M6P and bis-M6P than conventional rhGAA products. Recombinant human GAA produced by these cells, for example, as exemplified by rhGAA ATB-200 as described in the Examples, has significantly more M6P and bis-M6P groups that target muscle cells than conventional GAA (e.g., MyoGAA® ) and has been shown to effectively bind to CIMPR and be taken up efficiently by skeletal and cardiac muscle. The glycosylation pattern has also been shown to provide a favorable pharmacokinetic profile and reduce non-productive clearance in vivo.

[0066] The rhGAA according to the present application can be formulated into a pharmaceutical composition or used to produce a medicament for treating Pompe disease or other conditions associated with a deficiency in GAA. These compositions can be formulated with a physiologically acceptable carrier or excipient. The carrier and composition can be sterile and otherwise suitably adapted for the mode of administration.

[0067] Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solution (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or other, glucose, magnesium stearate, talc, silicic acid, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like, and combinations thereof. If desired, the pharmaceutical preparation can be mixed with auxiliary agents such as surfactants (e.g., polysorbates, like polysorbate 80), lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatics, etc., which do not have a deleterious reaction with the active composition. In preferred embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0068] The composition or medicament can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can also be formulated as a suppository, utilizing a

[0069] The composition or medicament can be formulated according to conventional procedures, as appropriate, into a pharmaceutical composition suitable for administration to humans. For example, in a preferred embodiment, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or aqueous solution in a hermetically sealed container such as an ampule or a small vial. Where the composition is to be administered by infusion, it can be dispensed by a pressurized container containing the composition in a sterile pharmaceutical grade water, saline, or dextrose / water. Where the composition is to be administered by injection, an ampule of sterile injectable water or saline can be provided so that the ingredients can be mixed prior to administration.

[0070] The rhGAA can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include those formed from free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those derived from free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.

[0071] The rhGAA (or composition or medicament comprising GAA) is administered by an appropriate route. In one embodiment, the GAA is administered intravenously. In other embodiments, the GAA is administered by direct administration to the target tissue (e.g., to the heart or skeletal muscle (e.g., intramuscularly) or nervous system (e.g., direct injection into the brain; intracerebroventricularly; intrathecally). If desired, more than one route can be used simultaneously.

[0072] The rhGAA (or a composition or drug containing a GAA) is administered in a therapeutically effective amount (e.g., a dose sufficient to treat the disease when administered at regular intervals, as described above by alleviating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also reducing the severity or frequency of the disease's symptoms). The therapeutically effective amount in treating the disease will depend on the nature and extent of the disease's effects and can be determined using standard clinical techniques. Additionally, in vitro or in vivo assays may optionally be used to help identify the optimal dose range. The precise dose used will also depend on the route of administration and the severity of the disease and should be determined based on the practitioner's judgment and the individual patient's situation. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems. In preferred embodiments, the therapeutically effective amount is equal to or less than 20 mg enzyme / kg body weight, preferably in the range of about 1-10 mg enzyme / kg body weight, and even more preferably about 10 mg enzyme / kg body weight or about 5 mg enzyme / kg body weight. The effective dose for a particular individual may vary over time (e.g., increasing or decreasing) depending on the individual's needs. For example, the amount can increase during physical illness or stress, or if anti-GAA antibodies are present or increased, or if disease symptoms worsen.

[0073] A therapeutically effective dose of GAA (or a composition or drug containing GAA) is administered at regular intervals, depending on the nature and extent of the disease effect and the basis for its continuation. As used herein, "regular intervals" indicates that the therapeutically effective dose is administered periodically (as opposed to a single dose). This interval can be determined using standard clinical techniques. In preferred embodiments, GAA is administered monthly, every two months; weekly; twice weekly; or daily. The dosing interval for an individual does not need to be fixed but can vary over time depending on the individual's needs. For example, the interval between doses may be reduced during physiological illness or physical stress if anti-GAA antibodies are present or increased, or if disease symptoms worsen. In some embodiments, a therapeutically effective dose of 5, 10, 20, 50, 100, or 200 mg of enzyme / kg body weight is administered twice weekly, weekly, or every other week, with or without a molecular chaperone.

[0074] The GAA or rhGAA of the present invention can be prepared for later use, such as in unit-dose vials or syringes, or in bottles or bags for intravenous administration. Kits containing GAA or rhGAA, along with optional excipients or other active ingredients, such as molecular chaperones or other drugs, can be encapsulated in packaging materials and accompanied by instructions for rehydration, dilution, or dosing, for the treatment of subjects in need, such as patients with Pompe disease.

[0075] GAA (or a composition or medicament comprising GAA) can be administered alone or in combination with other agents, such as chaperones. rhGAA having varying degrees of glycosylation with mono- or bi-M6P or combinations of rhGAA having varying degrees of M6P or bi-M6P glycosylation can be administered.

[0076] In some embodiments, the rhGAA compositions of the present application will be complexed or mixed with a chaperone, such as AT-2220 or AT-2221. Chaperones, sometimes referred to as "pharmacological chaperones," are compounds that alter the pharmacokinetics and other pharmacological properties of rhGAA when complexed or co-administered therewith. Representative chaperones exemplified herein include AT2221 (miglustat, N-butyl-deoxynojirimycin) and AT2220 (duvoglustat HCl, 1-deoxynojirimycin). Such complexing or mixing can occur outside or inside the body, e.g., where separate doses of rhGAA and chaperone are administered. For example, the active rhGAA, fractions or derivatives of the present application can be improved in targeting to CIMPR and subsequently to the lysosome of the cell by combining it with duvoglustat-HCl (AT2220, deoxynojirimycin, AT2220) or miglustat (AT2221, N-butyl-deoxynojirimycin). The following example shows a significant reduction in glycogen substrate in key skeletal muscles of knock-out GAA mice that received rhGAA of the present application in combination with a chaperone that was well targeted.

[0077] Another aspect of the present application relates to CHO cells or their derivatives or other equivalents that produce rhGAA according to the present application. One example of such a CHO cell line is GA-ATB-200 or a subculture thereof that produces rhGAA compositions as described herein. Such a CHO cell line can comprise multiple copies of the gene for the polynucleotide encoding GAA, e.g., 5, 10, 15 or 20 or more copies.

[0078] The high M6P and bi-M6P rhGAA of the present application, such as ATB-200 rhGAA, can be produced by transforming CHO cells (Chinese hamster ovary cells) with a DNA construct encoding GAA. Although CHO cells have been used previously to make rhGAA, it was not appreciated that transformed CHO cells could be cultured and selected in a manner to produce rhGAA having high levels of M6P and bi-M6P glycan targeting to CIMPR.

[0079] Surprisingly, the inventors found that CHO cell lines can be transformed, selecting transformants producing rhGAA comprising high levels of glycans bearing M6P or bis-M6P targeting CIMPR, and stably expressing the high-M6P rhGAA. Accordingly, related aspects of the invention relate to methods for making these CHO cell lines. The methods involve transforming CHO cells with DNA encoding GAA or a GAA variant, selecting CHO cells that stably integrate the DNA encoding GAA into one or more of their chromosomes and stably express GAA, and selecting CHO cells that express GAA with high levels of glycans bearing M6P or bis-M6P, and optionally selecting CHO cells with high sialic acid content N-glycans, and / or with low non-phosphorylated high-mannose content N-glycans.

[0080] The CHO cell lines can be used to produce rhGAA and rhGAA compositions according to the invention by culturing the CHO cell lines, and recovering the compositions from the CHO cell cultures.

[0081] The rhGAA compositions of the invention, or fractions or derivatives thereof, are advantageously used to treat subjects having a disorder, disease or condition associated with lysosomal GAA deficiency by administering the rhGAA compositions. Subjects in need of treatment include those having Pompe disease (glycogen storage disease type II), and other disorders, diseases or conditions that would benefit from administration of rhGAA.

[0082] The following examples show that the rhGAA of the invention (ATB-200) is taken up by skeletal muscle cells, binds to CIMPR, and effectively removes glycogen from skeletal muscle cells when administered at significantly lower doses than conventional rhGAA products. A once every two week schedule of intravenous administration of ATB-200 in knock-out GAA mice resulted in up to 75% reduction of glycogen in skeletal muscle myoblasts. These reductions exceeded those obtained from the same amount of The rhGAA of the invention having enhanced levels of N-glycans bearing M6P and bis-M6P provide greater reduction of glycogen substrates, due to improved targeting. The pharmacodynamics and pharmacokinetics of the rhGAA compositions of the invention can be administered at lower doses than conventional rhGAA products (such as or ).

[0083] It can be used to degrade, reduce, or remove glycogen from myocardium, smooth muscle, or striated muscle. Examples of skeletal muscle or striated muscle subjects subjected to treatment include at least one muscle selected from the group consisting of abductor digiti minimi (foot), abductor digiti minimi (hand), abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor longus, adductor magnus, adductor pollicis, anconeus, articularis cubiti, articularis genus, aryepiglotticus, aryjordanicus, auricularis, biceps brachii, biceps femoris, biceps, brachioradialis, buccinator, bulbospongiosus, constrictor pharyngis inferior, constrictor pharyngis medius, constrictor pharyngis superior, coracobrachialis, corrugator supercilii, cremaster, cricothyreoideus, diaphragm, diaphragm (anterior view), erector spinae- spinalis, erector spinae-iliocostalis, erector spinae-longissimus, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi (hand), extensor digitorum (hand), extensor digitorum brevis (foot), extensor digitorum longus (foot), extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, external oblique, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis (foot), flexor digiti minimi (hand), flexor digitorum brevis, flexor digitorum longus (foot), flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, frontalis, gastrocnemius, gemellus inferior, gemellus superior, genioglossus, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, hyoglossus, iliocapsularis, inferior oblique, inferior rectus, infraspinatus, intercostales externi, intercostales profundii, intercostales interni, internal oblique, interossei-dorsal of hand, interossei-dorsal of foot, palmaris, plantaris, intertransversarii, intrinsic muscles of tongue, ischiocavernosus, lateral cricoarytenoid, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator ani- coccygeus, levator ani-iliococcygeus, levator ani- pubococcygeus, levator ani- pubovaginalis, levator labii superioris, levator labii superioris, alaeque nasi, levator palpebrae superioris, levator scapulae, levator veli palatine, levatores costarum, longissimus capitis, longissimus colli, lumbrical muscles (4), lumbricales manus, masseter, medial pterygoid, medial rectus, mentalis, palatoglossus,uvulae), mylohyoid muscle, nasal muscle, arytenoid muscle, inferior oblique capitis muscle, superior oblique capitis muscle, obturator externus muscle, obturator internus muscle (A), obturator internus muscle (B), omohyoid muscle, pinna minor (hand), pinna pollicis, orbicularis oculi muscle, orbicularis oris muscle, palatoglossus muscle, palatopharynx muscle, palmaris brevis muscle, palmaris longus muscle, pectineus muscle, pectoralis major muscle, pectoralis minor muscle, peroneus brevis muscle, peroneus longus muscle, peroneus tertiary muscle, piriformis muscle (A), piriformis muscle (B), plantar muscles, platysma muscle, popliteal muscle, posterior cricoarytenoid muscle, depressor supercilii muscle, pronator quadratus muscle, pronator teres muscle, psoas major muscle, psoas minor muscle, pyramidal muscle, quadratus femoris muscle, quadratus lumborum muscle, quadratus plantaris muscle, rectus abdominis muscle, rectus capitis anterior muscle (rectus) (capitusanterior), rectus capitis posterior, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, eustachian tube pharynx, sartorius, anterior scalene, middle scalene, minor scalene, posterior scalene, semimembranosus, semitendinosus, serratus anterior, serratus posterior inferior, serratus posterior superior, soleus, anal sphincter, urethral sphincter, splenius capitis, splenius cervicis, stapedius, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, stylohyoid (anterior), stylopharynx, subclavian, subcostalis, subscapularis, superficial transverse perineum. The following muscles are involved in the muscle grouping: perinei (superior oblique), superior rectus, supinator, supraspinatus, temporalis, temporoparietal, tensor fasciae latae, tensor tympani, tensor veli palatini, teres major, teres minor, thyroarytenoid, vocal cord muscles, thyroepiglotticus, thyrohyoid, tibialis anterior, tibialis posterior, transverse arytenoid, transverse spinae (multifidus), transverse spinae (semispinalis), transverse abdominis, transverse thoracic, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus major.

[0084] The GAA compositions of the present invention can also be administered to or used to treat type 1 (slow contraction) or type 2 (fast contraction) muscle fibers or subjects with glycogen accumulation in these muscle fibers. Type I, slow contraction or “red” muscle, is rich in capillaries and mitochondria and myoglobin, which gives muscle tissue its characteristic red color. It can carry more oxygen and uses fat or carbohydrates as fuel to sustain aerobic activity. Slow contraction muscle fibers contract for long periods but with little force. Type II, fast contraction muscle has three main subtypes (IIa, IIx, and IIb) that differ in both contraction speed and the force produced. Fast contraction fibers contract rapidly and powerfully, but fatigue very quickly, lasting only briefly, with an anaerobic burst of activity before muscle contraction becomes painful. They contribute most to muscle strength and have the potential for greater mass increase. Type IIb is anaerobic, glycolytic, and the “white” muscle with the lowest density of mitochondria and myoglobin. In small animals (e.g., rodents), this is the predominant fast muscle type, which explains the pale color of their flesh.

[0085] The rhGAA compositions, fractions or derivatives of the application can be administered systemically, for example by intravenous (IV) infusion, or directly to the desired site, such as into the myocardium or skeletal muscle (such as quadriceps, triceps, or diaphragm). It can be administered to muscle cells, specific muscle tissue, muscle, or muscle groups. For example, such treatment can administer the rhGAA composition directly intramuscularly to the subject's quadriceps or triceps or diaphragm.

[0086] As mentioned above, the rhGAA compositions, fractions or derivatives of the application can be complexed or mixed with a chaperone such as AT-2220 (Duvoglustat HC1, 1- deoxynojirimycin) or AT-2221 (Meglitinide, N-Butyl-deoxynojirimycin) or salts thereof to improve the pharmacokinetics of the rhGAA administration. The rhGAA and chaperone can be administered together or separately. When administered simultaneously, the GAA in the composition can be pre-loaded with the chaperone. Alternatively, the GAA and chaperone can be administered at the same time or separately at different times.

[0087] Representative dosage ranges for AT2221 are from 0.25 to 400 mg / kg, preferably from 0.5-200 mg / kg, and most preferably from 2 to 50 mg / kg. Specific dosages for AT2221 include 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / kg. These dosages can be combined with rhGAA (such as ATB-200 rhGAA) in molar ratios of AT2221 to rhGAA ranging from 15: 1 to 150: 1. Specific ratios include 15: 1, 20: 1, 25: 1, 50: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 100: 1, 125: 1, and 150: 1. The rhGAA and AT2221 can be co-administered simultaneously, sequentially, or separately in these amounts or molar ratios. The above ranges include all intervening sub-ranges and values, such as all integer values between the range endpoints.

[0088] Representative dosage ranges for AT2220 are from 0.1 to 120 mg / kg, preferably 0.25 to 60 mg / kg, and most preferably from 0.6 to 15 mg / kg. Specific dosages of AT2220 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, and 30 mg / kg. These dosages can be combined with rhGAA (such as ATB-200 rhGAA) in molar ratios of AT2220 to rhGAA ranging from 15: 1 to 150: 1. Specific ratios include 15: 1, 20: 125: 1, 50: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 100: 1, 125: 1, and 150: 1. The rhGAA and AT2220 can be co-administered simultaneously, sequentially, or separately in these amounts or molar ratios. The above ranges include all intervening sub-ranges and values, such as all integer values between the range endpoints.

[0089] The rhGAA compositions, fractions, or derivatives of the application can also be used to metabolize, degrade, remove, or otherwise reduce glycogen in tissues, muscle, muscle fibers, muscle cells, lysosomes, organelles, cellular compartments, or cytoplasm. This is accomplished by administering the rhGAA composition to a subject, optionally with a chaperone or a drug that reduces the immune response to the rhGAA.

[0090] In another embodiment of its method of use, the rhGAA of the application can be used to modulate lysosomal proliferation, autophagy, or exocytosis in a cell by administering it, a fraction or derivative thereof, to a cell, tissue, or subject in need of such modulation, optionally in combination with a chaperone or optionally as a conjugate with another targeting moiety. Autophagy is a catabolic mechanism that allows a cell to degrade glycogen or other unnecessary or dysfunctional cellular components through the action of its lysosomes. This method can also involve systemically or locally administering the GAA composition to a subject in need of treatment.

[0091] The rhGAA according to the application (in combination with and Myozyme, which is enriched in mono- and bi-M6P and which has advantageous pharmacokinetic properties conferred by its glycosylation pattern) can also be used to treat other disorders in which complex carbohydrates are broken down, such as other disorders in which glycogen or other carbohydrates degraded by rhGAA accumulate in lysosomes or other parts of cells, such as in the cytoplasm of cells accessible to rhGAA, e.g., glycogen storage disease III. It can also be used for non-therapeutic purposes, such as for the production of food, beverage, chemical, and pharmaceutical products that require the breakdown of complex carbohydrates, such as starches and glycogen, into their monomers.

[0092] EXAMPLES

[0093] The following non-limiting examples illustrate various aspects of the application.

[0094] Section I: ATB-200 rhGAA and its properties

[0095] Prior Limitations of rhGAA products FIG. 4B

[0096] To assess the ability of rhGAA in and (only approved treatment for Pompe disease) to bind to CIMPR, these rhGAA preparations were injected onto a CIMPR column, which binds rhGAA with M6P groups, and subsequently eluted with a free M6 gradient. Fractions were collected in 96 well plates and GAA activity was determined by a 4MU-a-glucose substrate assay. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a fraction of total enzyme.

[0097] Figure 4 depicts the problem associated with conventional ERT (MyoKardia®) and ) in 73% of rhGAA (MyoKardia®) FIG. 4A ) and 78% of rhGAA (MyoKardia®) Generation of ATB-200 rhGAA CHO cells with high content of N-glycans with mono- or bi-M6P ) did not bind to CIMPR, see the left most peak in each figure. In only 27% of rhGAA and in 22% of rhGAA contained M6P, which can productively target it to CIMPR on muscle cells, see Figure 2, which depicts productive drug targeting and non-productive drug clearance.

[0098] and The effective dose of these two conventional products corresponds to the amount of rhGAA that contains M6P, which targets CIMPR on muscle cells. However, the majority of rhGAA in both of these conventional products does not target CIMPR receptors on the target muscle cells. Administration of conventional rhGAA, in which the majority of rhGAA does not target muscle cells, increases the risk of anaphylaxis or immune induction to the non-targeted rhGAA.

[0099] Preparation. FIG. 5

[0100] CHO cells were transfected with DNA expressing rh-GAA, followed by selection of transformants producing rhGAA. The DNA construct used to transform CHO cells with DNA encoding rh-GAA is shown in Purification of rhGAA ATB-200 rhGAA CHO cells were transfected with DNA expressing rh-GAA, followed by selection of transformants producing rhGAA.

[0101] Following transfection, DG44 CHO (DHFR-) cells containing stably integrated GAA gene were selected using hypoxanthine / thymidine deficient (-HT) media. Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools expressing high levels of GAA were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid media plates, picked by ClonePix system, and transferred to 24 deep well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. Conditioned media for determining GAA activity used 4-MU-a-glucosidase substrate. Clones producing higher levels of GAA as measured by GAA enzyme assay were further evaluated for viability, growth ability, GAA productivity, N-glycan structure, and stable protein expression. Using this procedure, CHO cell lines were isolated, including CHO cell line GA-ATB-200 expressing rhGAA with enhanced mono-M6P or bi-M6P N-glycans.

[0102] FIG. 6B

[0103] Multiple batches of rhGAA according to the application were produced in shake flasks, and binding was measured in perfusion bioreactors using CHO cell line GA-ATB-200 and CIMPR. Similar CIMPR receptor binding (about 70%) was observed for purified ATB-200 rhGAA from different production batches compared to those shown in FIG. 6A and Figure 7, indicating that ATB-200 rhGAA can be consistently produced. As shown in Analytical comparison of ATB-200 vs. Lumizyme and 6B ATB200 rhGAA compared to and rhGAA showed significantly less CIMPR binding.

[0104] FIG. 8

[0105] ATB-200 rhGAA was fractionated using weak anion exchange (“WAX”) liquid chromatography based on the terminal phosphate group. Elution profiles were generated by eluting ERT with incremental salt increments. These profiles were monitored by UV (A280 nm). ATB-200 rhGAA was obtained from CHO cells and purified. Obtained from commercial sources. A peak is shown on the left side of its elution curve. ATB-200 rhGAA shows elution at... The four prominent peaks on the right ( Oligosaccharide characterization of ATB-200 rhGAA Since this assessment is conducted via terminal charge rather than CIMPR affinity, this confirms that ATB-200 rhGAA is phosphorylated to a higher degree than... To a greater extent.

[0106] FIG. 9

[0107] The purified ATB-200 rhGAA and [other components] were evaluated using MALDI-TOF. Glycans were used to determine the individual glycan structures found on each ERT. FIG. 10A The ATB-200 sample was found to contain more than A slightly smaller amount of non-phosphorylated, high-mannose N-glycans. Compared to Lumizyme, the higher M6P glycan content in ATB-200 more effectively targets ATB-200 rhGAA to myocytes. The high percentage of mono- and di-phosphorylated structures determined by MALDI are consistent with the CIMPR curve, indicating a significantly greater binding of ATB-200 to the CIMPR receptor. N-glycan analysis by MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contains at least one native bis-M6P N-glycan structure. This higher bis-M6P N-glycan content on ATB-200 rhGAA is directly correlated with the high affinity binding to CIMPR in the M6P receptor plate binding assay (K). D Approximately 2-4 nM Characterization of CIMPR affinity of ATB-200 ).

[0108] FIG. 10A

[0109] In addition to having a larger percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of this interaction. This was determined using CIMPR plate binding assays. It binds to the ATB200 rhGAA receptor. In short, CIMPR-coated plates are used to capture GAA. Different concentrations of rhGAA are applied to the immobilized receptor, and unbound rhGAA is washed away. The amount of remaining rhGAA is determined by GAA activity.FIG. 10B As shown, ATB-200 rhGAA bound to CIMPR is significantly superior to Lumizyme.

[0110] ATB-200 rhGAA is more efficiently internalized by fibroblasts than Lumizyme. The relative contents of bis-M6P polysaccharides in Lumizyme, conventional rhGAA, and ATB-200 according to the present invention are shown. For On average, only 10% of the molecules have diphosphorylated glycans. This is compared to ATB-200, where on average each rhGAA molecule has at least one diphosphorylated glycan.

[0111] FIG. 11A

[0112] ATB-200 and normal and Pompeii fibroblast cell lines were used to compare ATB-200 and Relative cellular uptake of rhGAA. A comparison was made between 5-100 nM of ATB-200 rhGAA according to the present invention and 10-500 nM of conventional rhGAA. After 16 hours of incubation, external rhGAA was inactivated with TRIS base, and cells were washed three times with PBS before harvest. Internalized GAA was measured by 4 MU-α-glucosidase hydrolysis and plotted against total cellular protein; the results are shown in Figure 11.

[0113] It also shows that ATB-200 rhGAA is effectively internalized in cells ( FIG. 11C and 11B The images show that ATB-200rhGAA is internalized into normal and Pompeii fibroblasts, and that it is more effective than conventional ATB-200rhGAA. rhGAA is internalized to a greater extent. ATB-200 rhGAA saturates cellular receptors at approximately 20 nM, however, approximately 250 nM is required. The uptake efficiency constant (K) extrapolated from these results 摄取 For ATB-200, it is 2-3nm, and for It is 56 nM, such as Section II: Preclinical studies As shown, these results indicate that ATB-200 rhGAA is a well-targeted treatment for Pompe disease.

[0114] ATB-200 rhGAA with superior glycosylation significantly outperforms the care ERT standard for glycogen clearance in skeletal muscle of GAA KO mice

[0115] FIG. 12A FIG. 12B

[0116] As explained above, enzyme replacement therapy (ERT) using recombinant human GAA (rhGAA) is the only approved treatment for Pompe disease. This ERT requires a specific carbohydrate, mannose-6-phosphate (M6P), for cellular uptake and subsequent delivery to lysosomes via the cell surface cation-independent M6P receptor (CIMPR). However, current rhGAA ERTs contain low levels of M6P, limiting drug targeting and efficacy in disease-related tissues. The inventors have developed cell lines and manufacturing methods that produce rhGAA (designated ATB-200rhGAA) with superior glycosylation and higher M6P content (specifically, a high-affinity bis-M6P N-glycan structure) than conventional rhGAA, to improve drug targeting. ATB-200 rhGAA binds to the CI-MPR with high affinity (KD approximately 2-4 nM) and is efficiently internalized by Pompe fibroblasts and skeletal myoblasts (KD approximately 2-4 nM). 摄取 (Approximately 7-14 nM).

[0117] ATB-200 rhGAA showed significantly better glycogen clearance in skeletal muscle than... Assessment given Effects of ATB-200 rhGAA on glycogen clearance in GAA KO mice. Animals were administered two intravenous bolus injections (weekly intervals); tissues were harvested two weeks after the last dose, and GAA activity and glycogen content were analyzed (Figure 12). ATB-200 rhGAA and rhGAA is also effective in clearing glycogen from the heart. Rationale for co-administration of ATB-200 rhGAA with AT2221 (Myozyme®) ).like FIG. 13A and 12C As shown, for reducing glycogen in skeletal muscle, 5 mg / kg of ATB-200 rhGAA is equivalent to 20 mg / kg of [unclear - possibly a specific drug or treatment]. rhGAA; ATB-200 administered at 10 and 20 mg / kg was significantly superior for clearing glycogen from skeletal muscle.

[0118] Comparison of co-administration of ATB-200 rhGAA with Myozyme® vs. Myozyme®

[0119] Molecular chaperones bind to and stabilize rhGAA ERT, increasing the uptake of active enzymes into tissues, improving tolerance, and potentially reducing immunogenicity. As shown above, using CHART... TM This significantly improves the protein stability of ERT under adverse conditions. CHART: Advanced Alternative Therapies for Molecular Chaperones, see http: / / www.amicusrx.com / chaperone.aspx (last accessed September 22, 2015), which will be cited here. Co-administration with AT2221 (Myozyme®) and13B As shown, AT2221 (Miglustat, N-butyl-deoxynojirimycin) significantly improved the stability of ATB-200. The folding of rhGAA protein was monitored by heat denaturation in neutral (pH 7.4 - plasma environment) or acidic (pH 5.2 - lysosomal environment) buffers at 37°C. In neutral pH buffer, AT2220 stabilized rhGAA protein after 24 hours.

[0120] FIG. 14 FIG. 15 FIG. 16A

[0121] 12-week-old GAA KO mice were used Alternatively, ATB200 treatment, 20 mg / kg IV injections four times every other week; 30 minutes before rhGAA as shown, mirtegafur is co-administered with 10 mg / kg PO. Tissue is collected 14 days after the last enzyme dose for glycogen measurement. FIG. 16C It shows a relative reduction in glycogen in the quadriceps and triceps skeletal muscles.

[0122] ATB-200 rhGAA, administered in combination with the pharmacological molecular chaperone AT2221 (Miglustat), reduces tissue glycogen.

[0123] The combination of a pharmacological molecular chaperone and ATB-200 rhGAA was found to enhance glycogen clearance in vivo. GAA KO mice were administered rhGAA twice weekly via intravenous bolus at 20 mg / kg. The pharmacological molecular chaperone AT2221 was orally administered at doses of 0, 1, 2, and 10 mg / kg 30 minutes prior to the rhGAA administration. Tissues were harvested two weeks after the last dose of ERT, and GAA activity, glycogen content, cell-specific glycogen, and lysosomal proliferation were analyzed.

[0124] like FIG. 16C As shown, animals receiving ATB200+ molecular chaperone AT2221 exhibited enhanced glycogen clearance from the quadriceps muscle. ATB-200 rhGAA (20 mg / kg) reduced glycogen levels more than the same dose. Furthermore, when ATB-200rhGAA is combined with 10 mg / kg of AT2220, near-normal levels of glycogen are achieved in the muscles.

[0125] like FIG. 16D and 16BAs shown, unlike conventional rhGAA, which shows limited glycogen reduction (indicated by the abundant punctate PAS signal), ATB-200 rhGAA alone shows a marked reduction in PAS signal. Co-administration with 10 mg / kg magistan leads to a substantial further reduction in substrate. TEM reveals that glycogen in most lysosomes is membrane-bound, electron-dense material corresponding to the punctate PAS signal. Co-administration of ATB-200 rhGAA with magistan reduces the number, size, and density of lysosomes containing substrate, indicating targeted delivery of ATB-200 rhGAA to muscle cells and subsequent delivery to lysosomes.

[0126] From the studies shown above (2 IV bolus injections every other week), it is seen that treatment of tissue with the LAMP 1 marker for lysosome proliferation, upregulation is another marker of Pompe disease. LAMP: lysosome-associated membrane protein. From the studies shown above (2 IV bolus injections EOW injections), it is seen that the treatment of soleus muscle tissue for LAMP 1 staining in adjacent sections, and addition of type I fiber-specific antibody (NOQ7.5.4D) in adjacent sections. FIG. 16C and 16D ) compared to conventional rhGAA. ATB-200 rhGAA results in a greater reduction in LAMP1 compared to conventional rhGAA, which results in levels seen in WT animals. FIG. 17A

[0127] Furthermore, unlike rhGAA, where this effect is largely restricted to type I fibers (slow twitch, marked with asterisks), ATB-200 rhGAA also results in a marked reduction in LAMP1 signal in a subset of type II (fast twitch) fibers (red arrows). FIG. 17B ​ and 16D ) Co-administration with magistan further improves the ATB-200-mediated reduction in LAMP1 proliferation in most type II fibers. As a result, it appears that there is no significant fiber type-specific difference in LAMP1 signal levels. Similar conclusions were reached from the quadriceps and diaphragm (data not shown).

[0128] In a separate and similarly designed study, the effect of ATB-200 ± AT2221 was examined with 4 biweekly IV bolus injections over a longer period. In the heart, the predominant glycogen accumulation in cardiomyocytes was readily cleared by repeated administration of rhGAA or ATB-200 to levels seen in wild-type (WT) animals. ​ ​​). However, the substrate in cardiac smooth muscle cells appears to be preferentially cleared by ATB-200 rhGAA, suggesting a potentially broader biodistribution of ATB-200 compared to rhGAA (asterisk marks the lumen of the cardiac blood vessels). Importantly, co-administration with maglestatat further improved the reduction in LAMP1 proliferation mediated by ATB-200.

[0129] These results demonstrate that ATB-200 rhGAA, which has higher levels of M6P and bis-M6P on its N-glycans, effectively targets CIMPR in skeletal muscle. ATB-200 rhGAA also has well-processed complex-type N-glycans that minimize non-productive clearance in vivo, has pharmacokinetic properties that are favorable for its use in vivo, and exhibits good targeting to key muscle tissues in vivo. They also demonstrate that ATB-200 rhGAA is superior to the conventional-of-care standard Lumizyme for reducing glycogen in muscle tissue, and that the combination of ATB-200 rhGAA and the molecular chaperone AT2221 further improves glycogen removal from target tissues and improves muscle pathology. SEQUENCE LISTING <110> AMICUS THERAPEUTICS, INC. <120> High-strength acid alpha-glucosidase with enhanced carbohydrates <130> TWLB13957-21P1 <150> US 62 / 135,345 <151> 2015-03-19 <150> US 62 / 112,643 <151> 2015-02-05 <150> US 62 / 057,847 <151> 2014-09-30 <150> US 62 / 057,842 <151> 2014-09-30 <160> 4 <170> PatentIn 3.5 version <210> 1 <211> 952 <212> PRT <213> Homo sapiens <220> <221> Features not yet classified <222> (1)..(952) <223> SEQ ID NO: 4 from Patent US 8592362 GENBANK: AHE24104.1 <400> 1 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 Cys 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 Arg 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 His 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 Gin 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 Gin Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gin Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gin Gin Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gin 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 Gin Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gin Thr Val Pro lie Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala lie His Ser 785 790 795 800 Glu Gly Gin Trp Val Thr Leu Pro Ala Pro Leu Asp Thr lie Asn Val 805 810 815 His Leu Arg Ala Gly Tyr lie lie Pro Leu Gin Gly Pro Gly lie Thr 820 825 830 Thr Thr Glu Ser Arg Gin Gin 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 Gin Val lie Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr lie Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gin Leu Gin Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gin Gin 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> 2 <211> 3624 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (220)..(3078) <223> Homo sapiens GAA mRNA for lysosomal alpha-glucosidase (acid maltase); Genbank: Y00839.1 <400> 2 cagttgggaa agctgaggtt gtcgccgggg ccgcgggtgg aggtcgggga tgaggcagca 60 ggtaggacag tgacctcggt gacgcgaagg accccggcca cctctaggtt ctcctcgtcc 120 gcccgttgtt cagcgaggga ggctctgggc ctgccgcagc tgacggggaa actgaggcac 180 ggagcgggcc tgtaggagct gtccaggcca tctccaacc atg gga gtg agg cac 234 Met Gly Val Arg His 1 5 ccg ccc tgc tcc cac cgg ctc ctg gcc gtc tgc gcc ctc gtg tcc ttg 282 Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys Ala Leu Val Ser Leu 10 15 20 gca acc gct gca ctc ctg ggg cac atc cta ctc cat gat ttc ctg ctg 330 Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu His Asp Phe Leu Leu 25 30 35 gtt ccc cga gag ctg agt ggc tcc tcc cca gtc ctg gag gag act cac 378 Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val Leu Glu Glu Thr His 40 45 50 cca gct cac cag cag gga gcc agc aga cca ggg ccc cgg gat gcc cag 426 Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly Pro Arg Asp Ala Gln 55 60 65 gca cac ccc ggc cgt ccc aga gca gtg ccc aca cag tgc gac gtc ccc 474 Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr Gln Cys Asp Val Pro 70 75 80 85 ccc aac agc cgc ttc gat tgc gcc cct gac aag gcc atc acc cag gaa 522 Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys Ala Ile Thr Gln Glu 90 95 100 cag tgc gag gcc cgc ggc tgc tgc tac atc cct gca aag cag ggg ctg 570 Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro Ala Lys Gln Gly Leu 105 110 115 cag gga gcc cag atg ggg cag ccc tgg tgc ttc ttc cca ccc agc tac 618 Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe Phe Pro Pro Ser Tyr 120 125 130 ccc agc tac aag ctg gag aac ctg agc tcc tct gaa atg ggc tac acg 666 Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser Glu Met Gly Tyr Thr 135 140 145 gcc acc ctg acc cgt acc acc ccc acc ttc ttc ccc aag gac atc ctg 714 Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe Pro Lys Asp Ile Leu 150 155 160 165 acc ctg cgg ctg gac gtg atg atg gag act gag aac cgc ctc cac ttc 762 Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu Asn Arg Leu His Phe 170 175 180 acg atc aaa gat cca gct aac agg cgc tac gag gtg ccc ttg gag acc 810 Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu Val Pro Leu Glu Thr 185 190 195 ccg cgt gtc cac agc cgg gca ccg tcc cca ctc tac agc gtg gag ttc 858 Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu Tyr Ser Val Glu Phe 200 205 210 tcc gag gag ccc ttc ggg gtg atc gtg cac cgg cag ctg gac ggc cgc 906 Ser Glu Glu Pro Phe Gly Val Ile Val His Arg Gln Leu Asp Gly Arg 215 220 225 gtg ctg ctg aac acg acg gtg gcg ccc ctg ttc ttt gcg gac cag ttc 954 Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe Phe Ala Asp Gln Phe 230 235 240 245 ctt cag ctg tcc acc tcg ctg ccc tcg cag tat atc aca ggc ctc gcc 1002 Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr Ile Thr Gly Leu Ala 250 255 260 gag cac ctc agt ccc ctg atg ctc agc acc agc tgg acc agg atc acc 1050 Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser Trp Thr Arg Ile Thr 265 270 275 ctg tgg aac cgg gac ctt gcg ccc acg ccc ggt gcg aac ctc tac ggg 1098 Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly Ala Asn Leu Tyr Gly 280 285 290 tct cac cct ttc tac ctg gcg ctg gag gac ggc ggg tcg gca cac ggg 1146 Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly Gly Ser Ala His Gly 295 300 305 gtg ttc ctg cta aac agc aat gcc atg gat gtg gtc ctg cag ccg agc 1194 Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val Val Leu Gln Pro Ser 310 315 320 325 cct gcc ctt agc tgg agg tcg aca ggt ggg atc ctg gat gtc tac atc 1242 Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile Leu Asp Val Tyr Ile 330 335 340 ttc ctg ggc cca gag ccc aag agc gtg gtg cag cag tac ctg gac gtt 1290 Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln Gln Tyr Leu Asp Val 345 350 355 gtg gga tac ccg ttc atg ccg cca tac tgg ggc ctg ggc ttc cac ctg 1338 Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly Leu Gly Phe His Leu 360 365 370 tgc cgc tgg ggc tac tcc tcc acc gct atc acc cgc cag gtg gtg gag 1386 Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr Arg Gln Val Val Glu 375 380 385 aac atg acc agg gcc cac ttc ccc ctg gac gtc caa tgg aac gac ctg 1434 Asn Met Thr Arg Ala His Phe Pro Leu Asp Val Gln Trp Asn Asp Leu 390 395 400 405 gac tac atg gac tcc cgg agg gac ttc acg ttc aac aag gat ggc ttc 1482 Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe Asn Lys Asp Gly Phe 410 415 420 cgg gac ttc ccg gcc atg gtg cag gag ctg cac cag ggc ggc cgg cgc 1530 Arg Asp Phe Pro Ala Met Val Gln Glu Leu His Gln Gly Gly Arg Arg 425 430 435 tac atg atg atc gtg gat cct gcc atc agc agc tcg ggc cct gcc ggg 1578 Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser Ser Gly Pro Ala Gly 440 445 450 agc tac agg ccc tac gac gag ggt ctg cgg agg ggg gtt ttc atc acc 1626 Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg Gly Val Phe Ile Thr 455 460 465 aac gag acc ggc cag ccg ctg att ggg aag gta tgg ccc ggg tcc act 1674 Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val Trp Pro Gly Ser Thr 470 475 480 485 gcc ttc ccc gac ttc acc aac ccc aca gcc ctg gcc tgg tgg gag gac 1722 Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu Ala Trp Trp Glu Asp 490 495 500 atg gtg gct gag ttc cat gac cag gtg ccc ttc gac ggc atg tgg att 1770 Met Val Ala Glu Phe His Asp Gln Val Pro Phe Asp Gly Met Trp Ile 505 510 515 gac atg aac gag cct tcc aac ttc atc aga ggc tct gag gac ggc tgc 1818 Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly Ser Glu Asp Gly Cys 520 525 530 ccc aac aat gag ctg gag aac cca ccc tac gtg cct ggg gtg gtt ggg 1866 Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val Pro Gly Val Val Gly 535 540 545 ggg acc ctc cag gcg gcc acc atc tgt gcc tcc agc cac cag ttt ctc 1914 Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser Ser His Gln Phe Leu 550 555 560 565 tcc aca cac tac aac ctg cac aac ctc tac ggc ctg acc gaa gcc atc 1962 Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly Leu Thr Glu Ala Ile 570 575 580 gcc tcc cac agg gcg ctg gtg aag gct cgg ggg aca cgc cca ttt gtg 2010 Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly Thr Arg Pro Phe Val 585 590 595 atc tcc cgc tcg acc ttt gct ggc cac ggc cga tac gcc ggc cac tgg 2058 Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg Tyr Ala Gly His Trp 600 605 610 acg ggg gac gtg tgg agc tcc tgg gag cag ctc gcc tcc tcc gtg cca 2106 Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu Ala Ser Ser Val Pro 615 620 625 gaa atc ctg cag ttt aac ctg ctg ggg gtg cct ctg gtc ggg gcc gac 2154 Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro Leu Val Gly Ala Asp 630 635 640 645 gtc tgc ggc ttc ctg ggc aac acc tca gag gag ctg tgt gtg cgc tgg 2202 Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu Leu Cys Val Arg Trp 650 655 660 acc cag ctg ggg gcc ttc tac ccc ttc atg cgg aac cac aac agc ctg 2250 Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg Asn His Asn Ser Leu 665 670 675 ctc agt ctg ccc cag gag ccg tac agc ttc agc gag ccg gcc cag cag 2298 Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser Glu Pro Ala Gln Gln 680 685 690 gcc atg agg aag gcc ctc acc ctg cgc tac gca ctc ctc ccc cac ctc 2346 Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala Leu Leu Pro His Leu 695 700 705 tac aca ctg ttc cac cag gcc cac gtc gcg ggg gag acc gtg gcc cgg 2394 Tyr Thr Leu Phe His Gln Ala His Val Ala Gly Glu Thr Val Ala Arg 710 715 720 725 ccc ctc ttc ctg gag ttc ccc aag gac tct agc acc tgg act gtg gac 2442 Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser Thr Trp Thr Val Asp 730 735 740 cac cag ctc ctg tgg ggg gag gcc ctg ctc atc acc cca gtg ctc cag 2490 His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile Thr Pro Val Leu Gln 745 750 755 gcc ggg aag gcc gaa gtg act ggc tac ttc ccc ttg ggc aca tgg tac 2538 Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro Leu Gly Thr Trp Tyr 760 765 770 gac ctg cag acg gtg cca ata gag gcc ctt ggc agc ctc cca ccc cca 2586 Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly Ser Leu Pro Pro Pro 775 780 785 cct gca gct ccc cgt gag cca gcc atc cac agc gag ggg cag tgg gtg 2634 Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser Glu Gly Gln Trp Val 790 795 800 805 acg ctg ccg gcc ccc ctg gac acc atc aac gtc cac ctc cgg gct ggg 2682 Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val His Leu Arg Ala Gly 810 815 820 tac atc atc ccc ctg cag ggc cct ggc ctc aca acc aca gag tcc cgc 2730 Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr Thr Thr Glu Ser Arg 825 830 835 cag cag ccc atg gcc ctg gct gtg gcc ctg acc aag ggt gga gag gcc 2778 Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr Lys Gly Gly Glu Ala 840 845 850 cga ggg gag ctg ttc tgg gac gat gga gag agc ctg gaa gtg ctg gag 2826 Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser Leu Glu Val Leu Glu 855 860 865 cga ggg gcc tac aca cag gtc atc ttc ctg gcc agg aat aac acg atc 2874 Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala Arg Asn Asn Thr Ile 870 875 880 885 gtg aat gag ctg gta cgt gtg acc agt gag gga gct ggc ctg cag ctg 2922 Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly Ala Gly Leu Gln Leu 890 895 900 cag aag gtg act gtc ctg ggc gtg gcc acg gcg ccc cag cag gtc ctc 2970 Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala Pro Gln Gln Val Leu 905 910 915 tcc aac ggt gtc cct gtc tcc aac ttc acc tac agc ccc gac acc aag 3018 Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr Ser Pro Asp Thr Lys 920 925 930 gtc ctg gac atc tgt gtc tcg ctg ttg atg gga gag cag ttt ctc gtc 3066 Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly Glu Gln Phe Leu Val 935 940 945 agc tgg tgt tag ccgggcggag tgtgttagtc tctccagagg gaggctggtt 3118 Ser Trp Cys 950 ccccagggaa gcagagcctg tgtgcgggca gcagctgtgt gcgggcctgg gggttgcatg 3178 tgtcacctgg agctgggcac taaccattcc aagccgccgc atcgcttgtt tccacctcct 3238 gggccggggc tctggccccc aacgtgtcta ggagagcttt ctccctagat cgcactgtgg 3298 gccggggcct ggagggctgc tctgtgttaa taagattgta aggtttgccc tcctcacctg 3358 ttgccggcat gcgggtagta ttagccaccc ccctccatct gttcccagca ccggagaagg 3418 gggtgctcag gtggaggtgt ggggtatgca cctgagctcc tgcttcgcgc ctgctgctct 3478 gccccaacgc gaccgcttcc cggctgccca gagggctgga tgcctgccgg tccccgagca 3538 agcctgggaa ctcaggaaaa ttcacaggac ttgggagatt ctaaatctta agtgcaatta 3598 ttttaataaa aggggcattt ggaatc 3624 <210> 3 <211> 952 <212> PRT <213> Homo sapiens <400> 3 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 ​​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 Arg 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 His 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 Gin Phe Leu Gin Leu Ser Thr Ser Leu Pro Ser Gin 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 Gin 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 Gin 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 Gin 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 Gin 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 Gin Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gin 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 Gin Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gin Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gin Gin Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gin 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 Gin Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gin 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 Gin 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 Gin Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gin Gin 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 Gin 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 Gin Leu Gin Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gin Gin 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 Gin Phe Leu Val Ser Trp Cys 945 950 <210> 4 <211> 952 <212> PRT <213> Homo sapiens <220> <221> Unclassified characteristic <222> (1)..(952) <223> Lysosomal alpha-glucosidase preproprotein [Homo sapiens]; NCBI Reference Sequence: NP_000143.2 <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 Cys 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 lie Asp Met Asn Glu Pro Ser Asn Phe lie 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 Gin Ala Ala Thr lie Cys Ala Ser 545 550 555 560 Ser His Gin Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala lie Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val lie 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 Gin Leu 610 615 620 Ala Ser Ser Val Pro Glu lie Leu Gin 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 Gin Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gin Gin Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gin Gin Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gin 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 Gin Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gin Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gin Thr Val Pro Val Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Gin Pro Ala He His Ser 785 790 795 800 Glu Gly Gin Trp Val Thr Leu Pro Ala Pro Leu Asp Thr He Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gin Gly Pro Gly Leu Thr 820 825 830 Thr Thr Gin Ser Arg Gin Gin Pro Met Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Gin Ala Arg Gly Gin Leu Phe Trp Asp Asp Gly Gin Ser 850 855 860 Leu Gin Val Leu Gin Arg Gly Ala Tyr Thr Gin Val Ile Phe Leu Ala 865 870 875 880 Arg Asn Asn Thr Ile Val Asn Gin Leu Val Arg Val Thr Ser Gin Gly 885 890 895 Ala Gly Leu Gin Leu Gin Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gin Gin 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 Gin Phe Leu Val Ser Trp Cys 945 950

Claims

1. A composition comprising recombinant human acidic α-glucosidase (rhGAA) derived from Chinese hamster ovary (CHO) cells, wherein the CHO cells express rhGAA of SEQ ID NO:4, wherein the rhGAA contains 2.0 mol to 8.0 mol of sialic acid residues per mol of rhGAA, and wherein the rhGAA contains 3.0 mol to 7.0 mol of mannose-6-phosphate (M6P) residues per mol of rhGAA.

2. The composition of claim 1, wherein the rhGAA contains 4.0 mol to 7.0 mol of M6P residues per mol of rhGAA.

3. The composition of claim 1, wherein the rhGAA contains 5.0 mol to 7.0 mol of M6P residues per mol of rhGAA.

4. The composition of claim 1, wherein the rhGAA contains 7.0 mol of M6P residues per mol of rhGAA.

5. A composition comprising rhGAA derived from Chinese hamster ovary (CHO) cells, wherein the CHO cells express rhGAA of SEQ ID NO:4, wherein the rhGAA contains 2.0 mol to 8.0 mol of sialic acid residues per mol of rhGAA, and wherein at least 8% of the total polysaccharide on the rhGAA is mono-phosphorylated mannose-6-phosphate (mono-M6P) polysaccharide.

6. The composition of claim 5, wherein at least 9% of the total polysaccharides on the rhGAA are mono-M6P polysaccharides.

7. The composition of claim 5, wherein at least 10% of the total polysaccharides on the rhGAA are mono-M6P polysaccharides.

8. A composition comprising rhGAA derived from Chinese hamster ovary (CHO) cells, wherein the CHO cells express rhGAA of SEQ ID NO:4, wherein the rhGAA contains 2.0 mol to 8.0 mol of sialic acid residues per mol of rhGAA, and wherein at least 2% of the total polysaccharide on the rhGAA is bis-phosphorylated mannose-6-phosphate (bis-M6P) polysaccharide.

9. The composition of claim 8, wherein at least 2.5% of the total polysaccharides on the rhGAA are bis-M6P polysaccharides.

10. The composition of claim 8, wherein at least 3.0% of the total polysaccharides on the rhGAA are bis-M6P polysaccharides.

11. A composition comprising rhGAA derived from Chinese hamster ovary (CHO) cells, wherein the CHO cells express rhGAA of SEQ ID NO:4, wherein the rhGAA contains 2.0 mol to 8.0 mol of sialic acid residues per mol of rhGAA, and wherein each rhGAA molecule contains, on average, at least one bis-M6P polysaccharide.

12. A composition comprising rhGAA derived from Chinese hamster ovary (CHO) cells, wherein the CHO cells express rhGAA of SEQ ID NO:4, wherein the rhGAA comprises 6.0 mol-7.0 mol M6P residues / mol rhGAA.

13. The composition of claim 12, wherein the rhGAA comprises 7.0 mol M6P residues / mol rhGAA.

14. The composition of any one of claims 1 to 13, wherein, on average, the rhGAA comprises greater than 4 mol sialic acid residues / mol of rhGAA.

15. The composition of any one of claims 1 to 13, wherein, on average, the rhGAA comprises at least 1 mol of bis-M6P / mol of rhGAA.

16. The composition of any one of claims 1 to 13, wherein, on average, the rhGAA comprises 1.3 mol bis-M6P / mol rhGAA.

17. The composition according to any one of claims 1 to 13, wherein 40% to 60% of the N-glycans on the rhGAA are complex N-glycans.

18. The composition according to any one of claims 1 to 13, wherein 45% to 55% of the N-glycans on the rhGAA are complex N-glycans.

19. The composition according to any one of claims 1 to 13, wherein 50% of the N-glycans on the rhGAA are complex N-glycans.

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