HK1235433B - Highly potent acid alpha-glucosidase with enhanced carbohydrates - Google Patents

Description

High strength acidic alpha-glucosidase with enhanced carbohydrate

Cross Reference to Related Applications

This application claims priority rights to U.S. temporary 62/057,842 filed on month 9 and 30 of 2014, U.S. temporary 62/057,847 filed on month 9 and 30 of 2014, U.S. temporary 62/112,463 filed on month 2 and 5 of 2015, and U.S. temporary 62/135,345 filed on month 3 and 19 of 2015, each of which is incorporated by reference in its entirety.

Background

Technical Field

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 efficiently target CIMPR on myocytes and subsequently deliver rhGAA to lysosomes where it can break down abnormally high levels of accumulated glycogen. The rhGAA of the present invention exhibits superior targeting and subsequent delivery to lysosomes for muscle cells compared to conventional rhGAA products, and exhibits other pharmacokinetic properties that make it particularly effective for enzyme replacement therapy in subjects with Pompe disease.

Description of the related Art

Existing enzyme replacement therapies for pompe disease use conventional rhGAA products with low total content of glycans with M6P and bis-M6P. It is known thatAnd the conventional product under the alpha name of arabinosidase. "Lumizyme" and "Myozyme" are conventional forms of rhGAA produced or sold by Genzyme as a biological agent and approved by the U.S. food and drug administration, and are used by Reference to the physicians' Desk Reference 2014 (which is incorporated herein by Reference) or approved by the FDA in the United states for use 10.1.2014 under the name "Lumizyme" and "Myozyme" as used in the United statesOrThe product of (a). Alpha-glucosidase was identified as the chemical name [ 199-arginine, 223-histidine]Prepro-alpha-glucosidase (human); the molecular formula is as follows: c₄₇₅₈H₇₂₆₂N₁₂₇₄O₁₃₆₉S₃₅(ii) a CAS number 420794-05-0. These products are administered to a subject suffering from 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 the lysosome, thereby restoring the ability of the cell to break down lysosomal glycogen.

Pompe disease is an inherited lysosomal storage disorder due to a deficiency in acid alpha-Glucosidase (GAA) activity. People with pompe disease have no or reduced levels of acid alpha-Glucosidase (GAA), which breaks down glycogen and the substances the body uses as an energy source. This enzyme deficiency results in excessive glycogen accumulation in lysosomes, which are internal organelles containing enzymes that normally break down glycogen and other cellular debris or waste. Glycogen accumulation in certain tissues (especially muscles) of subjects with pompe disease impairs the ability of cells to function properly. In pompe disease, glycogen is not properly metabolized and gradually accumulates in lysosomes, particularly in skeletal muscle cells, and in infant-onset forms of the disease, glycogen is not properly metabolized and gradually accumulates in cardiac muscle cells. Accumulation of glycogen damages muscle and nerve cells as well as those in other affected tissues.

Traditionally, pompe disease is clinically identified as either an early infant form or an advanced onset form, depending on the age of onset. The age of onset tends to correspond to the severity of the genetic mutation leading to pompe disease. The most severe gene mutations result in a complete loss of GAA activity, manifested 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 delayed onset and progression. Infant-onset pompe disease manifests shortly after birth and is characterized by muscle weakness, respiratory insufficiency, and heart failure. Untreated, it is usually fatal within two years. Pompe disease, both childhood and adult onset, manifests late in life and often progresses more slowly than infant onset. This form of disease, while it does not normally affect the heart, can also lead to death due to weakening of skeletal muscles and those involved in breathing.

Current non-palliative treatment of Pompe disease involves the use of recombinant human GAA (rhGAA) such asOrEnzyme Replacement Therapy (ERT). rhGAA is administered in an attempt to replace or supplement the missing or deficient GAA in subjects with pompe disease. However, since most of rhGAA in conventional rhGAA products does not target muscle tissue, it is non-productively eliminated after administration.

This condition occurs because conventional rhGAA lacks a high total content of M6P and bis-M6P bearing glycans that target the rhGAA molecule to the CIMPR on target muscle cells where it is subsequently transported to the lysosomes of the cells. This cellular uptake of rhGAA for enzyme replacement therapy is facilitated by a specialized carbohydrate (mannose-6-phosphate (M6P)), which M6P binds to cation-independent mannose 6-phosphate receptor (CIMPR) present on the cell surface for subsequent delivery of exogenous enzymes to lysosomes.

There are seven potential N-linked glycosylation sites on rhGAA. Since each glycosylation site is heterogeneous among the types of N-linked oligosaccharides (N-glycans) present, rhGAA consists of a complex mixture of proteins with N-glycans having different binding affinities for the M6P receptor and other carbohydrate receptors. rhGAA containing high mannose N-glycans with one M6P group (mono-M6P) binds with low (about 6,000nM) affinity to CIMPR, while rhGAA containing two M6P groups (bis-M6P) on the same N-glycan binds with high (about 2nM) affinity. Representative structures of non-phosphorylated, mono-M6P, and bis-M6P glycans are shown in fig. 1A. The mannose-6-P group is shown in FIG. 1B. Once in the lysosome, rhGAA can enzymatically degrade accumulated glycogen. However, conventional rhGAA has low total levels of glycans with M6P and bis-M6P, and thus targeting of muscle cells is poor, resulting in poor delivery of rhGAA to lysosomes. Most of these conventional products have no phosphorylated N-glycans and thus lack affinity for CIMPR. Non-phosphorylated high mannose glycans can also be cleared by mannose receptors that result in non-productive clearance of ERT (fig. 2).

Other types of N-glycans, complex carbohydrates, comprising galactose and sialic acid are also present on rhGAA. Since the complexed N-glycans were not phosphorylated, they had no affinity for CIMPR. However, complex N-glycans with exposed galactose residues have moderate to high affinity for asialoglycoprotein receptors on liver hepatocytes, which leads to rapid non-productive clearance of rhGAA (fig. 2).

Glycosylation of GAA or rhGAA can be enzymatically modified in vitro by phosphotransferase and an uncovering enzyme (uncovered enzyme) described in candelilla et al, U.S. patent No. 6,534,300, to produce the M6P group. Enzymatic glycosylation cannot be adequately controlled and rhGAA is produced with undesirable immunological and pharmacological properties. Enzymatically modified rhGAA may comprise only high-mannose N-glycans, all of which can potentially be enzymatically phosphorylated in vitro with phosphotransferases/uncovering enzymes, and may comprise an average of 5-6M 6P groups per GAA. The glycosylation pattern produced by in vitro enzymatic treatment of GAA is problematic because the additional terminal mannose residues, particularly the non-phosphorylated terminal mannose residues, negatively affect the pharmacokinetics of the modified rhGAA. When such enzymatically modified products are administered in vivo, these mannosyl groups increase the non-productive clearance of GAA, increase the uptake of the enzymatically modified GAA by immune cells, and decrease rhGAA therapeutic efficacy due to less GAA reaching target tissues (e.g., 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 enzymatically modified rhGAA and reduced targeting of rhGAA to target tissues. In addition, the glycosylation pattern of enzymatically modified GAA with high mannose N-glycans containing terminal non-phosphorylated mannose residues resembles that on glycoproteins produced in yeast, molds and functions that increase the risk of eliciting an immune or allergic reaction to the enzymatically modified rhGAA, such as a life threatening severe allergy (anaphylaxis) or hypersensitivity reaction.

As explained above, conventional rhGAA products are as followsWith low levels of monophosphorylated glycans and even lower bisphosphorylated glycans. For pompe disease therapy to be effective, rhGAA must be delivered to lysosomes in muscle cells. The low total amount of mono-M6P and bis-M6P targeting groups on conventional rhGAA limits cellular uptake via CIMPR and lysosomal delivery, thus making conventional enzyme replacement therapy inefficient. For example, while conventional rhGAA products at doses of 20mg/kg or higher 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.

Due to the inefficiency of delivering conventional enzyme replacement therapies to lysosomes, these therapies are often associated with other problems, including the generation of an immune response to GAA. The GAA in most conventional rhGAA does not contain glycans with mono-or bis-M6P, which mono-or bis-M6P targets rhGAA to muscle cells. The subject's immune system is exposed to this excess of non-phosphorylated GAA and can generate an adverse 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 immune inactivation of the administered rhGAA and increases the risk of the patient experiencing an adverse autoimmune or allergic reaction to rhGAA treatment. The rhGAA according to the invention contains significantly less of this non-targeted, non-phosphorylated rhGAA, thereby reducing the exposure of the patient's immune system to it.

Logically, larger doses place additional burdens on the subject and medical professionals treating the subject, such as prolonging the infusion time required for intravenous administration of rhGAA. This is because conventional rhGAA contains higher levels of non-phosphorylated rhGAA, which does not target CIMPR on muscle cells. rhGAA that does not bind CIMPR on muscle cells and then enters lysosomes does not enzymatically degrade glycogen there. When equal doses of conventional rhGAA and rhGAA according to the invention were administered, more rhGAA in the composition according to the invention bound to CIMPR on the myocytes and was then delivered to the lysosomes. The rhGAA of the present invention provides the physician with the option of administering lower amounts of rhGAA while delivering the same or more rhGAA to the lysosome.

For preparing conventional rhGAA (e.g. usingOr alpha) of the glucosaccharase, does not significantly increase the content of M6P or bis-M6P, since the cellular carbohydrate processing is naturally complex and extremely difficult to handle. In view of these deficiencies of conventional rhGAA products, the inventors sought and identified methods to effectively target rhGAA to muscle cells and deliver it to lysosomes, minimize non-productive clearance of rhGAA once administered, and thereby more productively target rhGAA to muscle tissue.

Summary of The Invention

In response to the problems associated with targeting and administering conventional forms of rhGAA, and in response to the difficulties associated with producing this well-targeted form of rhGAA, the inventors have investigated and developed procedures for preparing rhGAA that more efficiently targets CIMPR and delivers rhGAA to lysosomes in muscle tissue because it has a higher content of M6P-glycans and bis-M6P glycans than conventional rhGAA compositions. In addition, the rhGAA of the present invention has well-processed complex N-glycans that minimize non-productive clearance of rhGAA by non-target tissues.

Considering and using conventional rhGAA products (e.g. the rhGAA product)) With diligent research and investigation, the inventors have developed methods for producing rhGAA in CHO cells with significantly higher total content of mono-M6P and bis-M6P glycans, which target the CIMPR on muscle cells and then deliver rhGAA to lysosomes.

The rhGAA produced by this method also has favorable pharmacokinetic properties by virtue of its overall glycosylation pattern, which increases target tissue uptake and reduces non-productive clearance upon administration to subjects with pompe disease. The inventors show that the rhGAA of the present invention, as exemplified by rhGAA designated ATB-200, is more targeted to skeletal muscle tissue than conventional rhGAA (e.g., as exemplified by) More powerful and more efficient. As illustrated by fig. 2, rhGAA according to the present invention has an excellent ability to productively target muscle tissue in patients with pompe disease and reduce non-productive clearance of rhGAA.

The superior rhGAA according to the invention may be further completed or conjugated with chaperone combinations or with other moieties that target CIMPR in muscle tissue (e.g., the moiety of IGF2 that binds the receptor). The following examples show the use of conventional rhGAA productsCompared to the existing protocol, rhGAA of the present invention (exemplified by ATB-200 rhGAA) exceeded the existing standard of care for enzyme replacement therapy by providing significantly better glycogen clearance in skeletal muscle.

Brief description of the drawings

The application file comprises at least one drawing executed in color.

Figure 1A shows non-phosphorylated high mannose glycans, mono-M6P glycans, and bis-M6P glycans. Fig. 1B shows the chemical structure of the M6P group.

Figure 2A depicts the productive targeting of rhGAA to a target tissue (e.g., muscle tissue of a subject with pompe disease) via glycans carrying M6P. Figure 2B depicts non-productive drug clearance to non-target tissues (e.g., liver and spleen) or by binding of non-M6P glycans to non-target tissues.

Figure 3A illustrates the CIMPR receptor (also known as IGF2 receptor) and the domains of this receptor. FIG. 3B is a table showing the binding affinity (nanomolar) of di-and mono-M6P-bearing glycans to CIMPR, the binding affinity of high mannose glycans to mannose receptors, and the binding affinity of desialylated complex glycans to asialoglycoprotein receptors. RhGAA having glycans with M6P and bis-M6P can bind productively to CIMPR on muscle target cells. RhGAA with high mannose glycans and desialylated glycans can bind non-productively to non-target cells with the corresponding receptors.

FIG. 4 FIGS. 4A and 4B show respectivelyAndresults of the CIMPR affinity chromatography of (1). The dashed line refers to the M6P elution gradient. Elution with M6P replaced GAA molecules bound to CIMPR via glycans containing M6P. As shown in figure 4A of the drawings,medium 78% GAA activity was eluted before M6P was added. FIG. 4B shows 73% GAAActivity was eluted before addition of M6P. In thatOr only 22% or 27% of rhGAA in Myozyme, eluted with M6P, respectively. These figures show that most rhGAA in these two conventional rhGAA products lack the glycans of M6P required to target CIMPR in the target muscle tissue.

FIG. 5A DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA (SEQ ID NO: 4).

FIG. 6 FIGS. 6A and 6B show the results of CIMPR affinity chromatography of Myozyme and ATB-200 rhGAA. As shown in FIG. 6B, about 70% of the rhGAA in ATB-200rhGAA contained M6P.

FIG. 7 ATB-200rhGAA purification, examples 1 and 2.

FIG. 8And ATB-200.rhGAA, Polivix elution profile.

FIG. 9 compares three different formulations of ATB200rhGAA identified as BP-rhGAA, ATB200-1 and ATB200-2,summary of the N-glycan structures of (a).

FIG. 10A compares ATB-200rhGAA (left trace) andCIMPR binding affinity (right trace). FIG. 10B depictsAnd bis-M6P content of ATB-200 rhGAA.

FIG. 11A compares ATB-200rhGAA activity (left trace) in normal fibroblasts with that of GAA at different concentrationsrhGAA activity (right trace). FIG. 11B compares ATB-200rhGAA activity (left trace) in fibroblasts from subjects with Pompe disease at different GAA concentrations withrhGAA activity (right trace). FIG. 11C compares (K) of fibroblasts from normal subjects and subjects with Pompe disease_{Intake of})。

FIG. 12A shows the amount of glycogen relative to protein in the myocardium after contact with vehicle (negative control), with 20mg/ml of arabinosidase α or with 5mg/kg, 10mg/kg, or 20mg/kg ATB-200 rhGAA. FIG. 12B shows the comparison of the concentration of the antibody in the sample with the vector (negative control) and 20mg/mlOr 5mg/kg, 10mg/kg or 20mg/kg ATB-200rhGAA, relative to the amount of glycogen of protein in the quadriceps muscle. FIG. 12C shows comparison of control with vehicle (negative control), and comparison with 20mg/mlOr 5mg/kg, 10mg/kg or 20mg/kg ATB-200rhGAA, relative to the amount of glycogen of protein in triceps muscle. Compared with a negative control and withIn contrast, ATB-200rhGAA produced a significant glycogen reduction in quadriceps and triceps.

FIG. 13 ATB-200rhGAA stability was improved in the presence of the molecular partner AT 2221. The first left trace in fig. 13A shows the percentage of unfolded ATB-200rhGAA protein at various temperatures at pH 7.4 (blood pH). The last right trace shows the percentage of unfolded ATB-200rhGAA protein at various temperatures at pH 5.2 (lysosomal pH). The three middle traces show the effect of 10, 30 or 100 μ g of the AT2221 chaperone on protein folding. These data show that AT2221 prevents unfolding of ATB-200rhGAA AT blood pH compared to control samples. The improvement in Tm AT2221 AT neutral pH is summarized in fig. 13B.

FIG. 14 this table shows that the combination of ATB-200rhGAA and the chaperone AT2221 was used in GAA knockout mice compared to the use in GAA knockout miceAnd AT2221 or without AT2221 chaperonesTreatment with the control of ATB200rhGAA provided significantly better glycogen clearance.

FIG. 15 residual glycogen in quadriceps after treatment with Lumizyme, ATB-200rhGAA, or ATB-200rhGAA and various concentrations of AT2221 chaperones.

Figure 16 improvement in skeletal myopathy in mice treated with ATB200+ Miglustat (AT2221) over those treated with ERT alone. PAS glycogen staining of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200rhGAA and Magcistat (AT-2221) (FIG. 16A) and EM (FIG. 16B). FIG. 16C; assessment of lysosomal proliferation by LAMP-1 marker. Figure 16 identification of type 16D I and type II muscle fibers.

FIG. 17 improvement in skeletal myopathy in mice treated with ATB-200+ Magcistat (AT2221) over those treated with ERT alone. PAS glycogen staining of muscle tissue from GAA KO mice treated with conventional rhGAA or ATB-200rhGAA and Magcistat (AT-2221) (FIG. 17A). FIG. 17B; assessment of lysosomal proliferation by LAMP-1 marker.

Detailed Description

Definition of: in the context of the present invention and in the specific context of each term's usage, 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 describing the compositions and methods of the invention and how to make and use them.

The term "GAA" refers to human acid alpha-Glucosidase (GAA) (an enzyme that catalyzes the hydrolysis of the alpha-1, 4-and alpha-1, 6-glycosidic linkages of lysosomal glycogen), as well as to insertions, related or substituted variants of the GAA amino acid sequence, as well as fragments of longer GAA sequences that exert enzymatic activity. The term "rhGAA" is used to distinguish endogenous GAA from synthetically or recombinantly produced GAA, such as that 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. The GAA and rhGAA may be present in a composition comprising a mixture of GAA molecules having different glycosylation patterns, such as a mixture of rhGAA molecules with mono-M6P or bis-M6P groups on their glycans and GAA molecules without M6P or bis-M6P. GAA and rhGAA can also be accomplished with other compounds (e.g., chaperones), or can be bound to other moieties in the GAA or rhGAA conjugate, such as to the IGF2 moiety that targets the conjugate to CIMPR and then delivers it to lysosomes.

The "subject" or "patient" is preferably a human, although other mammals and non-human animals suffering from conditions involving glycogen accumulation may also be treated. The subject may be a fetus, neonate, toddler, child, or adult suffering from pompe disease or other disorders of glycogen storage or accumulation. An example of an individual being treated is an individual (fetus, neonate, toddler, child, adolescent, or adult) having GSD-II (e.g., infant GSD-II, pediatric GSD-II, or adult-onset GSD-II). The individual may have residual GAA activity, or no measurable activity. For example, an individual with GSD-II may have GAA activity of less than about 1% of normal GAA activity (infant GSD-II), about 1% -10% of normal GAA activity (pediatric GSD-II), or about 10% -40% of normal GAA activity (adult GSD-II).

The term "treating" (treat or treatment) as used herein refers to amelioration of one or more symptoms associated with a disease, prevention or delay of onset of one or more symptoms of a disease, and/or reduction in severity or frequency of one or more symptoms of a disease. For example, treatment may refer to an improvement in cardiac condition (e.g., an increase in end-diastolic and/or end-systolic volume, or a reduction, amelioration, or prevention of progressive cardiomyopathy commonly found in GSD-II) or an improvement in pulmonary function (e.g., an increase in crying lung capacity above baseline capacity, and/or a normalization of oxygen desaturation during crying); improvement in neurodevelopmental and/or motor skills (e.g., increase in AIMS score); a decrease in glycogen levels in a tissue of the individual affected by the disease; or any combination of these effects. In a preferred embodiment, the treatment comprises improving a cardiac condition, in particular reducing or preventing GSD-II related cardiomyopathy.

The terms "improve," "increase," or "decrease," as used herein, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein. Control individuals are individuals with the same form of GSD-II (infant, pediatric or adult onset) as the treated individual, which are about the same age as the treated individual (to ensure that the disease stage is comparable in the treated and control individuals).

The term "purified" as used herein refers to a material that is isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including natural materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it binds in a cell, and a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally in the context of analytical testing of a material. Preferably, the 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, composition analysis, bioassay, enzymatic assay, and other methods known in the art. In particular embodiments, purified means that the contaminant levels are below levels acceptable by regulatory agencies for safe administration to humans or non-human animals. 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.

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) following 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 may be accomplished by any method known in the art, including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered (e.g., by gene activation techniques) to express or overexpress an endogenous gene or gene product that is not normally expressed by such cells.

"pompe disease" refers to an autosomal recessive LSD characterized by a lack of acid alpha Glucosidase (GAA) activity that impairs lysosomal glycogen metabolism. Enzyme deficiency leads to lysosomal glycogen accumulation and to progressive skeletal muscle weakness, decreased cardiac function, respiratory insufficiency, and/or CNS injury in advanced stages of the disease. Genetic mutations in the GAA gene result in lower expression or produce mutant forms of the enzyme with altered stability and/or biological activity that ultimately leads to disease. (see generally, Helichun (Hirschhorn) R, 1995, Glycogen Storage Disease Type II: Acid alpha-glucosidase (Acid maltase) Deficiency (Glycogen Storage Disease Type II: Acid alpha-glucosidase (Acid maltase) Deficiency), Metabolic and Molecular basis of genetic diseases (The Metabolic and Molecular Bases of Inherited diseases), Sclerville (Scriver) et al, eds., Megrahicle Press (McGraw-Hill), New York, 7 th edition, p. 2443-2464). Three recognized clinical forms of pompe disease (infants, children and adults) are associated with residual alpha-glucosidase activity levels (Reuser A J et al, 1995, glycogen storage disease type II (acid maltase deficiency), Muscle and Nerve supplementation (Muscle & Nerve Supplement)3, S61-S69). Pompe disease in infants (type I or type a) is the most common and severe, characterized by the inability to thrive, systemic hypotony, cardiac hypertrophy, and cardiopulmonary failure in the second year of life. The severity of pompe disease (type II or B) in children is moderate and is characterized by the absence of the preponderance of muscular symptoms of cardiac hypertrophy. Children with pompe disease typically die before the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) is commonly manifested as slowly progressive myopathy during adolescence or at the latest to sixty years (Ferrisia (Felicia) K J et al, 1995, Clinical variation in Adult Onset Acid Maltase Deficiency: reports and Literature reviews of Affected siblings (Clinical value in Adult-Onset Acid malt Deficiency: Report of Affected Sibs and Review of the same), Medicine (Medicine)74, 131-. In pompe, it has been shown that α -glucosidase is extensively modified post-translationally through glycosylation, phosphorylation, and proteolytic processing. Conversion of 110 kilodalton (young child) precursors to the 76 and 70 young mature forms by proteolysis in lysosomes is required for optimal glycogen catalysis. As used herein, the term "pompe disease" refers to all types of pompe disease. The formulations and dosing regimens disclosed herein can be used to treat, for example, type I, type II, or type III pompe disease.

Non-limiting examples of the invention

A rhGAA composition derived from CHO cells, the rhGAA composition comprising rhGAA(alpha glucosidase; CAS 420794-05-0) higher amounts of rhGAA comprising N-glycans carrying mono-mannose-6-phosphate (M6P) or bis-M6P. An exemplary rhGAA composition according to the invention is ATB-200 (sometimes designated as ATB-200, ATB-200 or CBP-rhGAA) as described in the examples. The rhGAA (ATB-200) of the present invention has been shown to have high affinity (K)_DAbout 2-4nM) bind to CIMPR and are bound by Pompe fibroblast (Pompe fibroplast) and skeletal myoblast (K)_{Intake of}About 7-14 nM). ATB-200 was characterized in vivo and was shown to have a greater than current rhGAA ERT (t)_1/2About 60min) shorter apparent plasma half-life (t)_1/2About 45 min).

The amino acid sequence of rhGAA may have at least 70%, 75%, 80%, 85%, 95%, or 99% identity to the amino acid sequence described by SEQ ID No. 1, 3, or 4, or comprise 1, 2,3, 4, 5, 6, 7,8, 9, 10, or more deletions, substitutions, or additions to the amino acid sequence described by SEQ ID No. 1, 3, or 4. In some embodiments of GAA or rhGAA of the invention, such as in ATB-200rhGAA, the GAA or rhGAA will comprise a wild-type GAA amino acid sequence, such as that 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 an active site for substrate binding and/or substrate reduction. In one embodiment, the rhGAA is glucosidase α, which is the human enzyme acid α -Glucosidase (GAA), encoded by the most prominent nine observed haplotypes of the gene. The rhGAA of the invention, including ATB-200rhGAA, may include an amino acid sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of human alpha glucosidase, as given by accession number AHE24104.1(GI:568760974) (SEQ ID NO:1) and incorporated by reference to the amino acid sequence of U.S. Pat. No. 8,592,362 or to NP-000143.2 (SEQ ID NO: 4). The nucleotide and amino acid sequences of GAA are also given by SEQ ID NO 2 and 3, respectively. Variants of this 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 to the GAA amino acid sequence. Polynucleotide sequences encoding GAA and such variant human GAA are also contemplated and may be used for recombinant expression of rhGAA according to the invention.

Different alignment algorithms and/or programs can be used to calculate identity between two sequences, including FASTA or BLAST, which can be used as part of the GCG sequence analysis package (university of wisconsin, madison, wisconsin), and can be used with, for example, default settings. For example, polypeptides that are at least 70%, 85%, 90%, 95%, 98%, or 99% identical to a particular polypeptide described herein, and preferably exhibit substantially the same function, are contemplated, as are polynucleotides encoding such polypeptides. Unless otherwise stated, the similarity score will be based on the use of BLOSUM 62. When BLASTP is used, the percent similarity is based on the BLASTP positive score and the percent sequence identity is based on the BLASTP identity score. BLASTP "identity" shows the number and fraction of total residues in the same high-scoring sequence pair; and BLASTP "positive" shows the number and fraction of residues that have positive alignment scores 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. The polynucleotide sequence of a similar polypeptide deduced using the genetic code and obtainable by conventional means, in particular by reverse transcription of its amino acid sequence using the genetic code.

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 invention lacks N-glycans with M6P or bis-M6P or lacks the ability to bind cation-independent mannose-6-phosphate receptors (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 with M6P and/or bis-M6P or has the ability to bind to CIMPR.

The rhGAA molecule in the rhGAA composition of the invention may have 1, 2,3, or 4M 6P groups on its glycans. For example, the only one N-glycan on the rhGAA molecule may carry M6P (mono-phosphorylated), a single N-glycan may carry two M6P groups (di-phosphorylated), or two different N-glycans on the same rhGAA molecule may carry a single M6P group. The rhGAA molecule in the rhGAA composition may also have an N-glycan with no M6P group. In another embodiment, the N-glycans comprise greater than 3mol/mol of M6P and greater than 4mol/mol of sialic acid, on average. On average, at least about 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total glycans on rhGAA may be in the mono-M6P glycan form, e.g., about 6.25% of the total glycans may 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 rhGAA are in the bis-M6P glycan form, and on average less than 25% of the total rhGAA of the invention does not comprise phosphorylated glycans bound to CIMPR.

The rhGAA composition according to the invention may have an average content of M6P-bearing N-glycans ranging from 0.5 to 7.0mol/mol rhGAA, or any intermediate value of a subrange 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 7.0mol/mol rhGAA. As shown in the examples, the rhGAA of the present invention can be fractionated to provide rhGAA compositions having different average numbers of glycans with M6P or with bis-M6P on rhGAA, thereby allowing further customization of rhGAA targeted to lysosomes in target tissues by selecting specific fractions or by selectively combining different fractions.

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

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

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

In other embodiments of the invention, 40%, 45%, 50%, 55% to 60% of the total N-glycans on rhGAA in the composition are complex N-glycans; or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7% of the total N-glycans on rhGAA in the composition are hybrid N-glycans; no more than 5%, 10%, or 15% of the high mannose type N-glycans on rhGAA in the composition are non-phosphorylated; at least 5% or 10% of the high mannose type N-glycans on rhGAA in the composition are mono-M6P phosphorylated; and/or at least 1% or 2% of the high mannose type N-glycans on rhGAA in the composition are bis-M6P phosphorylated. These values include all intermediate values and subranges. The rhGAA composition according to the present invention may satisfy one or more of the content ranges described above.

In some embodiments, the rhGAA composition of the invention will have an average of 2.0 to 8.0 sialic acid residues per mole of rhGAA. This range includes all intermediate values and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 residues per mole of rhGAA. Sialic acid residues may prevent unproductive clearance of asialoglycoprotein receptors.

The rhGAA composition of the invention is 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 or other variant GAA amino acid sequences of GAA (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 the production of such DNA constructs.

The inventors have discovered that Chinese Hamster Ovary (CHO) cells can be used to produce rhGAA with excellent ability to target CIMPR and cellular lysosomes as well as glycosylation patterns that reduce non-productive clearance thereof in vivo. 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, e.g., as exemplified by rhGAA ATB-200 described in the examples, has greater than conventional GAA (e.g., as exemplified by) Significantly more M6P and bis-M6P groups targeted to muscle cells, and have been shown to bind effectively to CIMPR and be taken up efficiently by skeletal and cardiac muscle. It has also been shown to have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces non-productive clearance in vivo.

The rhGAA according to the invention may be formulated as a pharmaceutical composition or for the manufacture of a medicament for the treatment of pompe disease or other disorders associated with the absence of GAA. These compositions may be formulated with physiologically acceptable carriers or excipients. The carriers and compositions may be sterile and are otherwise suitable for administration.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl carbinol, polyethylene glycols, gelatin, carbohydrates (e.g., lactose, amylose, or starch), sugars (e.g., mannitol, sucrose, or others), glucose, magnesium stearate, talc, silicic acid, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like, and combinations thereof. If desired, the pharmaceutical preparations can be mixed with auxiliaries, such as, for example, surfactants (e.g. polysorbates, like polysorbate 80), lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors, and/or aromatic substances, which do not deleteriously react with the active composition. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

The composition or medicament may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The compositions may also be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like. In a preferred embodiment, the rhGAA is administered by intravenous infusion.

The composition or medicament may be formulated according to conventional procedures as a pharmaceutical composition suitable for administration to a human. For example, in a preferred embodiment, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If necessary, the composition may further include a solubilizing agent and a local anesthetic to relieve pain at the injection site. Typically, the ingredients are provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. Where the composition is administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the compositions are administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

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

rhGAA (or a composition or medicament comprising GAA) is administered by an appropriate route. In one embodiment, the GAA is administered intravenously. In other embodiments, GAA is administered by direct administration to a target tissue (such as to the heart or skeletal muscle (e.g., intramuscularly)) or to the nervous system (e.g., direct injection into the brain; intracerebroventricular; intrathecal). More than one route may be used simultaneously if desired.

The rhGAA (or a composition or medicament comprising 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 reducing symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also reducing the severity or frequency of symptoms of the disease). The amount therapeutically effective in treating a disease will depend on the nature and extent of the effect of the disease and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. The precise dose employed will also depend on the route of administration and the severity of the disease and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. In a preferred embodiment, the therapeutically effective amount is equal to or less than 20mg enzyme/kg body weight of the individual, preferably in the range of about 1-10mg enzyme/kg body weight, and even more preferably about 10mg enzyme/kg body weight or about 5mg enzyme/kg body weight. The effective dose for a particular individual may vary (e.g., increase or decrease) over time, depending on the needs of the individual. For example, the amount may be increased at the time of a physiological disorder or physical stress, or if anti-GAA antibodies are present or increased, or if the symptoms of the disorder worsen.

A therapeutically effective amount of GAA (or a composition or medicament comprising GAA) is administered at regular intervals, depending on the nature and extent of the disease effect, as well as the ongoing basis. As used herein, "administering at" regular intervals "means that the therapeutically effective amount is administered periodically (as distinguished from a single dose). The interval may be determined by standard clinical techniques. In a preferred embodiment, monthly, every two months; weekly; twice a week; or GAA is administered daily. The administration interval for a single individual need not be a fixed interval, but may vary over time, depending on the needs of the individual. For example, in the case of a physiological disorder or physical stress, if an anti-GAA antibody is present or increases, or if the symptoms of the disorder worsen, the interval between doses may be reduced. In some embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200mg enzyme per kg body weight is administered twice weekly, or every other week, with or without a chaperone.

The GAA or rhGAA of the invention may be prepared for later use, such as in unit dose vials or syringes, or in bottles or bags for intravenous administration. A kit comprising GAA or rhGAA, together with optional excipients or other active ingredients, such as a chaperone or other drug, may be enclosed in a packaging material and accompanied by instructions for rehydration, dilution or dosing for use in the treatment of a subject in need thereof, such as a patient with pompe disease.

GAA (or a composition or medicament comprising GAA) may be administered alone or in combination with other agents, such as chaperones. rhGAA with different degrees of glycosylation from mono-M6P or bis-M6P or a combination of rhGAA with different degrees of M6P or bis-M6P glycosylation can be administered.

In some embodiments, the rhGAA compositions of the invention will be complexed or mixed with a chaperone (e.g., AT-2220 or AT-2221). Chaperones, sometimes referred to as "pharmacological chaperones," are compounds that alter their pharmacokinetic and other pharmacological properties when complexed or co-administered with rhGAA. Representative chaperones exemplified herein include AT2221 (McGrestat, N-butyl-deoxynojirimycin) and AT2220(duvoglustat HCl, 1-deoxynojirimycin). This complexing or mixing can occur outside or inside the body, for example, where separate doses of rhGAA and chaperone are administered. For example, the targeting of the active rhGAA, fraction or derivative thereof, to the CIMPR and subsequently to the lysosomes of cells of the invention can be improved by combining it with duvoglustat-HCl (AT2220, deoxynojirimycin, AT2220) or magastrat (AT2221, N-butyl-deoxynojirimycin). The following example shows a significant reduction in glycogen substrate in critical skeletal muscle of GAA knockout mice that received the well-targeted rhGAA of the present invention in combination with a chaperone.

Another aspect of the invention relates to a CHO cell producing rhGAA according to the invention or a derivative or other equivalent thereof. An example of such a CHO cell line is GA-ATB-200 or its progeny culture producing a rhGAA composition as described herein. Such CHO cell lines may comprise multiple copies, e.g., 5, 10, 15 or 20 or more copies, of the gene of the polynucleotide encoding GAA.

The high M6P and bis-M6P rhGAA (e.g., ATB-200 rhGAA) of the invention can be produced by transforming CHO cells (Chinese hamster ovary cells) with a GAA-encoding DNA construct. Although CHO cells were previously used to prepare rhGAA, it was not appreciated that transformed CHO cells could be cultured and selected in a manner that produced rhGAA with high levels of CIMPR-targeted M6P and bis-M6P glycans.

Surprisingly, the inventors found that it was possible to transform CHO cell lines, select transformants that produce rhGAA comprising high content of glycans with CIMPR-targeted M6P or bis-M6P, and stably express the high-M6P rhGAA. Accordingly, a related aspect of the invention relates to methods for producing these CHO cell lines. The method involves transforming CHO cells with DNA encoding GAA or GAA variants, selecting CHO cells that stably integrate the DNA encoding GAA into one or more chromosomes thereof and stably express GAA, and selecting CHO cells that express GAA with a high content of glycans with M6P or bis-M6P, and optionally selecting CHO cells with N-glycans with a high sialic acid content, and/or with N-glycans with a low non-phosphorylated high-mannose content.

The rhGAA and rhGAA compositions according to the invention can be produced using CHO cell lines which are cultured and the compositions recovered from the CHO cell culture.

The rhGAA composition of the invention, or a fraction or derivative thereof, is advantageously used to treat a subject having a condition, disorder or disease associated with lysosomal GAA deficiency by administering the rhGAA composition. Subjects in need of treatment include those with glycogen storage disease type II (pompe disease) as well as other conditions, disorders, or diseases that would benefit from administration of rhGAA.

The following example shows that rhGAA (ATB-200) of the present invention 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. Up to 75% reduction in glycogen in skeletal muscle myoblasts was obtained using a biweekly regimen of intravenous ATB-200 administration in knockout GAA mice. These reductions are more than the same amountProvided, this indicates that rhGAA of the present invention having enhanced content of N-glycans with M6P and bis-M6P provides greater reduction of glycogen substrates. Due to the improved targeting, the pharmacodynamics and pharmacokinetics of the rhGAA compositions of the invention can be compared to conventional rhGAA products (e.g., in the case of conventional rhGAA products)Or) Lower doses were administered.

It can be used to degrade, reduce or remove glycogen from cardiac muscle, smooth muscle, or striated muscle. Examples of skeletal or striated muscle subjects undergoing treatment include at least one muscle selected from the group consisting of: little toe extensor (foot), little finger extensor (hand), thumb extensor, short thumb extensor, long thumb extensor, adductor brevis, adductor, adductor longus, adductor magnus, adductor hallucis thumb, elbow, muscle of the elbow joint, knee joint, arytenosynovitis (aryteglotticus), aryjordanicus, ear muscle, biceps brachii, biceps femoris, brachii, muscle of the brachium, radial muscle, buccoderm, hypopharynx, constrictor pharyngis, suprapharyngeal constrictor, brachial muscle, rugus, levator testimonium, cricothyroid, sarcolemma, profunda transection, deltoid muscle, hypogastrium, diaphragmatic muscle, digastrus, digastrium (anterior), erector-spinalis muscle, erector-ilium muscle, erector costalis-longissimus muscle, short radial extensor, brachialis radialis, extensor ulnar muscle, extensor carpus ulnar muscle, extensor longus, extensor brachium, brachium elongator (short wrist) brachium, extensor index (extensor index), extensor pollicis brevis, extensor pollicis longus, flexor abdominis, flexor radialis, flexor ulnaris, flexor digitorum minor, flexor hallucis major, frontalis, gastrocnemius, flexor inferior, flexor superior, genioglossus, musculus genioglossus, gluteus maximus, gluteus medius gluteus, gluteus minimus, gracilis, glossoglossus, ilius, flexor digitorum inferior, rectus, infraspinatus, extracostalis, profundalis, internus, intestinalis, oblique, dorsi-dorsum of hand, latissimus, dorsi-dorsum of foot, dorsum manus, periforamina, peridactylus, dorsi, levator dorsi, vastus indicus, vastus, levator dorsum, vastus indicus, vastus indicus, levator dorsum, vastus indicus, vastus dorsum, vastus indicus, vastus indicus, vastus indicus, vastus indicus, vastus indictus indicus, vastus indicus, vastus indicus, vastus indicus, vastus indicus, vastus, levator ani-cauiliacus muscle, levator ani-pubococcus muscle, levator ani-puborectalis muscle, levator ani-pubovaginalis muscle, levator labialis, alar (alaeque nasi), levator palpebrae, levator scapulae, levator veli palatini (levator veli palatine), levator costalis, levator capitalis, levator cervicales, lumbricus (4), lumbricus, masseter muscle, infradentalis, uvula palatoses, mylohyoid, nasopharyngis, oblique arytenosis, oblique subtopictus, oblique cephaleus, external obturator foramen, internal obturator foramen (a), internal obturator foramen (B), hyoglossus scapulae muscle, metacarpus little finger, metacarpus, eye orbicularis muscle, peroneus oralis muscle, peronosus palatopharyngeusialis, palatopharyngeusis, pubis muscle, peripharaotus brevis muscle, vastus manus, vastus palmaris, periapical flexor palatus muscle, peroneus palmaris, peroneus, levator palmaris, peroneus versis, peroneus, levator palmaris, peroneus, levator palmaris, vastus palmaris, vastus palmaris, vastus palmaris, vastus palm, Metatarsus, platysma, popliteal, critenoid posterior, glabellar glabellus, quadratus pronator, teres supinator, psoas major, psoas minor, pyramidal, quadratus femoris, psoas, quadratus plantaris, rectus abdominis, rectus capitis rectus (rectus capitis orientalis), rectus capitis postcephalus, rectus capitis posterosus, rectus femoris, rhombus major, smilus major, eustachyopharynx, sartorius, trapezius, canthus, semimembranosus, semiaponeurosis, serratus inferior posterior, superior posterior, serratus, sphincter ani, sphincter sternum, sphincter ani, levator, musculus cervicis, stapes, papilla, musculus hyoglossus, musculus stylosus, stylus, stylus hyoglossus, musculus stylus, hyoglossus, hyoid (bcid), anterior hyoplastus), supraspina muscle, and supraspina muscle (superior), supraspina muscle, musculus striatus, musculus, hyoplastic muscle, supraspina, and superior muscle (superior), anterior striatus), superior scaphis, superior muscle, superior scapus striatus, superior muscle, superior costus striatus, superior muscle, Superior oblique, superior rectus, supinator, superior spinatus, temporal, parietal, tensor fasciae latae, tensor tympanic membrane, tensor palatine, greater circular, small circular, arytenoid and vocal cords, thyroid-epiglottitis, thyroid hyoid, tibialis anterior, tibialis posterior, arytenosynovium, transverse spinatus-multifidus, transverse spinatus-gyrus, transverse spinatus-hemispinatus, abdominus, transverse pectoralis, trapezius, triceps, intermedius femoris, vastus lateralis, vastus medialis, zygosus, and vastus zygosus.

The GAA compositions of the invention can also be administered to or used to treat type 1 (slow contraction) muscle fibers or type 2 (fast contraction) muscle fibers or subjects that accumulate glycogen in these muscle fibers. Type I, slowly contracting or "red" muscle, is enriched in capillaries, and is enriched in mitochondria and myoglobin, which gives muscle tissue its characteristic red color. It can carry more oxygen and use fats or carbohydrates as fuel to maintain aerobic activity. Slow contracting muscle fibers contract for a long time, but with little strength. Type II, the tachyconstrictor muscle has three major subtypes (IIa, IIx and IIb) that differ in both contraction velocity and force produced. The fast-shrinking fibers shrink rapidly and powerfully, but fatigue is very rapid, only persisting briefly, with an explosion of anaerobic activity before muscle contraction becomes painful. They contribute most to muscle strength and have the potential for greater mass gain. Type IIb is anaerobic, glycolytic, and is the "white" muscle with the least density in mitochondria and myoglobin. In small animals (e.g., rodents), this is the major fast muscle type, which explains the pale color of their flesh.

The rhGAA composition, fraction or derivative thereof, of the invention can be administered systemically, e.g., by Intravenous (IV) infusion, or directly to a desired site, e.g., into the cardiac or skeletal muscle (e.g., quadriceps, triceps, or diaphragm). It can be administered to a muscle cell, a specific muscle tissue, a muscle or a group of muscles. For example, such treatment may administer the rhGAA composition intramuscularly directly to the quadriceps or triceps or diaphragm of the subject.

As mentioned above, the rhGAA composition of the present invention, a fraction or derivative thereof, may be complexed or mixed with a chaperone such as AT-2220(Duvoglustat HCl, 1-deoxynojirimycin) or AT-2221 (megrasta, N-butyl-deoxynojirimycin) or a salt thereof to improve the pharmacokinetics of the administration of the rhGAA. The rhGAA and the chaperone may be administered together or separately. When administered simultaneously, the GAA in the composition may be pre-loaded with the chaperone. Alternatively, the GAA and chaperone may be administered separately at the same time or at different times.

Representative doses of AT2221 range from 0.25 to 400mg/kg, preferably from 0.5-200mg/kg, and most preferably from 2 to 50 mg/kg. Specific doses of AT2221 include 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mg/kg. These doses can be combined with rhGAA (e.g., ATB-200 rhGAA) AT a molar ratio 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. rhGAA and AT2221 may be co-administered simultaneously, sequentially or separately in these amounts or molar ratios. The above ranges include all intermediate sub-ranges and values, such as all integer values between the range endpoints.

Representative doses of AT2220 range from 0.1 to 120mg/kg, preferably 0.25 to 60mg/kg, and most preferably from 0.6 to 15 mg/kg. Specific doses of AT2220 include 1, 2,3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25 and 30 mg/kg. These doses can be combined with rhGAA (e.g., ATB-200 rhGAA) AT a molar ratio 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. rhGAA and AT2220 may be co-administered simultaneously, sequentially or separately in these amounts or molar ratios. The above ranges include all intermediate sub-ranges and values, such as all integer values between the range endpoints.

The rhGAA composition of the invention, fractions or derivatives thereof, can also be used to metabolize, degrade, remove, or otherwise reduce glycogen in tissues, muscle fibers, muscle cells, lysosomes, organelles, cellular compartments, or cytoplasm. By administering the rhGAA composition to a subject, optionally with a chaperone or drug that reduces an immune response to rhGAA.

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

The rhGAA according to the invention may also be used (withEnriched in mono-M6P and bis-M6P compared to Myozyme, and which have favorable pharmacokinetic properties conferred by their glycosylation pattern) for use in the treatment of other disorders requiring breakdown of complex carbohydrates, such as other disorders in which glycogen or other carbohydrates degraded by rhGAA accumulate in lysosomes or other parts of the cell (such as in the cytoplasm accessible to rhGAA), e.g., glycogen storage disease III. It can also be used for non-therapeutic purposes, such as in the production of food, beverages, chemicals and pharmaceutical products that require the breakdown of complex carbohydrates (such as starch and glycogen) into their monomers.

Examples of the invention

The following non-limiting examples illustrate various aspects of the invention.

Part I: ATB-200 rhGAA and its Properties

Existing And limitation of hGAA products

To evaluate rhGAA inAnd(currently the only approved treatment for pompe disease), these rhGAA formulations were injected onto a CIMPR column (which binds rhGAA with the M6P group) and subsequently eluted with a free M6 gradient. Fractions were collected in 96-well plates and GAA activity was determined by 4 MU-alpha-glucose substrate. The relative amounts of bound and unbound rhGAA were determined based on GAA activity and reported as a fraction of total enzyme.

FIG. 5 depicts a graph relating to conventional ERT (R) ((R))And) The related problems are as follows: in that73% of rhGAA (FIG. 5B) and78% of rhGAA (fig. 5A) did not bind to CIMPR, see the leftmost peak in each figure. In thatOnly 27% of rhGAA in the composition and in22% of rhGAA contained M6P, which M6P could productively target it to CIMPR on myocytes, see fig. 2, which fig. 2 depicts productive drug targeting and non-productive drug clearance.

Andthe effective dose of (a) corresponds to an amount of rhGAA comprising M6P, which M6P targets CIMPR on muscle cells. However, most of the rhGAA in these two conventional products did not target the CIMPR receptor on the target myocytes. Administration of conventional rhGAA in which most of the rhGAA is not targeted to muscle cells increases the risk of allergic reactions or induction of immunity to non-targeted rhGAA.

Production of ATB-200 with high content of N-glycans with mono-M6P or bis-M6P Of CHO cells of rhGAA And (4) preparation.

CHO cells were transfected with rh-GAA expressing DNA, and rhGAA-producing transformants were subsequently selected. The DNA construct used to transform CHO cells with DNA encoding rh-GAA is shown in FIG. 5. CHO cells were transfected with rh-GAA expressing DNA, and rhGAA-producing transformants were subsequently selected.

Following transfection, hypoxanthine/thymidine deficient (-HT) medium was used to select DG44CHO (DHFR-) cells containing a stably integrated GAA gene. Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Pools of cells expressing large amounts of GAA were identified by GAA enzyme activity assays and used to establish single clones producing rhGAA. Single clones were generated on semi-solid culture plates, picked by the clonipax system, and transferred to 24-deep well plates. Individual clones were assayed for GAA enzyme activity to identify clones expressing high levels of GAA. The conditioned media used to determine GAA activity used a 4-MU-alpha-glucosidase substrate. Clones producing higher levels of GAA as measured by GAA enzyme assay were further evaluated for viability, growth capacity, GAA productivity, N-glycan structure and stable protein expression. This procedure was used to isolate CHO cell lines, including the CHO cell line GA-ATB-200 expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans.

rhGAA ATB-200 Purification of rhGAA

Batches of rhGAA according to the invention were produced in shake flasks and binding was measured in a perfusion bioreactor using the CHO cell lines GA-ATB-200 and CIMPR. For purified ATB-200rhGAA from different production batches, similar CIMPR receptor binding (about 70%) to those shown in fig. 6B and fig. 7 was observed, indicating that ATB-200rhGAA could be consistently produced. As shown in fig. 6A and 6B, compared to ATB200rhGAA,andrhGAA showed significantly less CIMPR binding.

Analytical comparison of ATB-200 with Lumizyme

Fractionation of ATB-200rhGAA was performed according to terminal phosphate using weak anion exchange ("WAX") liquid chromatography. The elution profile was generated by eluting ERT with increasing amounts of salt. The curve was monitored by UV (a280 nm). ATB-200rhGAA was obtained from CHO cells and purified.Obtained from commercial sources.A high peak is shown on the left side of the elution curve. ATB-200rhGAA showed elution inFour prominent peaks on the right (fig. 8). Since this assessment was by terminal charge rather than by CIMPR affinity, this confirmed that ATB-200rhGAA was phosphorylated to a specific valueTo a greater extent.

Oligosaccharide characterization of ATB-200rhGAA

Evaluation of purified ATB-200rhGAA and by MALDI-TOFGlycans to determine the single glycan structure found on each ERT (figure 9). The ATB-200 sample was found to contain the ratioA slightly smaller amount of non-phosphorylated high mannose type N-glycans. The higher content of M6P glycan in ATB-200 more efficiently targets ATB-200rhGAA to muscle cells than Lumizyme. The high percentage of mono-phosphorylated and di-phosphorylated structures determined by MALDI were consistent with the CIMPR curve, which illustrates significantly greater binding of ATB-200 to the CIMPR receptor. N-glycan analysis via MALDI-TOF mass spectrometry confirmed that, on average, each ATB200 molecule contained at least one native bis-M6P N-glycan structure. This higher bis-M6P N-glycan content on ATB-200rhGAA was directly related to high affinity binding to CIMPR in the M6P receptor plate binding assay (K-200 rhGAA)_DApproximately 2-4nM) (FIG. 10A).

Characterization of CIMPR affinity of ATB-200

In addition to having a greater percentage of rhGAA that can bind to CIMPR, it is important to understand the quality of this interaction. Determination using CIMPR plate binding assaysAnd ATB200rhGAA receptor. Briefly, a CIMPR-coated plate was used to capture GAA. Applying rhGAA of different concentrationsTo the immobilized receptor and washing away unbound rhGAA. The amount of rhGAA remaining was determined by GAA activity. As shown in FIG. 10A, ATB-200rhGAA bound to CIMPR is significantly superior to Lumizyme.

FIG. 10B shows the relative amounts of bis-M6P glycans in Lumizyme, conventional rhGAA, and ATB-200 according to the present invention. For theOn average, only 10% of the molecules have di-phosphorylated glycans. This was compared to ATB-200 (where there was an average of at least one di-phosphorylated glycan per rhGAA molecule).

ATB-200 rhGAA is more efficiently internalized by fibroblasts than Lumizyme.

Comparison of ATB-200 and PONPE fibroblast cell linesrelative cellular uptake of rhGAA. Comparison of ATB-200rhGAA according to the invention with 5-100nM and conventional rhGAA with 10-500nMAfter 16-hr incubation, the external rhGAA was inactivated with TRIS base and the cells were washed 3 times with PBS before harvest. Internalized GAA as measured by 4 MU-alpha-glucoside hydrolysis and plotted against total cell protein, and the results appear in fig. 11.

Efficient internalization of ATB-200rhGAA in cells is also shown (FIGS. 11A and 11B), which shows that ATB-200rhGAA internalizes into normal and Pompe fibroblasts, respectively, and is more conventional than conventionalTo a greater extent, rhGAA internalizes. ATB-200rhGAA saturates the cellular receptor at about 20nM, however about 250nM is requiredFrom these results, the followingExtrapolated uptake efficiency constant (K)_{Intake of}) 2-3nm for ATB-200 andat 56nM, as shown in FIG. 11C. These results indicate that ATB-200rhGAA is a well-targeted treatment for Pompe disease.

Part II: preclinical study

ATB-200rhGAA with excellent glycosylation is significantly superior to glycogen for use in skeletal muscle of GAA KO mice Standard of care ERT for clearance

As explained above, Enzyme Replacement Therapy (ERT) using recombinant human gaa (rhgaa) is the only approved treatment available for pompe disease. This ERT requires the specialized carbohydrate mannose 6-phosphate (M6P) for cellular uptake and subsequent delivery to lysosomes via cell surface cation-independent M6P receptors (CIMPR). However, current rhGAA ERT contains a small amount of M6P, which limits drug targeting and efficacy in disease-related tissues. The inventors developed production cell lines and manufacturing methods that produce rhGAA (named ATB-200 rhGAA) with superior glycosylation and higher M6P content (in particular high affinity bis-M6P N-glycan structures) than conventional rhGAA for improved drug targeting. ATB-200rhGAA binds CI-MPR with high affinity (KD about 2-4nM) and is efficiently internalized by Pompe fibroblasts and skeletal myoblasts (K)_{Intake of}About 7-14 nM).

ATB-200rhGAA cleared glycogen significantly better in skeletal muscle thanAssessment administrationAnd the effect of ATB-200rhGAA on glycogen clearance in GAA KO mice. Two bolus intravenous administrations (every other week) to the animals; tissues were harvested two weeks after the last dose and analyzed for GAA activity and glycogen content (fig. 12). ATB-200rhGAA andrhGAA was also effective in clearing glycogen in the heart (fig. 12A). As shown in FIGS. 12B and 12C, 5mg/kg of ATB-200rhGAA corresponds to 20mg/kg for reducing glycogen in skeletal musclerhGAA; ATB-200 administered at 10 and 20mg/kg is significantly superior to that used to clear glycogen in skeletal muscle

Basic principles of co-administration of ATB-200rhGAA and AT2221 (CHART technique)

The chaperone binds to rhGAA ERT and stabilizes rhGAA ERT, increasing the uptake of active enzyme into the tissue, improving tolerance and potentially reducing immunogenicity. As indicated above, CHART was used^TMSubstantially improving the protein stability of ERT under adverse conditions. CHART: advanced chaperone replacement therapy, see http:// _ www.amicusrx.com/chaperone. aspx (last visit of 9/22/2015), which was incorporated by reference. As shown in fig. 13A and 13B, the stability of ATB-200 was significantly improved by AT2221 (megestrol, N-butyl-deoxynojirimycin). The folding of rhGAA protein was monitored by thermal denaturation in neutral (pH 7.4-plasma environment) or acidic (pH 5.2-lysosomal environment) buffers at 37 ℃. AT2220 stabilizes the rhGAA protein in neutral pH buffer over 24 hours.

Compared to co-administration of ATB-200rhGAA and megestrol And AT2221 (Mgesitat) Co-administration of

The 12-week-old GAA KO mice were usedOr ATB200 treatment, 20mg/kg every other weekIV injection is carried out for 4 times; magsitaxel was co-administered at 10mg/kg PO 30 minutes prior to rhGAA as indicated. Tissues were collected for glycogen measurements 14 days after the last enzyme dose. Figure 14 shows the relative reduction in glycogen in quadriceps and triceps skeletal muscle.

Tissue glycogen reduction was achieved with ATB-200rhGAA co-administered with the pharmacological chaperone AT2221 (Magcistat).

The combination of pharmacological chaperones and ATB-200rhGAA was found to enhance glycogen clearance in vivo. GAA KO mice were given two IV bolus administrations of rhGAA at 20mg/kg every other week. Pharmacological chaperone AT2221 was administered orally 30 minutes prior to rhGAA AT doses of 0, 1, 2 and 10 mg/kg. Tissues were harvested two weeks after the last dose of ERT and analyzed for GAA activity, glycogen content cell-specific glycogen, and lysosomal proliferation.

As shown in figure 15, animals receiving the ATB200+ chaperone AT2221 exhibited enhanced glycogen clearance from the quadriceps muscle. ATB-200rhGAA (20mg/kg) reduced glycogen over the same doseAnd when ATB-200rhGAA was combined with 10mg/kg AT2220, near normal levels of glycogen in muscle were obtained.

As shown in fig. 16A and 16B, ATB-200rhGAA alone showed a significant decrease in PAS signal, unlike conventional rhGAA which showed limited glycogen reduction (indicated by abundant dotted PAS signals). Co-administration with 10mg/kg of megestrol resulted in a substantial further reduction of the substrate. TEM revealed that most glycogen in lysosomes is a membrane-bound, electron dense material corresponding to a punctate PAS signal. Co-administration of ATB-200rhGAA with megestrol reduced the number, size and density of lysosomes containing the substrate, suggesting targeted delivery of ATB-200rhGAA to muscle cells and subsequently to lysosomes.

From the studies shown above (2 IV boluses injected every other week), treatment of tissue with the LAMP1 marker for lysosomal proliferation, upregulation is another marker of pompe disease. LAMP: lysosomal associated membrane proteins. From the study shown above (2 IV bolus EOW injections), soleus muscle tissue was treated in adjacent sections for LAMP1 staining and type I fiber-specific antibodies (NOQ7.5.4D) were added to adjacent sections (fig. 16C and 16D). Compared to conventional rhGAA, ATB-200rhGAA resulted in a greater reduction of LAMP1, which resulted in levels seen in WT animals (fig. 16C).

Furthermore, unlike rhGAA, where the effect is primarily confined to type I fibers (slow shrinkage, marked with an asterisk), ATB-200rhGAA also resulted in a significant reduction in LAMP1 signal in a portion of type II (fast shrinkage) fibers (red arrows) (fig. 16D). Importantly, co-administration with magastrat further improved the ATB-200 mediated reduction of proliferation of LAMP1 in most type II fibers (fig. 16C and 16D). As a result, it appears that no significant fiber type specific differences appear at the LAMP1 signal level. Similar conclusions were obtained from the quadriceps and diaphragm muscles (data not shown).

In a separate and similarly designed study, the effect of ATB-200. + -. AT2221 was examined over a longer period with 4 biweekly IV bolus injections. In the heart, major glycogen stores in cardiomyocytes were readily cleared by repeated administration of rhGAA or ATB-200 to levels seen in Wild Type (WT) animals (fig. 17A). However, it appears that the substrate in cardiac smooth muscle cells is preferentially cleared by ATB-200rhGAA, suggesting a potentially broader biodistribution of ATB-200 compared to rhGAA (asterisk marks the lumen of the heart vessels). Importantly, co-administration with megestrol further improved the ATB-200 mediated reduction of proliferation of LAMP 1.

These results indicate that ATB-200rhGAA (which has higher levels of M6P and bis-M6P on its N-glycans) effectively targets CIMPR in skeletal muscle. ATB-200rhGAA also has well-processed complex N-glycans (which minimize non-productive clearance in vivo), has pharmacokinetic properties that facilitate its use in vivo, and exhibits good targeting of critical muscle tissues in vivo. They also showed that ATB-200rhGAA was superior to the conventional standard of care Lumizyme for reducing glycogen in muscle tissue, and that the combination of ATB-200rhGAA and the molecular chaperone AT2221 further improved removal of glycogen from the target tissue and improved muscle pathology.

Sequence listing

<110> Amikuis THERAPEUTICS, Inc. (AMICUS THERAPEUTICS, INC.)

<120> high strength acidic alpha-glucosidase with enhanced carbohydrate

<130> TWLB7360-17P8

<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 version 3.5

<210> 1

<211> 952

<212> PRT

<213> Intelligent people

<220>

<221> features not yet classified

<222> (1)..(952)

<223> sequence 4 from the gene bank of patent US 8592362: 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 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 Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 2

<211> 3624

<212> DNA

<213> Intelligent people

<220>

<221> CDS

<222> (220)..(3078)

<223> homo sapiens GAA mRNA of lysosomal α -glucosidase (acid maltase); gene bank: 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> Intelligent people

<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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

<210> 4

<211> 952

<212> PRT

<213> Intelligent people

<220>

<221> features not yet classified

<222> (1)..(952)

<223> lysosomal α -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 Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Glu Gln Phe Leu Val Ser Trp Cys

945 950

Claims (22)

1. A composition comprising recombinant human alpha glucosidase (rhGAA) produced by Chinese Hamster Ovary (CHO) cells, wherein the CHO cells express rhGAA as set forth in SEQ ID No. 4, which is a 110kDa precursor form of the rhGAA; wherein 40% to 60% of the N-glycans on the rhGAA are complex N-glycans; and the rhGAA comprises 3.0 to 5.0mol of M6P residues per mol of rhGAA.

2. The composition of claim 1, wherein 90% to 100% of the rhGAA binds to cation-independent mannose-6-phosphate receptor (CIMPR).

3. The composition of claim 1, wherein 45% to 55% of the N-glycans on rhGAA in the composition are complex N-glycans.

4. The composition of claim 1 wherein the rhGAA comprises 3.0 to 4.0mol M6P/mol rhGAA.

5. The composition of claim 1 wherein the rhGAA comprises 4.0 to 5.0mol M6P/mol rhGAA.

6. The composition of claim 1, wherein the rhGAA comprises at least 4mol sialic acid residues per mol rhGAA.

7. The composition of claim 1, wherein each rhGAA comprises at least one bisphosphorylated N-glycan.

8. The composition of claim 1 wherein the rhGAA comprises 2.0 to 8.0mol sialic acid residues per molrhGAA.

9. The composition of claim 1, further comprising a pharmacological chaperone.

10. A method for making the composition of claim 1, comprising culturing a CHO cell line that produces the rhGAA and recovering the rhGAA from the CHO cell culture.

11. A CHO cell line producing the composition of claim 1.

12. A method of making a CHO cell line according to claim 11, comprising transforming CHO cells with DNA encoding rhGAA, selecting CHO cells stably integrating the DNA encoding rhGAA into one or more chromosomes thereof and stably expressing rhGAA, and selecting CHO cells expressing rhGAA having a high content of glycans with mono-M6P or bis-M6P, and optionally selecting CHO cells having N-glycans with a high sialic acid content and/or having N-glycans with a low non-phosphorylated high-mannose content.

13. Use of the composition of claim 1 in the manufacture of a medicament for treating a disorder, condition, or disease associated with a lysosomal rhGAA deficiency in a subject in need thereof.

14. The use of claim 13, wherein the subject has glycogen storage disease type II (pompe disease).

15. The use of claim 13, wherein the medicament is administered to the myocardium of the subject.

16. The use of claim 13, wherein said medicament is administered to the quadriceps, triceps, or other skeletal muscle of the subject.

17. The use of claim 13, wherein the medicament is administered to the diaphragm muscle of the subject.

18. The use of claim 13, wherein said medicament further comprises a pharmacological chaperone, or wherein said medicament is co-administered or separately administered with a pharmacological chaperone.

19. The use of claim 18, wherein the pharmacological chaperone is AT2220 or a pharmaceutically acceptable salt thereof.

20. The use of claim 18, wherein the pharmacological chaperone is AT2221 or a pharmaceutically acceptable salt thereof.

21. Use of the composition of claim 1 in the manufacture of a medicament for metabolizing, degrading, removing or otherwise reducing glycogen in a tissue, muscle fiber, muscle cell, lysosome, organelle, cellular compartment, or cytoplasm in a subject in need thereof, wherein the medicament is optionally administered with a pharmacological chaperone and/or an agent that reduces the immune response to rhGAA.

22. Use of the composition of claim 1 in the manufacture of a medicament for modulating lysosomal proliferation or autophagy in a cell of a subject in need thereof, wherein the medicament is administered to the subject, optionally with a pharmacological chaperone.